Haoxing Xu

The view of the lysosome as the terminal end of cellular catabolic pathways has been challenged by recent studies showing a central role of this organelle in the control of cell function. Here we show that a lysosomal Ca2+ signalling mechanism controls the activities of the phosphatase calcineurin and of its substrate TFEB, a master transcriptional regulator of lysosomal biogenesis and autophagy. Lysosomal Ca2+ release through mucolipin 1 (MCOLN1) activates calcineurin, which binds and dephosphorylates TFEB, thus promoting its nuclear translocation. Genetic and pharmacological inhibition of calcineurin suppressed TFEB activity during starvation and physical exercise, while calcineurin overexpression and constitutive activation had the opposite effect. Induction of autophagy and lysosomal biogenesis through TFEB required MCOLN1-mediated calcineurin activation. These data link lysosomal calcium signalling to both calcineurin regulation and autophagy induction and identify the lysosome as a hub for the signalling pathways that regulate cellular homeostasis. Medina, Ballabio and colleagues report that calcium release from the lysosome stimulates calcineurin, which dephosphorylates and activates TFEB. These findings reveal a central role for calcium signalling in autophagy and lysosome homeostasis.

Lysosomes are acidic compartments filled with more than 60 different types of hydrolases. They mediate the degradation of extracellular particles from endocytosis and of intracellular components from autophagy. The digested products are transported out of the lysosome via specific catabolite exporters or via vesicular membrane trafficking. Lysosomes also contain more than 50 membrane proteins and are equipped with the machinery to sense nutrient availability, which determines the distribution, number, size, and activity of lysosomes to control the specificity of cargo flux and timing (the initiation and termination) of degradation. Defects in degradation, export, or trafficking result in lysosomal dysfunction and lysosomal storage diseases (LSDs). Lysosomal channels and transporters mediate ion flux across perimeter membranes to regulate lysosomal ion homeostasis, membrane potential, catabolite export, membrane trafficking, and nutrient sensing. Dysregulation of lysosomal channels underlies the pathogenesis of many LSDs and possibly that of metabolic and common neurodegenerative diseases.

Membrane fusion and fission events in intracellular trafficking are controlled by both intraluminal Ca(2+) release and phosphoinositide (PIP) signalling. However, the molecular identities of the Ca(2+) release channels and the target proteins of PIPs are elusive. In this paper, by direct patch-clamping of the endolysosomal membrane, we report that PI(3,5)P(2), an endolysosome-specific PIP, binds and activates endolysosome-localized mucolipin transient receptor potential (TRPML) channels with specificity and potency. Both PI(3,5)P(2)-deficient cells and cells that lack TRPML1 exhibited enlarged endolysosomes/vacuoles and trafficking defects in the late endocytic pathway. We find that the enlarged vacuole phenotype observed in PI(3,5)P(2)-deficient mouse fibroblasts is suppressed by overexpression of TRPML1. Notably, this PI(3,5)P(2)-dependent regulation of TRPML1 is evolutionarily conserved. In budding yeast, hyperosmotic stress induces Ca(2+) release from the vacuole. In this study, we show that this release requires both PI(3,5)P(2) production and a yeast functional TRPML homologue. We propose that TRPMLs regulate membrane trafficking by transducing information regarding PI(3,5)P(2) levels into changes in juxtaorganellar Ca(2+), thereby triggering membrane fusion/fission events.

Mutations in the human TRPML1 gene, a member of the transient receptor potential (TRP) superfamily of ion channels, cause mucolipidosis type IV disease. Symptoms of the condition include anaemia, psychomotor retardation and retinal degeneration. Xian-ping Dong et al. now show that TRPML1 acts as a Fe2+-permeable channel in lysosomes, and that disease-associated mutations impair Fe2+ transport. The work suggests that impaired iron transport underlies symptoms of mucolipidosis, including neurodegeneration, and that lysosome-targeting chelators might alleviate the degenerative symptoms of patients with mucolipidosis type IV. TRPML1 is a member of the Transient Receptor Potential (TRP) superfamily of ion channels, and mutation in the human TRPML1 gene causes mucolipidosis, symptoms of which include anaemia. It is shown that TRPML1 functions as a Fe2+-permeable channel in lysosomes, and that disease-associated mutations impair Fe2+transport, suggesting that impaired iron transport may underlie symptoms of mucolipidosis. TRPML1 (mucolipin 1, also known as MCOLN1) is predicted to be an intracellular late endosomal and lysosomal ion channel protein that belongs to the mucolipin subfamily of transient receptor potential (TRP) proteins1,2,3. Mutations in the human TRPML1 gene cause mucolipidosis type IV disease (ML4)4,5. ML4 patients have motor impairment, mental retardation, retinal degeneration and iron-deficiency anaemia. Because aberrant iron metabolism may cause neural and retinal degeneration6,7, it may be a primary cause of ML4 phenotypes. In most mammalian cells, release of iron from endosomes and lysosomes after iron uptake by endocytosis of Fe3+-bound transferrin receptors6, or after lysosomal degradation of ferritin–iron complexes and autophagic ingestion of iron-containing macromolecules6,8, is the chief source of cellular iron. The divalent metal transporter protein DMT1 (also known as SLC11A2) is the only endosomal Fe2+ transporter known at present and it is highly expressed in erythroid precursors6,9. Genetic studies, however, suggest the existence of a DMT1-independent endosomal and lysosomal Fe2+ transport protein9. By measuring radiolabelled iron uptake, by monitoring the levels of cytosolic and intralysosomal iron and by directly patch-clamping the late endosomal and lysosomal membrane, here we show that TRPML1 functions as a Fe2+ permeable channel in late endosomes and lysosomes. ML4 mutations are shown to impair the ability of TRPML1 to permeate Fe2+ at varying degrees, which correlate well with the disease severity. A comparison of TRPML1-/-ML4 and control human skin fibroblasts showed a reduction in cytosolic Fe2+ levels, an increase in intralysosomal Fe2+ levels and an accumulation of lipofuscin-like molecules in TRPML1-/- cells. We propose that TRPML1 mediates a mechanism by which Fe2+ is released from late endosomes and lysosomes. Our results indicate that impaired iron transport may contribute to both haematological and degenerative symptoms of ML4 patients.

The pore-forming subunits of canonical voltage-gated sodium and calcium channels are encoded by four repeated domains of six-transmembrane (6TM) segments. We expressed and characterized a bacterial ion channel (NaChBac) from Bacillus halodurans that is encoded by one 6TM segment. The sequence, especially in the pore region, is similar to that of voltage-gated calcium channels. The expressed channel was activated by voltage and was blocked by calcium channel blockers. However, the channel was selective for sodium. The identification of NaChBac as a functionally expressed bacterial voltage-sensitive ion-selective channel provides insight into both voltage-dependent activation and divalent cation selectivity.

Mammalian two-pore channel proteins (TPC1, TPC2; TPCN1, TPCN2) encode ion channels in intracellular endosomes and lysosomes and were proposed to mediate endolysosomal calcium release triggered by the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). By directly recording TPCs in endolysosomes from wild-type and TPC double-knockout mice, here we show that, in contrast to previous conclusions, TPCs are in fact sodium-selective channels activated by PI(3,5)P(2) and are not activated by NAADP. Moreover, the primary endolysosomal ion is Na(+), not K(+), as had been previously assumed. These findings suggest that the organellar membrane potential may undergo large regulatory changes and may explain the specificity of PI(3,5)P(2) in regulating the fusogenic potential of intracellular organelles.

Abstract Cellular stresses trigger autophagy to remove damaged macromolecules and organelles. Lysosomes ‘host’ multiple stress-sensing mechanisms that trigger the coordinated biogenesis of autophagosomes and lysosomes. For example, transcription factor (TF)EB, which regulates autophagy and lysosome biogenesis, is activated following the inhibition of mTOR, a lysosome-localized nutrient sensor. Here we show that reactive oxygen species (ROS) activate TFEB via a lysosomal Ca 2+ -dependent mechanism independent of mTOR. Exogenous oxidants or increasing mitochondrial ROS levels directly and specifically activate lysosomal TRPML1 channels, inducing lysosomal Ca 2+ release. This activation triggers calcineurin-dependent TFEB-nuclear translocation, autophagy induction and lysosome biogenesis. When TRPML1 is genetically inactivated or pharmacologically inhibited, clearance of damaged mitochondria and removal of excess ROS are blocked. Furthermore, TRPML1’s ROS sensitivity is specifically required for lysosome adaptation to mitochondrial damage. Hence, TRPML1 is a ROS sensor localized on the lysosomal membrane that orchestrates an autophagy-dependent negative-feedback programme to mitigate oxidative stress in the cell.

Lysosomal lipid accumulation, defects in membrane trafficking and altered Ca2+ homoeostasis are common features in many lysosomal storage diseases. Mucolipin transient receptor potential channel 1 (TRPML1) is the principle Ca2+ channel in the lysosome. Here we show that TRPML1-mediated lysosomal Ca2+ release, measured using a genetically encoded Ca2+ indicator (GCaMP3) attached directly to TRPML1 and elicited by a potent membrane-permeable synthetic agonist, is dramatically reduced in Niemann–Pick (NP) disease cells. Sphingomyelins (SMs) are plasma membrane lipids that undergo sphingomyelinase (SMase)-mediated hydrolysis in the lysosomes of normal cells, but accumulate distinctively in lysosomes of NP cells. Patch-clamp analyses revealed that TRPML1 channel activity is inhibited by SMs, but potentiated by SMases. In NP-type C cells, increasing TRPML1's expression or activity was sufficient to correct the trafficking defects and reduce lysosome storage and cholesterol accumulation. We propose that abnormal accumulation of luminal lipids causes secondary lysosome storage by blocking TRPML1- and Ca2+-dependent lysosomal trafficking. Accumulation of lysosomal lipids is a feature of Niemann'-Picks (NP) disease, but how these lipids contribute to the disease is unclear. In this study, calcium released via the lysosomal TRPML1 channel is shown to be reduced in NP-type C cells, and sphingomyelins are found to inhibit the channel's activity.

Although the PI3K (phosphatidylinositol 3-kinase) pathway typically regulates cell growth and survival, increasing evidence indicates the involvement of this pathway in neural plasticity. It is unknown whether the PI3K pathway can mediate pain hypersensitivity. Intradermal injection of capsaicin and NGF produce heat hyperalgesia by activating their respective TRPV1 (transient receptor potential vanilloid receptor-1) and TrkA receptors on nociceptor sensory nerve terminals. We examined the activation of PI3K in primary sensory DRG neurons by these inflammatory agents and the contribution of PI3K activation to inflammatory pain. We further investigated the correlation between the PI3K and the ERK (extracellular signal-regulated protein kinase) pathway. Capsaicin and NGF induce phosphorylation of the PI3K downstream target AKT (protein kinase B), which is blocked by the PI3K inhibitors LY294002 and wortmannin, indicative of the activation of PI3K by both agents. ERK activation by capsaicin and NGF was also blocked by PI3K inhibitors. Similarly, intradermal capsaicin in rats activated PI3K and ERK in C-fiber DRG neurons and epidermal nerve fibers. Injection of PI3K or MEK (ERK kinase) inhibitors into the hindpaw attenuated capsaicin- and NGF-evoked heat hyperalgesia but did not change basal heat sensitivity. Furthermore, PI3K, but not ERK, inhibition blocked early induction of hyperalgesia. In acutely dissociated DRG neurons, the capsaicin-induced TRPV1 current was strikingly potentiated by NGF, and this potentiation was completely blocked by PI3K inhibitors and primarily suppressed by MEK inhibitors. Therefore, PI3K induces heat hyperalgesia, possibly by regulating TRPV1 activity, in an ERK-dependent manner. The PI3K pathway also appears to play a role that is distinct from ERK by regulating the early onset of inflammatory pain.

Camphor is a naturally occurring compound that is used as a major active ingredient of balms and liniments supplied as topical analgesics. Despite its long history of common medical use, the underlying molecular mechanism of camphor action is not understood. Capsaicin and menthol, two other topically applied agents widely used for similar purposes, are known to excite and desensitize sensory nerves by acting on two members of transient receptor potential (TRP) channel superfamily: heat-sensitive TRP vanilloid subtype 1 (TRPV1) and cold-sensitive TRP channel M8, respectively. Camphor has recently been shown to activate TRPV3, and here we show that camphor also activates heterologously expressed TRPV1, requiring higher concentrations than capsaicin. Activation was enhanced by phospholipase C-coupled receptor stimulation mimicking inflamed conditions. Similar camphor-activated TRPV1-like currents were observed in isolated rat DRG neurons and were strongly potentiated after activation of protein kinase C with phorbol-12-myristate-13-acetate. Camphor activation of rat TRPV1 was mediated by distinct channel regions from capsaicin, as indicated by camphor activation in the presence of the competitive inhibitor capsazepine and in a capsaicin-insensitive point mutant. Camphor did not activate the capsaicin-insensitive chicken TRPV1. TRPV1 desensitization is believed to contribute to the analgesic actions of capsaicin. We found that, although camphor activates TRPV1 less effectively, camphor application desensitized TRPV1 more rapidly and completely than capsaicin. Conversely, TRPV3 current sensitized after repeated camphor applications, which is inconsistent with the analgesic role of camphor. We also found that camphor inhibited several other related TRP channels, including ankyrin-repeat TRP 1 (TRPA1). The camphor-induced desensitization of TRPV1 and block of TRPA1 may underlie the analgesic effects of camphor.

To mediate the degradation of biomacromolecules, lysosomes must traffic towards cargo-carrying vesicles for subsequent membrane fusion or fission. Mutations of the lysosomal Ca2+ channel TRPML1 cause lysosomal storage disease (LSD) characterized by disordered lysosomal membrane trafficking in cells. Here we show that TRPML1 activity is required to promote Ca2+-dependent centripetal movement of lysosomes towards the perinuclear region (where autophagosomes accumulate) following autophagy induction. ALG-2, an EF-hand-containing protein, serves as a lysosomal Ca2+ sensor that associates physically with the minus-end-directed dynactin–dynein motor, while PtdIns(3,5)P2, a lysosome-localized phosphoinositide, acts upstream of TRPML1. Furthermore, the PtdIns(3,5)P2–TRPML1–ALG-2–dynein signalling is necessary for lysosome tubulation and reformation. In contrast, the TRPML1 pathway is not required for the perinuclear accumulation of lysosomes observed in many LSDs, which is instead likely to be caused by secondary cholesterol accumulation that constitutively activates Rab7–RILP-dependent retrograde transport. Ca2+ release from lysosomes thus provides an on-demand mechanism regulating lysosome motility, positioning and tubulation. Following autophagy induction, lysosomes move to the perinuclear region. Xu and colleagues delineate a pathway involving PtdIns(3,5)P2-mediated activation of the TRPML1 channel and the Ca2+ sensor ALG-2 in this process.

A plethora of growth factors regulate keratinocyte proliferation and differentiation that control hair morphogenesis and skin barrier formation. Wavy hair phenotypes in mice result from naturally occurring loss-of-function mutations in the genes for TGF-alpha and EGFR. Conversely, excessive activities of TGF-alpha/EGFR result in hairless phenotypes and skin cancers. Unexpectedly, we found that mice lacking the Trpv3 gene also exhibit wavy hair coat and curly whiskers. Here we show that keratinocyte TRPV3, a member of the transient receptor potential (TRP) family of Ca(2+)-permeant channels, forms a signaling complex with TGF-alpha/EGFR. Activation of EGFR leads to increased TRPV3 channel activity, which in turn stimulates TGF-alpha release. TRPV3 is also required for the formation of the skin barrier by regulating the activities of transglutaminases, a family of Ca(2+)-dependent crosslinking enzymes essential for keratinocyte cornification. Our results show that a TRP channel plays a role in regulating growth factor signaling by direct complex formation.

Trace metals, such as iron, copper, zinc, manganese and cobalt, are essential cofactors for many cellular enzymes. Extensive research on iron, the most abundant transition metal in biology, has contributed to an increased understanding of the molecular machinery involved in maintaining its homeostasis in mammalian peripheral tissues. However, the cellular and intercellular iron-transport mechanisms in the CNS are still poorly understood. Accumulating evidence suggests that impaired iron metabolism is an initial cause of neurodegeneration and several common genetic and sporadic neurodegenerative disorders have been proposed as being associated with dysregulated CNS iron homeostasis. This review aims to provide a summary of the molecular mechanisms of brain iron transport. Our discussion is focused on iron transport across endothelial cells of the blood–brain barrier and within the neuro- and glial-vascular units of the brain, with the aim of revealing novel therapeutic targets for neurodegenerative and CNS disorders.

Our understanding of the intrinsic mechanosensitive properties of human pluripotent stem cells (hPSCs), in particular the effects that the physical microenvironment has on their differentiation, remains elusive. Here, we show that neural induction and caudalization of hPSCs can be accelerated by using a synthetic microengineered substrate system consisting of poly(dimethylsiloxane) micropost arrays (PMAs) with tunable mechanical rigidities. The purity and yield of functional motor neurons derived from hPSCs within 23 days of culture using soft PMAs were improved more than fourfold and tenfold, respectively, compared with coverslips or rigid PMAs. Mechanistic studies revealed a multi-targeted mechanotransductive process involving Smad phosphorylation and nucleocytoplasmic shuttling, regulated by rigidity-dependent Hippo/YAP activities and actomyosin cytoskeleton integrity and contractility. Our findings suggest that substrate rigidity is an important biophysical cue influencing neural induction and subtype specification, and that microengineered substrates can thus serve as a promising platform for large-scale culture of hPSCs.

Phagocytosis of large extracellular particles such as apoptotic bodies requires delivery of the intracellular endosomal and lysosomal membranes to form plasmalemmal pseudopods. Here, we identified mucolipin TRP channel 1 (TRPML1) as the key lysosomal Ca2+ channel regulating focal exocytosis and phagosome biogenesis. Both particle ingestion and lysosomal exocytosis are inhibited by synthetic TRPML1 blockers and are defective in macrophages isolated from TRPML1 knockout mice. Furthermore, TRPML1 overexpression and TRPML1 agonists facilitate both lysosomal exocytosis and particle uptake. Using time-lapse confocal imaging and direct patch clamping of phagosomal membranes, we found that particle binding induces lysosomal PI(3,5)P2 elevation to trigger TRPML1-mediated lysosomal Ca2+ release specifically at the site of uptake, rapidly delivering TRPML1-resident lysosomal membranes to nascent phagosomes via lysosomal exocytosis. Thus phagocytic ingestion of large particles activates a phosphoinositide- and Ca2+-dependent exocytosis pathway to provide membranes necessary for pseudopod extension, leading to clearance of senescent and apoptotic cells in vivo.

Vertebrate cells have evolved elaborate cell-autonomous defense programs to monitor subcellular compartments for infection and to evoke counter-responses. These programs are activated by pathogen-associated pattern molecules and by various strategies intracellular pathogens employ to alter cellular microenvironments. Here, we show that, when uropathogenic E. coli (UPEC) infect bladder epithelial cells (BECs), they are targeted by autophagy but avoid degradation because of their capacity to neutralize lysosomal pH. This change is detected by mucolipin TRP channel 3 (TRPML3), a transient receptor potential cation channel localized to lysosomes. TRPML3 activation then spontaneously initiates lysosome exocytosis, resulting in expulsion of exosome-encased bacteria. These studies reveal a cellular default system for lysosome homeostasis that has been co-opted by the autonomous defense program to clear recalcitrant pathogens.

The structure of mouse transient receptor potential mucolipin 1 (TRPML1), a cation channel located within endosomal and lysosomal membranes, is resolved using single-particle electron cryo-microscopy. Numerous ion channels sit in the membranes of intracellular organelles and are responsible for maintaining concentration gradients and ionic signalling. The transient receptor potential mucolipin (TRPML) channels are Ca(II)-releasing channels that are crucial to endolysosomal function. While TRPML channels regulate physiological processes including membrane trafficking and exocytosis, mutations of TRPML1 cause the lysosomal storage disorder mucolipidosis type IV. Three papers in this issue of Nature report the structure of TRPML channels by cryo-electron microscopy. Seok-Yong Lee and colleagues report the structure of TRPML3, while studies from teams led by Xiaochun Li and Youxing Jiang present the structure of TRPML1. Together, these studies reveal the open and closed states of the TRPML family, indicating the regulatory mechanisms of these channels. As with most TRP channels, TRPML can be gated by specific lipids, and these studies provide insights into substrate binding and channel activation. Transient receptor potential mucolipin 1 (TRPML1) is a cation channel located within endosomal and lysosomal membranes. Ubiquitously expressed in mammalian cells1,2, its loss-of-function mutations are the direct cause of type IV mucolipidosis, an autosomal recessive lysosomal storage disease3,4,5,6. Here we present the single-particle electron cryo-microscopy structure of the mouse TRPML1 channel embedded in nanodiscs. Combined with mutagenesis analysis, the TRPML1 structure reveals that phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2) binds to the N terminus of the channel—distal from the pore—and the helix–turn–helix extension between segments S2 and S3 probably couples ligand binding to pore opening. The tightly packed selectivity filter contains multiple ion-binding sites, and the conserved acidic residues form the luminal Ca2+-blocking site that confers luminal pH and Ca2+ modulation on channel conductance. A luminal linker domain forms a fenestrated canopy atop the channel, providing several luminal ion passages to the pore and creating a negative electrostatic trap, with a preference for divalent cations, at the luminal entrance. The structure also reveals two equally distributed S4–S5 linker conformations in the closed channel, suggesting an S4–S5 linker-mediated PtdInsP2 gating mechanism among TRPML channels7,8.

The mucolipin family of Transient Receptor Potential (TRPML) proteins is predicted to encode ion channels expressed in intracellular endosomes and lysosomes. Loss-of-function mutations of human TRPML1 cause type IV mucolipidosis (ML4), a childhood neurodegenerative disease. Meanwhile, gain-of-function mutations in the mouse TRPML3 result in the varitint-waddler (Va) phenotype with hearing and pigmentation defects. The broad spectrum phenotypes of ML4 and Va appear to result from certain aspects of endosomal/lysosomal dysfunction. Lysosomes, traditionally believed to be the terminal "recycling center" for biological "garbage", are now known to play indispensable roles in intracellular signal transduction and membrane trafficking. Studies employing animal models and cell lines in which TRPML genes have been genetically disrupted or depleted have uncovered roles of TRPMLs in multiple cellular functions including membrane trafficking, signal transduction, and organellar ion homeostasis. Physiological assays of mammalian cell lines in which TRPMLs are heterologously overexpressed have revealed the channel properties of TRPMLs in mediating cation (Ca(2+)/Fe(2+)) efflux from endosomes and lysosomes in response to unidentified cellular cues. This review aims to summarize these recent advances in the TRPML field and to correlate the channel properties of endolysosomal TRPMLs with their biological functions. We will also discuss the potential cellular mechanisms by which TRPML deficiency leads to neurodegeneration.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands ( www.guidetopharmacology.org ), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point‐in‐time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15539 . Ion channels are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein‐coupled receptors, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid‐2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC‐IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Upon nutrient starvation, autophagy digests unwanted cellular components to generate catabolites that are required for housekeeping biosynthesis processes. A complete execution of autophagy demands an enhancement in lysosome function and biogenesis to match the increase in autophagosome formation. Here, we report that mucolipin-1 (also known as TRPML1 or ML1), a Ca(2+) channel in the lysosome that regulates many aspects of lysosomal trafficking, plays a central role in this quality-control process. By using Ca(2+) imaging and whole-lysosome patch clamping, lysosomal Ca(2+) release and ML1 currents were detected within hours of nutrient starvation and were potently up-regulated. In contrast, lysosomal Na(+)-selective currents were not up-regulated. Inhibition of mammalian target of rapamycin (mTOR) or activation of transcription factor EB (TFEB) mimicked a starvation effect in fed cells. The starvation effect also included an increase in lysosomal proteostasis and enhanced clearance of lysosomal storage, including cholesterol accumulation in Niemann-Pick disease type C (NPC) cells. However, this effect was not observed when ML1 was pharmacologically inhibited or genetically deleted. Furthermore, overexpression of ML1 mimicked the starvation effect. Hence, lysosomal adaptation to environmental cues such as nutrient levels requires mTOR/TFEB-dependent, lysosome-to-nucleus regulation of lysosomal ML1 channels and Ca(2+) signaling.

Impaired homeostasis of lysosomal Ca2+ causes lysosome dysfunction and lysosomal storage diseases (LSDs), but the mechanisms by which lysosomes acquire and refill Ca2+ are not known. We developed a physiological assay to monitor lysosomal Ca2+ store refilling using specific activators of lysosomal Ca2+ channels to repeatedly induce lysosomal Ca2+ release. In contrast to the prevailing view that lysosomal acidification drives Ca2+ into the lysosome, inhibiting the V-ATPase H+ pump did not prevent Ca2+ refilling. Instead, pharmacological depletion or chelation of Endoplasmic Reticulum (ER) Ca2+ prevented lysosomal Ca2+ stores from refilling. More specifically, antagonists of ER IP3 receptors (IP3Rs) rapidly and completely blocked Ca2+ refilling of lysosomes, but not in cells lacking IP3Rs. Furthermore, reducing ER Ca2+ or blocking IP3Rs caused a dramatic LSD-like lysosome storage phenotype. By closely apposing each other, the ER may serve as a direct and primary source of Ca2+for the lysosome.

Lysosomes require an acidic lumen between pH 4.5 and 5.0 for effective digestion of macromolecules. This pH optimum is maintained by proton influx produced by the V-ATPase and efflux through an unidentified "H+ leak" pathway. Here we show that TMEM175, a genetic risk factor for Parkinson's disease (PD), mediates the lysosomal H+ leak by acting as a proton-activated, proton-selective channel on the lysosomal membrane (LyPAP). Acidification beyond the normal range potently activated LyPAP to terminate further acidification of lysosomes. An endogenous polyunsaturated fatty acid and synthetic agonists also activated TMEM175 to trigger lysosomal proton release. TMEM175 deficiency caused lysosomal over-acidification, impaired proteolytic activity, and facilitated α-synuclein aggregation in vivo. Mutational and pH normalization analyses indicated that the channel's H+ conductance is essential for normal lysosome function. Thus, modulation of LyPAP by cellular cues may dynamically tune the pH optima of endosomes and lysosomes to regulate lysosomal degradation and PD pathology.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and over 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands ( https://www.guidetopharmacology.org/ ), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point‐in‐time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16178 . Ion channels are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein‐coupled receptors, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid‐2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC‐IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Insects rely on a family of seven transmembrane proteins called gustatory receptors (GRs) to encode different taste modalities, such as sweet and bitter. We report structures of Drosophila sweet taste receptors GR43a and GR64a in the apo and sugar-bound states. Both GRs form tetrameric sugar-gated cation channels composed of one central pore domain (PD) and four peripheral ligand-binding domains (LBDs). Whereas GR43a is specifically activated by the monosaccharide fructose that binds to a narrow pocket in LBDs, disaccharides sucrose and maltose selectively activate GR64a by binding to a larger and flatter pocket in LBDs. Sugar binding to LBDs induces local conformational changes, which are subsequently transferred to the PD to cause channel opening. Our studies reveal a structural basis for sugar recognition and activation of GRs.

The transient receptor potential (TRP) superfamily comprises a group of non-selective cation channels that sense and respond to changes in their local environments. TRP channels are found in many eukaryotes, from yeast to mammals. They are a diverse group of proteins organized into six families: classical (TRPC), vanilloid (TRPV), melastatin (TRPM), muclopins (TRPML), polycystin (TRPP), and ANKTM1 (TRPA). In the peripheral nervous system, stimuli including temperature, pressure, inflammatory agents, and receptor activation effect TRP-mediated responses. In the central nervous system, TRPs participate in neurite outgrowth, receptor signalling and excitotoxic cell death resulting from anoxia. TRP channels are emerging as essential cellular switches that allow animals to respond to their environments.

Transient receptor potential (TRP) genes of the mucolipin subfamily (TRPML1–3 and MCOLN1–3) are presumed to encode ion channel proteins of intracellular endosomes and lysosomes. Mutations in human TRPML1 (mucolipin 1/MCOLN1) result in mucolipidosis type IV, a severe inherited neurodegenerative disease associated with defective lysosomal biogenesis and trafficking. A mutation in mouse TRPML3 (A419P; TRPML3 Va ) results in the varitint–waddler ( Va ) phenotype. Va mice are deaf, exhibit circling behavior due to vestibular defects, and have variegated/dilute coat color as a result of pigmentation defects. Prior electrophysiological studies of presumed TRPML plasma membrane channels are contradictory and inconsistent with known TRP channel properties. Here, we report that the Va mutation produces a gain-of-function that allows TRPML1 and TRPML3 to be measured and identified as inwardly rectifying, proton-impermeant, Ca 2+ -permeant cation channels. TRPML3 is highly expressed in normal melanocytes. Melanocyte markers are lost in the Va mouse, suggesting that their variegated and hypopigmented fur is caused by severe alteration of melanocyte function or cell death. TRPML3 Va expression in melanocyte cell lines results in high resting Ca 2+ levels, rounded, poorly adherent cells, and loss of membrane integrity. We conclude that the Va phenotype is caused by mutation-induced TRPML3 gain-of-function, resulting in cell death.

NaChBac, a six-α-helical transmembrane-spanning protein cloned from <i>Bacillus halodurans</i>, is the first functionally characterized bacterial voltage-gated Na⁺-selective channel (Ren, D., Navarro, B., Xu, H., Yue, L., Shi, Q., and Clapham, D. E. (2001) <i>Science</i> 294, 2372-2375). As a highly expressing ion channel protein, NaChBac is an ideal candidate for high resolution structural determination and structure-function studies. The biological role of NaChBac, however, is still unknown. In this report, another 11 structurally related bacterial proteins are described. Two of these functionally expressed as voltage-dependent Na⁺ channels (Na_VPZ from <i>Paracoccus zeaxanthinifaciens</i> and Na_VSP from <i>Silicibacter pomeroyi</i>). Na_VPZ and Na_VSP share ∼40% amino acid sequence identity with NaChBac. When expressed in mammalian cell lines, both Na_VPZ and Na_VSP were Na⁺-selective and voltage-dependent. However, their kinetics and voltage dependence differ significantly. These single six-α-helical transmembrane-spanning subunits constitute a widely distributed superfamily (Na_VBac) of channels in bacteria, implying a fundamental prokaryotic function. The degree of sequence homology (22-54%) is optimal for future comparisons of Na_VBac structure and function of similarity and dissimilarity among Na_VBac proteins. Thus, the Na_VBac superfamily is fertile ground for crystallographic, electrophysiological, and microbiological studies.

Ion channels are classically understood to regulate the flux of ions across the plasma membrane in response to a variety of environmental and intracellular cues. Ion channels serve a number of functions in intracellular membranes as well. These channels may be temporarily localized to intracellular membranes as a function of their biosynthetic or secretory pathways, i.e., en route to their destination location. Intracellular membrane ion channels may also be located in the endocytic pathways, either being recycled back to the plasma membrane or targeted to the lysosome for degradation. Several channels do participate in intracellular signal transduction; the most well known example is the inositol 1,4,5-trisphosphate receptor (IP(3)R) in the endoplasmic reticulum. Some organellar intracellular membrane channels are required for the ionic homeostasis of their residing organelles. Several newly-discovered intracellular membrane Ca(2+) channels actually play active roles in membrane trafficking. Transient receptor potential (TRP) proteins are a superfamily (28 members in mammal) of Ca(2+)-permeable channels with diverse tissue distribution, subcellular localization, and physiological functions. Almost all mammalian TRP channels studied thus far, like their ancestor yeast TRP channel (TRPY1) that localizes to the vacuole compartment, are also (in addition to their plasma membrane localization) found to be localized to intracellular membranes. Accumulated evidence suggests that intracellularly-localized TRP channels actively participate in regulating membrane traffic, signal transduction, and vesicular ion homeostasis. This review aims to provide a summary of these recent works. The discussion will also be extended to the basic membrane and electrical properties of the TRP-residing compartments.

Global disruption of transient receptor potential-melastatin-like 7 (Trpm7) in mice results in embryonic lethality before embryonic day 7. Using tamoxifen-inducible disruption of Trpm7 and multiple Cre recombinase lines, we show that Trpm7 deletion before and during organogenesis results in severe tissue-specific developmental defects. We find that Trpm7 is essential for kidney development from metanephric mesenchyme but not ureteric bud. Disruption of neural crest Trpm7 at early stages results in loss of pigment cells and dorsal root ganglion neurons. In contrast, late disruption of brain-specific Trpm7 after embryonic day 10.5 does not alter normal brain development. We developed induced pluripotent stem cells and neural stem (NS) cells in which Trpm7 disruption could be induced. Trpm7(-/-) NS cells retained the capacities of self-renewal and differentiation into neurons and astrocytes. During in vitro differentiation of induced pluripotent stem cells to NS cells, Trpm7 disruption prevents the formation of the NS cell monolayer. The in vivo and in vitro results demonstrate a temporal requirement for the Trpm7 channel kinase during embryogenesis.

Lysosomes are acidic compartments in mammalian cells that are primarily responsible for the breakdown of endocytic and autophagic substrates such as membranes, proteins, and lipids into their basic building blocks. Lysosomal storage diseases (LSDs) are a group of metabolic disorders caused by genetic mutations in lysosomal hydrolases required for catabolic degradation, mutations in lysosomal membrane proteins important for catabolite export or membrane trafficking, or mutations in nonlysosomal proteins indirectly affecting these lysosomal functions. A hallmark feature of LSDs is the primary and secondary excessive accumulation of undigested lipids in the lysosome, which causes lysosomal dysfunction and cell death, and subsequently pathological symptoms in various tissues and organs. There are more than 60 types of LSDs, but an effective therapeutic strategy is still lacking for most of them. Several recent in vitro and in vivo studies suggest that induction of lysosomal exocytosis could effectively reduce the accumulation of the storage materials. Meanwhile, the molecular machinery and regulatory mechanisms for lysosomal exocytosis are beginning to be revealed. In this paper, we first discuss these recent developments with the focus on the functional interactions between lipid storage and lysosomal exocytosis. We then discuss whether lysosomal exocytosis can be manipulated to correct lysosomal and cellular dysfunction caused by excessive lipid storage, providing a potentially general therapeutic approach for LSDs.

Phosphoinositides serve as address labels for recruiting peripheral cytoplasmic proteins to specific subcellular compartments, and as endogenous factors for modulating the activity of integral membrane proteins. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is a plasma-membrane (PM)-specific phosphoinositide and a positive cofactor required for the activity of most PM channels and transporters. This requirement for phosphoinositide cofactors has been proposed to prevent PM channel/transporter activity during passage through the biosynthetic/secretory and endocytic pathways. To determine whether intracellularly localized channels are similarly "inactivated" at the PM, we studied PIP(2) modulation of intracellular TRPML1 channels. TRPML1 channels are primarily localized in lysosomes, but can also be detected temporarily in the PM upon lysosomal exocytosis. By directly patch-clamping isolated lysosomes, we previously found that lysosomal, but not PM-localized, TRPML1 is active with PI(3,5)P(2), a lysosome-specific PIP(2), as the underlying positive cofactor. Here we found that "silent" PM-localized TRPML1 could be activated by depleting PI(4,5)P(2) levels and/or by adding PI(3,5)P(2) to inside-out membrane patches. Unlike PM channels, surface-expressed TRPML1 underwent a unique and characteristic run-up upon patch excision, and was potently inhibited by a low micromolar concentration of PI(4,5)P(2). Conversely, depletion of PI(4,5)P(2) by either depolarization-induced activation or chemically induced translocation of 5'-phosphatase potentiated whole-cell TRPML1 currents. PI(3,5)P(2) activation and PI(4,5)P(2) inhibition of TRPML1 were mediated by distinct basic amino acid residues in a common PIP(2)-interacting domain. Thus, PI(4,5)P(2) may serve as a negative cofactor for intracellular channels such as TRPML1. Based on these results, we propose that phosphoinositide regulation sets compartment-specific activity codes for membrane channels and transporters.

Significance Phosphatidylinositol polyphosphates (PIPs) are transiently generated at specific membrane subdomains. Changes of PIP levels regulate the trafficking of vesicles and the activity of membrane transport proteins. To directly visualize the intracellular dynamics of phosphatidylinositol 3,5-bisphosphate [PI(3,5)P 2 ], a key phosphoinositide in the endosome and lysosome, we have engineered a PI(3,5)P 2 probe by fusing fluorescent proteins directly to the lipid-binding domain of TRPML1, a lysosomal ion channel that is potently and specifically activated by PI(3,5)P 2 . This PI(3,5)P 2 probe binds to PI(3,5)P 2 with biochemical specificity in vitro and responds quickly to changes in the intracellular PI(3,5)P 2 level in living cells. With this biosensor, rapid changes of PI(3,5)P 2 on single vesicle membranes are captured prior to membrane fusion of two vesicles.

The scavenging of extracellular macromolecules by engulfment can sustain cell growth in a nutrient-depleted environment. Engulfed macromolecules are contained within vacuoles that are targeted for lysosome fusion to initiate degradation and nutrient export. We have shown that vacuoles containing engulfed material undergo mTORC1-dependent fission that redistributes degraded cargo back into the endosomal network. Here we identify the lipid kinase PIKfyve as a regulator of an alternative pathway that distributes engulfed contents in support of intracellular macromolecular synthesis during macropinocytosis, entosis, and phagocytosis. We find that PIKfyve regulates vacuole size in part through its downstream effector, the cationic transporter TRPML1. Furthermore, PIKfyve promotes recovery of nutrients from vacuoles, suggesting a potential link between PIKfyve activity and lysosomal nutrient export. During nutrient depletion, PIKfyve activity protects Ras-mutant cells from starvation-induced cell death and supports their proliferation. These data identify PIKfyve as a critical regulator of vacuole maturation and nutrient recovery during engulfment.

Lysosomes, the degradation center of the cell, are filled with acidic hydrolases. Lysosomes generate nutrient-sensitive signals to regulate the import of H+, hydrolases, and endocytic and autophagic cargos, as well as the export of their degradation products (catabolites). In response to environmental and cellular signals, lysosomes change their positioning, number, morphology, size, composition, and activity within minutes to hours to meet the changing cellular needs. Ion channels in the lysosome are essential transducers that mediate signal-initiated Ca2+/Fe2+/Zn2+ release and H+/Na+/K+-dependent changes of membrane potential across the perimeter membrane. Dysregulation of lysosomal ion flux impairs lysosome movement, membrane trafficking, nutrient sensing, membrane repair, organelle membrane contact, and lysosome biogenesis and adaptation. Hence, activation and inhibition of lysosomal channels by synthetic modulators may tune lysosome function to maintain cellular health and promote cellular clearance in lysosome storage disorders.

The integrity of the plasma membrane is maintained through an active repair process, especially in skeletal and cardiac muscle cells, in which contraction-induced mechanical damage frequently occurs in vivo. Muscular dystrophies (MDs) are a group of muscle diseases characterized by skeletal muscle wasting and weakness. An important cause of these group of diseases is defective repair of sarcolemmal injuries, which normally requires Ca(2+) sensor proteins and Ca(2+)-dependent delivery of intracellular vesicles to the sites of injury. MCOLN1 (also known as TRPML1, ML1) is an endosomal and lysosomal Ca(2+) channel whose human mutations cause mucolipidosis IV (ML4), a neurodegenerative disease with motor disabilities. Here we report that ML1-null mice develop a primary, early-onset MD independent of neural degeneration. Although the dystrophin-glycoprotein complex and the known membrane repair proteins are expressed normally, membrane resealing was defective in ML1-null muscle fibers and also upon acute and pharmacological inhibition of ML1 channel activity or vesicular Ca(2+) release. Injury facilitated the trafficking and exocytosis of vesicles by upmodulating ML1 channel activity. In the dystrophic mdx mouse model, overexpression of ML1 decreased muscle pathology. Collectively, our data have identified an intracellular Ca(2+) channel that regulates membrane repair in skeletal muscle via Ca(2+)-dependent vesicle exocytosis.

Lysosomes, the cell's recycling center, undergo nutrient-sensitive adaptive changes in function and biogenesis, i.e., lysosomal adaptation. We recently discovered that lysosomes also mediate the cell's "survival" response (i.e., autophagy) to oxidative stress through the activation of TFEB (transcription factor EB), a master regulator of lysosome biogenesis and autophagy. MCOLN1/TRPML1, the principal Ca2+ release channel on the lysosomal membrane, serves as the redox sensor in this process. Increasing reactive oxygen species (ROS) levels, either endogenously by mitochondrial damage or exogenously, directly activates MCOLN1 to induce lysosomal Ca2+ release, triggering PPP3/calcineurin-dependent TFEB nuclear translocation to enhance autophagy. Hence, ROS may induce autophagy by activating the MCOLN1-lysosome Ca2+-TFEB pathway, facilitating the removal of damaged mitochondria and excess ROS. Our findings have revealed a lysosomal signaling mechanism for cells to respond to oxidative bursts and adapt to oxidative stress.

Antiretroviral therapy extends the lifespan of human immunodeficiency virus (HIV)-infected patients, but many survivors develop premature impairments in cognition. These residual cognitive impairments may involve aberrant deposition of amyloid β-peptides (Aβ). By unknown mechanisms, Aβ accumulates in the lysosomal and autophagic compartments of neurons in the HIV-infected brain. Here we identify the molecular events evoked by the HIV coat protein gp120 that facilitate the intraneuronal accumulation of Aβ. We created a triple transgenic gp120/APP/PS1 mouse that recapitulates intraneuronal deposition of Aβ in a manner reminiscent of the HIV-infected brain. In cultured neurons, we found that the HIV coat protein gp120 increased the transcriptional expression of BACE1 through repression of PPARγ, and increased APP expression by promoting interaction of the translation-activating RBP heterogeneous nuclear ribonucleoprotein C with APP mRNA. APP and BACE1 were colocalized into stabilized membrane microdomains, where the β-cleavage of APP and Aβ formation were enhanced. Aβ-peptides became localized to lysosomes that were engorged with sphingomyelin and calcium. Stimulating calcium efflux from lysosomes with a TRPM1 agonist promoted calcium efflux, luminal acidification, and cleared both sphingomyelin and Aβ from lysosomes. These findings suggest that therapeutics targeted to reduce lysosomal pH in neurodegenerative conditions may protect neurons by facilitating the clearance of accumulated sphingolipids and Aβ-peptides.

Decades of intensive research have led to the discovery of most plasma membrane ion channels and transporters and the characterization of their physiological functions. In contrast, although over 80% of transport processes occur inside the cells, the ion flux mechanisms across intracellular membranes (the endoplasmic reticulum, Golgi apparatus, endosomes, lysosomes, mitochondria, chloroplasts, and vacuoles) are difficult to investigate and remain poorly understood. Recent technical advances in super-resolution microscopy, organellar electrophysiology, organelle-targeted fluorescence imaging, and organelle proteomics have pushed a large step forward in the research of intracellular ion transport. Many new organellar channels are molecularly identified and electrophysiologically characterized. Additionally, molecular identification of many of these ion channels/transporters has made it possible to study their physiological functions by genetic and pharmacological means. For example, organellar channels have been shown to regulate important cellular processes such as programmed cell death and photosynthesis, and are involved in many different pathologies. This special issue (SI) on organellar channels and transporters aims to provide a forum to discuss the recent advances and to define the standard and open questions in this exciting and rapidly developing field. Along this line, a new Gordon Research Conference dedicated to the multidisciplinary study of intracellular membrane transport proteins will be launched this coming summer.

Cells utilize calcium ions (Ca2+) to signal almost all aspects of cellular life, ranging from cell proliferation to cell death, in a spatially and temporally regulated manner. A key aspect of this regulation is the compartmentalization of Ca2+ in various cytoplasmic organelles that act as intracellular Ca2+ stores. Whereas Ca2+ release from the large-volume Ca2+ stores, such as the endoplasmic reticulum (ER) and Golgi apparatus, are preferred for signal transduction, Ca2+ release from the small-volume individual vesicular stores that are dispersed throughout the cell, such as lysosomes, may be more useful in local regulation, such as membrane fusion and individualized vesicular movements. Conceivably, these two types of Ca2+ stores may be established, maintained or refilled via distinct mechanisms. ER stores are refilled through sustained Ca2+ influx at ER-plasma membrane (PM) membrane contact sites (MCSs). In this review, we discuss the release and refilling mechanisms of intracellular small vesicular Ca2+ stores, with a special focus on lysosomes. Recent imaging studies of Ca2+ release and organelle MCSs suggest that Ca2+ exchange may occur between two types of stores, such that the small stores acquire Ca2+ from the large stores via ER-vesicle MCSs. Hence vesicular stores like lysosomes may be viewed as secondary Ca2+ stores in the cell.

Oxidative stress underlies a number of pathological conditions, including cancer, neurodegeneration, and aging. Antioxidant-rich foods help maintain cellular redox homeostasis and mitigate oxidative stress, but the underlying mechanisms are not clear. For example, sulforaphane (SFN), an electrophilic compound that is enriched in cruciferous vegetables such as broccoli, is a potent inducer of cellular antioxidant responses. NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2), a transcriptional factor that controls the expression of multiple detoxifying enzymes through antioxidant response elements (AREs), is a proposed target of SFN. NFE2L2/NRF2 is a target gene of TFEB (transcription factor EB), a master regulator of autophagic and lysosomal functions, which we show here to be potently activated by SFN. SFN induces TFEB nuclear translocation via a Ca2+-dependent but MTOR (mechanistic target of rapamycin kinase)-independent mechanism through a moderate increase in reactive oxygen species (ROS). Activated TFEB then boosts the expression of genes required for autophagosome and lysosome biogenesis, which are known to facilitate the clearance of damaged mitochondria. Notably, TFEB activity is required for SFN-induced protection against both acute oxidant bursts and chronic oxidative stress. Hence, by simultaneously activating macroautophagy/autophagy and detoxifying pathways, natural compound SFN may trigger a self-defense cellular mechanism that can effectively mitigate oxidative stress commonly associated with many metabolic and age-related diseases.Abbreviations: ANOVA: analyzes of variance; AREs: antioxidant response elements; Baf-A1: bafilomycin A1; BHA: butylhydroxyanisole; CAT: catechin hydrate; CCCP: carbonyl cyanide m- chlorophenylhydrazone; CLEAR: coordinated lysosomal expression and regulation; DCFH-DA: 2ʹ,7ʹ-dichlorofluorescin diacetate; FBS: fetal bovine serum; GFP: green fluorescent protein; HMOX1/HO-1: heme oxygenase 1; KD: knockdown; KEAP1: kelch like ECH associated protein 1; KO: knockout; LAMP1: lysosomal associated membrane protein 1; MCOLN1/TRPML1: mucolipin 1; ML-SA1: mucolipin-specific synthetic agonist 1; ML-SI3: mucolipin-specific synthetic inhibitor 3; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; NAC: N-acetylcysteine; NFE2L2/NRF2: nuclear factor: erythroid 2 like 2; NPC: Niemann–Pick type C; PBS: phosphate-buffered saline; PPP2/PP2A: protein phosphatase 2; Q-PCR: real time polymerase chain reaction; ROS: reactive oxygen species; RPS6KB1/S6K1/p70S6K: ribosomal protein S6 kinase B1; SFN: sulforaphane; TFEB: transcription factor EB; WT, wild-type

Rapamycin (Rap) and its derivatives, called rapalogs, are being explored in clinical trials targeting cancer and neurodegeneration. The underlying mechanisms of Rap actions, however, are not well understood. Mechanistic target of rapamycin (mTOR), a lysosome-localized protein kinase that acts as a critical regulator of cellular growth, is believed to mediate most Rap actions. Here, we identified mucolipin 1 (transient receptor potential channel mucolipin 1 [TRPML1], also known as MCOLN1), the principle Ca2+ release channel in the lysosome, as another direct target of Rap. Patch-clamping of isolated lysosomal membranes showed that micromolar concentrations of Rap and some rapalogs activated lysosomal TRPML1 directly and specifically. Pharmacological inhibition or genetic inactivation of mTOR failed to mimic the Rap effect. In vitro binding assays revealed that Rap bound directly to purified TRPML1 proteins with a micromolar affinity. In both healthy and disease human fibroblasts, Rap and rapalogs induced autophagic flux via nuclear translocation of transcription factor EB (TFEB). However, such effects were abolished in TRPML1-deficient cells or by TRPML1 inhibitors. Hence, Rap and rapalogs promote autophagy via a TRPML1-dependent mechanism. Given the demonstrated roles of TRPML1 and TFEB in cellular clearance, we propose that lysosomal TRPML1 may contribute a significant portion to the in vivo neuroprotective and anti-aging effects of Rap via an augmentation of autophagy and lysosomal biogenesis.

During tumor progression, lysosome function is often maladaptively upregulated to match the high energy demand required for cancer cell hyper-proliferation and invasion. Here, we report that mucolipin TRP channel 1 (TRPML1), a lysosomal Ca2+ and Zn2+ release channel that regulates multiple aspects of lysosome function, is dramatically upregulated in metastatic melanoma cells compared with normal cells. TRPML-specific synthetic agonists (ML-SAs) are sufficient to induce rapid (within hours) lysosomal Zn2+-dependent necrotic cell death in metastatic melanoma cells while completely sparing normal cells. ML-SA-caused mitochondria swelling and dysfunction lead to cellular ATP depletion. While pharmacological inhibition or genetic silencing of TRPML1 in metastatic melanoma cells prevents such cell death, overexpression of TRPML1 in normal cells confers ML-SA vulnerability. In the melanoma mouse models, ML-SAs exhibit potent in vivo efficacy of suppressing tumor progression. Hence, targeting maladaptively upregulated lysosome machinery can selectively eradicate metastatic tumor cells in vitro and in vivo.

The prokaryotic voltage-gated Na + channel, NaChBac, is one of a growing channel superfamily of unknown function. Here we show that Na V BP, the NaChBac homologue encoded by ncbA in alkaliphilic Bacillus pseudofirmus OF4, is a voltage-gated Na + channel potentiated by alkaline pH. Na V BP has roles in motility, chemotaxis, and pH homeostasis at high pH. Reduced motility of bacteria lacking functional Na V BP was reversed by restoration of the native channel but not by a mutant Na V BP engineered to be Ca 2+ -selective. Motile ncbA mutant cells and wild-type cells treated with a channel inhibitor exhibited behavior opposite to the wild type in response to chemoeffectors. Mutants lacking functional Na V BP were also defective in pH homeostasis in response to a sudden alkaline shift in external pH under conditions in which cytoplasmic [Na + ] is limiting for this crucial process. The defect was exacerbated by mutation of motPS , the motility channel genes. We hypothesize that activation of Na V BP at high pH supports diverse physiological processes by a combination of direct and indirect effects on the Na + cycle and the chemotaxis system.

The mucolipin TRP (TRPML) proteins are a family of endolysosomal cation channels with genetically established importance in humans and rodent. Mutations of human TRPML1 cause type IV mucolipidosis, a devastating pediatric neurodegenerative disease. Our recent electrophysiological studies revealed that, although a TRPML1-mediated current can only be recorded in late endosome and lysosome (LEL) using the lysosome patch clamp technique, a proline substitution in TRPML1 (TRPML1(V432P)) results in a large whole cell current. Thus, it remains unknown whether the large TRPML1(V432P)-mediated current results from an increased surface expression (trafficking), elevated channel activity (gating), or both. Here we performed systemic Pro substitutions in a region previously implicated in the gating of various 6 transmembrane cation channels. We found that several Pro substitutions displayed gain-of-function (GOF) constitutive activities at both the plasma membrane (PM) and endolysosomal membranes. Although wild-type TRPML1 and non-GOF Pro substitutions localized exclusively in LEL and were barely detectable in the PM, the GOF mutations with high constitutive activities were not restricted to LEL compartments, and most significantly, exhibited significant surface expression. Because lysosomal exocytosis is Ca(2+)-dependent, constitutive Ca(2+) permeability due to Pro substitutions may have resulted in stimulus-independent intralysosomal Ca(2+) release, hence the surface expression and whole cell current of TRPML1. Indeed, surface staining of lysosome-associated membrane protein-1 (Lamp-1) was dramatically increased in cells expressing GOF TRPML1 channels. We conclude that TRPML1 is an inwardly rectifying, proton-impermeable, Ca(2+) and Fe(2+)/Mn(2+) dually permeable cation channel that may be gated by unidentified cellular mechanisms through a conformational change in the cytoplasmic face of the transmembrane 5 (TM5). Furthermore, activation of TRPML1 in LEL may lead to the appearance of TRPML1 proteins at the PM.

The first member of the mammalian mucolipin TRP channel subfamily (TRPML1) is a cation-permeable channel that is predominantly localized on the membranes of late endosomes and lysosomes (LELs) in all mammalian cell types. In response to the regulatory changes of LEL-specific phosphoinositides or other cellular cues, TRPML1 may mediate the release of Ca2+ and heavy metal Fe2+/Zn2+ions into the cytosol from the LEL lumen, which in turn may regulate membrane trafficking events (fission and fusion), signal transduction, and ionic homeostasis in LELs. Human mutations in TRPML1 result in type IV mucolipidosis (ML-IV), a childhood neurodegenerative lysosome storage disease. At the cellular level, loss-of-function mutations of mammalian TRPML1 or its C. elegans or Drosophila homolog gene results in lysosomal trafficking defects and lysosome storage. In this chapter, we summarize recent advances in our understandings of the cell biological and channel functions of TRPML1. Studies on TRPML1’s channel properties and its regulation by cellular activities may provide clues for developing new therapeutic strategies to delay neurodegeneration in ML-IV and other lysosome-related pediatric diseases.

Resealing allows cells to mend damaged membranes rapidly when plasma membrane (PM) disruptions occur. Models of PM repair mechanisms include the “lipid-patch”, “endocytic removal”, and “macro-vesicle shedding” models, all of which postulate a dependence on local increases in intracellular Ca2+ at injury sites. Multiple calcium sensors, including synaptotagmin (Syt) VII, dysferlin, and apoptosis-linked gene-2 (ALG-2), are involved in PM resealing, suggesting that Ca2+ may regulate multiple steps of the repair process. Although earlier studies focused exclusively on external Ca2+, recent studies suggest that Ca2+ release from intracellular stores may also be important for PM resealing. Hence, depending on injury size and the type of injury, multiple sources of Ca2+ may be recruited to trigger and orchestrate repair processes. In this review, we discuss the mechanisms by which the resealing process is promoted by vesicular Ca2+ channels and Ca2+ sensors that accumulate at damage sites.

The resting membrane potential (Δψ) of the cell is negative on the cytosolic side and determined primarily by the plasma membrane’s selective permeability to K+. We show that lysosomal Δψ is set by lysosomal membrane permeabilities to Na+ and H+, but not K+, and is positive on the cytosolic side. An increase in juxta-lysosomal Ca2+ rapidly reversed lysosomal Δψ by activating a large voltage-dependent and K+-selective conductance (LysoKVCa). LysoKVCa is encoded molecularly by SLO1 proteins known for forming plasma membrane BK channels. Opening of single LysoKVCa channels is sufficient to cause the rapid, striking changes in lysosomal Δψ. Lysosomal Ca2+ stores may be refilled from endoplasmic reticulum (ER) Ca2+ via ER–lysosome membrane contact sites. We propose that LysoKVCa serves as the perilysosomal Ca2+ effector to prime lysosomes for the refilling process. Consistently, genetic ablation or pharmacological inhibition of LysoKVCa, or abolition of its Ca2+ sensitivity, blocks refilling and maintenance of lysosomal Ca2+ stores, resulting in lysosomal cholesterol accumulation and a lysosome storage phenotype.

Significance Cells encountering physiological stresses often become vacuolated, but the source of the vacuoles and their role in causing or ameliorating necrotic cell death are unclear. Studies of cells responding to hypoosmotic, hypoxic, or hypothermic stress revealed that 1) vacuoles are derived from lysosomes, 2) many vacuoles subsequently undergo exocytosis, and 3) blocking either vacuolation or vacuole exocytosis by genetic or pharmacological approaches led to greatly increased necrotic cell death. By storing and then expelling excess water, lysosome-derived vacuoles maintain cytosolic water homeostasis and relieve membrane stress. This basic cellular function was present in all mammalian cell types tested and required ionic strength-sensitive ion channels formed from LRRC8 proteins on lysosomal membranes (Lyso-VRACs) acting in parallel with plasma membrane VRAC channels.

Macroautophagy/autophagy is elevated to ensure the high demand for nutrients for the growth of cancer cells. Here we demonstrated that MCOLN1/TRPML1 is a pharmaceutical target of oncogenic autophagy in cancers such as pancreatic cancer, breast cancer, gastric cancer, malignant melanoma, and glioma. First, we showed that activating MCOLN1, by increasing expression of the channel or using the MCOLN1 agonists, ML-SA5 or MK6-83, arrests autophagic flux by perturbing fusion between autophagosomes and lysosomes. Second, we demonstrated that MCOLN1 regulates autophagy by mediating the release of zinc from the lysosome to the cytosol. Third, we uncovered that zinc influx through MCOLN1 blocks the interaction between STX17 (syntaxin 17) in the autophagosome and VAMP8 in the lysosome and thereby disrupting the fusion process that is determined by the two SNARE proteins. Furthermore, we demonstrated that zinc influx originating from the extracellular fluid arrests autophagy by the same mechanism as lysosomal zinc, confirming the fundamental function of zinc as a participant in membrane trafficking. Last, we revealed that activating MCOLN1 with the agonists, ML-SA5 or MK6-83, triggers cell death of a number of cancer cells by evoking autophagic arrest and subsequent apoptotic response and cell cycle arrest, with little or no effect observed on normal cells. Consistent with the in vitro results, administration of ML-SA5 in Patu 8988 t xenograft mice profoundly suppresses tumor growth and improves survival. These results establish that a lysosomal cation channel, MCOLN1, finely controls oncogenic autophagy in cancer by mediating zinc influx into the cytosol.Abbreviation: Abbreviations: 3-MA: 3-methyladenine; AA: amino acid; ATG12: autophagy related 12; Baf-A1: bafilomycin A1; BAPTA-am: 1,2-bis(2-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester; co-IP: coimmunoprecipitaion; CQ: chloroquine; DMEM: Dulbecco's Modified Eagle Medium; FBS: fetal bovine serum; GAPDH: glyceraldehyde- 3-phosphate dehydrogenase; HCQ: hydroxychloroquine; HEK: human embryonic kidney; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCOLN1/TRPML1: mucolipin TRP cation channel 1; MTORC1: mechanistic target of rapamycin kinase complex 1; NC: negative control; NRK: normal rat kidney epithelial cells; PBS: phosphate-buffered saline; PtdIns3K: phosphatidylinositol 3-kinase; RPS6KB/S6K: ribosomal protein S6 kinase B; shRNA: short hairpin RNA; siRNA: short interfering RNA; SNARE: soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TPEN: N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine; TTM: tetrathiomolybdate; ULK1: unc-51 like autophagy activating kinase 1; VAMP8: vesicle associated membrane protein 8; Zn2+: zinc.

CO2 chemoreception may be mediated by the modulation of certain ion channels in neurons. Kir4.1 and Kir5.1, two members of the inward rectifier K+ channel family, are expressed in several brain regions including the brainstem. To test the hypothesis that Kir4.1 and Kir5. 1 are modulated by CO2 and pH, we carried out experiments by expressing Kir4.1 and coexpressing Kir4.1 with Kir5.1 (Kir4.1-Kir5. 1) in Xenopus oocytes. K+ currents were then studied using two-electrode voltage clamp and excised patches. Exposure of the oocytes to CO2 (5, 10 and 15 %) produced a concentration-dependent inhibition of the whole-cell K+ currents. This inhibition was fast and reversible. Exposure to 15 % CO2 suppressed Kir4.1 currents by approximately 20 % and Kir4.1-Kir5.1 currents by approximately 60 %. The effect of CO2 was likely to be mediated by intracellular acidification, because selective intracellular, but not extracellular, acidification to the measured hypercapnic pH levels lowered the currents as effectively as hypercapnia. In excised inside-out patches, exposure of the cytosolic side of membranes to solutions with various pH levels brought about a dose-dependent inhibition of the macroscopic K+ currents. The pK value (-log of dissociation constant) for the inhibition was 6.03 in the Kir4.1 channels, while it was 7.45 in Kir4.1-Kir5.1 channels, an increase in pH sensitivity of 1.4 pH units. Hypercapnia without changing pH did not inhibit the Kir4.1 and Kir4.1-Kir5.1 currents, suggesting that these channels are inhibited by protons rather than molecular CO2. A lysine residue in the N terminus of Kir4.1 is critical. Mutation of this lysine at position 67 to methionine (K67M) completely eliminated the CO2 sensitivity of both the homomeric Kir4. 1 and heteromeric Kir4.1-Kir5.1. These results therefore indicate that the Kir4.1 channel is inhibited during hypercapnia by a decrease in intracellular pH, and the coexpression of Kir4.1 with Kir5.1 greatly enhances channel sensitivity to CO2/pH and may enable cells to detect both increases and decreases in PCO2 and intracellular pH at physiological levels.

Human embryonic kidney cells (HEK 293) are widely used as an expression system in studies of ion channels. However, their endogenous ionic currents remain largely unidentified. To characterize these currents, we performed patch clamp experiments on this expression system. In whole-cell voltage clamp mode, the HEK 293 cells showed mainly outward currents using physiological concentrations of Na+ and K+ and symmetric concentrations of Cl- (150 mM) across the plasma membranes. K+ currents contributed to a small portion of these outward currents, since a shift of the reversal potentials of only approximately 20 mV was seen with a change of extracellular K+ concentration from 3 to 150 mM. In contrast, the reversal potential shifted approximately 25 mV when extracellular Cl- was reduced to 50 mM, indicating that most of the outward currents are carried by Cl-. In inside-out patches, several distinct Cl- currents were identified. They were: (1) 350 pS Cl- current, which was voltage-activated and had a moderate outward rectification; (2) 240 pS Cl- current with a weak outward rectification; and (3) 55 pS Cl- current, which was voltage-activated, sensitive to DIDS, and showed a strong outward rectification. Activation of these Cl- currents did not require an elevation of free Ca2+ level in the cytosol. Besides these three currents, we observed two other Cl- currents with much smaller conductances (25 and 16 pS, respectively). Two different K+ currents were seen in the HEK 293 cells, with one of them (125 pS) showing inward rectification and the other (70 pS) outward rectification. Moreover, a 50 pS cation channel was recorded in these cells. The presence of a variety of ion channels in the HEK 293 cells suggests that a great precaution needs to be taken when this expression system is used in studies of several similar ion channels.

CO2 chemoreception may be related to modulation of inward rectifier K+ channels (Kir channels) in brainstem neurons. Kir4.1 is expressed predominantly in the brainstem and inhibited during hypercapnia. Although the homomeric Kir4.1 only responds to severe intracellular acidification, coexpression of Kir4.1 with Kir5.1 greatly enhances channel sensitivities to CO2 and pH. To understand the biophysical and molecular mechanisms underlying the modulation of these currents by CO2 and pH, heteromeric Kir4. 1-Kir5.1 were studied in inside-out patches. These Kir4.1-Kir5.1 currents showed a single channel conductance of 59 pS with open-state probability (P(open)) approximately 0.4 at pH 7.4. Channel activity reached the maximum at pH 8.5 and was completely suppressed at pH 6.5 with pKa 7.45. The effect of low pH on these currents was due to selective suppression of P(open) without evident effects on single channel conductance, leading to a decrease in the channel mean open time and an increase in the mean closed time. At pH 8.5, single-channel currents showed two sublevels of conductance at approximately 1/4 and 3/4 of the maximal openings. None of them was affected by lowering pH. The Kir4.1-Kir5.1 currents were modulated by phosphatidylinositol-4,5-bisphosphate (PIP2) that enhanced baseline P(open) and reduced channel sensitivity to intracellular protons. In the presence of 10 microM PIP2, the Kir4.1-Kir5.1 showed a pKa value of 7.22. The effect of PIP2, however, was not seen in homomeric Kir4.1 currents. The CO2/pH sensitivities were related to a lysine residue in the NH2 terminus of Kir4.1. Mutation of this residue (K67M, K67Q) completely eliminated the CO2 sensitivity of both homomeric Kir4.1 and heteromeric Kir4.1-Kir5.1. In excised patches, interestingly, the Kir4.1-Kir5.1 carrying K67M mutation remained sensitive to low pHi. Such pH sensitivity, however, disappeared in the presence of PIP2. The effect of PIP2 on shifting the titration curve of wild-type and mutant channels was totally abolished when Arg178 in Kir5.1 was mutated. Thus, these studies demonstrate a heteromeric Kir channel that can be modulated by both acidic and alkaline pH, show the modulation of pH sensitivity of Kir channels by PIP2, and provide information of the biophysical and molecular mechanisms underlying the Kir modulation by intracellular protons.

Abstract The direction and specificity of endolysosomal membrane trafficking is tightly regulated by various cytosolic and membrane‐bound factors, including soluble NSF attachment protein receptors (SNAREs), Rab GTPases, and phosphoinositides. Another trafficking regulatory factor is juxta‐organellar Ca 2+ , which is hypothesized to be released from the lumen of endolysosomes and to be present at higher concentrations near fusion/fission sites. The recent identification and characterization of several Ca 2+ channel proteins from endolysosomal membranes has provided a unique opportunity to examine the roles of Ca 2+ and Ca 2+ channels in the membrane trafficking of endolysosomes. SNAREs, Rab GTPases, and phosphoinositides have been reported to regulate plasma membrane ion channels, thereby suggesting that these trafficking regulators may also modulate endolysosomal dynamics by controlling Ca 2+ flux across endolysosomal membranes. In this paper, we discuss the roles of phosphoinositides, Ca 2+ , and potential interactions between endolysosomal Ca 2+ channels and phosphoinositides in endolysosomal dynamics.

Abstract Endosomal and lysosomal membrane trafficking requires the coordination of multiple signalling events to control cargo sorting and processing, and endosome maturation. The initiation and termination of signalling events in endosomes and lysosomes is not well understood, but several key regulators have been identified, which include small GTPases, phosphoinositides, and Ca 2+ . Small GTPases act as master regulators and molecular switches in a GTP‐dependent manner, initiating signalling cascades to regulate the direction and specificity of endosomal trafficking. Phosphoinositides are membrane‐bound lipids that indicate vesicular identities for recruiting specific cytoplasmic proteins to endosomal membranes, thus allowing specificity of membrane fusion, fission, and cargo sorting to occur within and between specific vesicle compartments. In addition, phosphoinositides regulate the function of membrane proteins such as ion channels and transporters in a compartment‐specific manner to mediate transport and signalling. Finally, Ca 2+ , a locally acting second messenger released from intracellular ion channels, may provide precise spatiotemporal regulation of endosomal signalling and trafficking events. Small GTPase signalling can regulate phosphoinositide conversion during endosome maturation, and electrophysiological studies on isolated endosomes have shown that endosomal and lysosomal Ca 2+ channels are directly modulated by endosomal lipids. Thus trafficking and maturation of endosomes and lysosomes can be precisely regulated by dynamic changes in GTPases and membrane lipids, as well as Ca 2+ signalling. Importantly, impaired phosphoinositide and Ca 2+ signalling can cause endosomal and lysosomal trafficking defects at the cellular level, and a spectrum of lysosome storage diseases.

Gastric acid secretion by parietal cells requires trafficking and exocytosis of H/K-ATPase-rich tubulovesicles (TVs) toward apical membranes in response to histamine stimulation via cyclic AMP elevation. Here, we found that TRPML1 (ML1), a protein that is mutated in type IV mucolipidosis (ML-IV), is a tubulovesicular channel essential for TV exocytosis and acid secretion. Whereas ML-IV patients are reportedly achlorhydric, transgenic overexpression of ML1 in mouse parietal cells induced constitutive acid secretion. Gastric acid secretion was blocked and stimulated by ML1 inhibitors and agonists, respectively. Organelle-targeted Ca2+ imaging and direct patch-clamping of apical vacuolar membranes revealed that ML1 mediates a PKA-activated conductance on TV membranes that is required for histamine-induced Ca2+ release from TV stores. Hence, we demonstrated that ML1, acting as a Ca2+ channel in TVs, links transmitter-initiated cyclic nucleotide signaling with Ca2+-dependent TV exocytosis in parietal cells, providing a regulatory mechanism that could be targeted to manage acid-related gastric diseases.

Mammalian transient receptor potential (TRP) channels mediate Ca2+ flux and voltage changes across membranes in response to environmental and cellular signals. At the plasma membrane, sensory TRPs act as neuronal detectors of physical and chemical environmental signals, and receptor-operated (metabotropic) TRPs decode extracellular neuroendocrine cues to control body homeostasis. In intracellular membranes, such as those in lysosomes, organellar TRPs respond to compartment-derived signals to control membrane trafficking, signal transduction, and organelle function. Complementing mouse and human genetics and high-resolution structural approaches, physiological studies employing natural agonists and synthetic inhibitors have become critical in resolving the in vivo functions of metabotropic, sensory, and organellar TRPs.

Mammalian two-pore-channels (TPC1, 2; TPCN1, TPCN2) are ubiquitously- expressed, PI(3,5)P2-activated, Na+-selective channels in the endosomes and lysosomes that regulate luminal pH homeostasis, membrane trafficking, and Ebola viral infection. Whereas the channel activity of TPC1 is strongly dependent on membrane voltage, TPC2 lacks such voltage dependence despite the presence of the presumed ‘S4 voltage-sensing’ domains. By performing high-throughput screening followed by lysosomal electrophysiology, here we identified a class of tricyclic anti-depressants (TCAs) as small-molecule agonists of TPC channels. TCAs activate both TPC1 and TPC2 in a voltage-dependent manner, referred to as Lysosomal Na+ channel Voltage-dependent Activators (LyNa-VAs). We also identified another compound which, like PI(3,5)P2, activates TPC2 independent of voltage, suggesting the existence of agonist-specific gating mechanisms. Our identification of small-molecule TPC agonists should facilitate the studies of the cell biological roles of TPCs and can also readily explain the reported effects of TCAs in the modulation of autophagy and lysosomal functions.

Duchenne muscular dystrophy (DMD) is a devastating disease caused by mutations in dystrophin that compromise sarcolemma integrity. Currently, there is no treatment for DMD. Mutations in transient receptor potential mucolipin 1 (ML1), a lysosomal Ca2+ channel required for lysosomal exocytosis, produce a DMD-like phenotype. Here, we show that transgenic overexpression or pharmacological activation of ML1 in vivo facilitates sarcolemma repair and alleviates the dystrophic phenotypes in both skeletal and cardiac muscles of mdx mice (a mouse model of DMD). Hallmark dystrophic features of DMD, including myofiber necrosis, central nucleation, fibrosis, elevated serum creatine kinase levels, reduced muscle force, impaired motor ability, and dilated cardiomyopathies, were all ameliorated by increasing ML1 activity. ML1-dependent activation of transcription factor EB (TFEB) corrects lysosomal insufficiency to diminish muscle damage. Hence, targeting lysosomal Ca2+ channels may represent a promising approach to treat DMD and related muscle diseases.

ABSTRACTThe acidic environment within lysosomes is maintained within a narrow pH range (pH 4.5-5.0) optimal for digesting autophagic cargo macromolecules so that the resulting building block metabolites can be reused. This pH homeostasis is a consequence of proton influx produced by a V-type H+-translocating ATPase (V-ATPase) and rapid proton efflux through an unidentified “leak” pathway. By performing a candidate expression screening, we discovered that the TMEM175 gene encodes a proton-activated, proton-selective channel (LyPAP) that is required for lysosomal H+ “leak” currents. The activity of LyPAP is most active when lysosomes are hyper-acidified, and cells lacking TMEM175 exhibit lysosomal hyper-acidification and impaired proteolytic degradation, both of which can be restored by optimizing lysosomal pH using pharmacological agents. Variants of TMEM175 that are associated with susceptibility to Parkinson disease (PD) cause a reduction in TMEM175-dependent LyPAP currents and lysosomal hyper-acidification. Hence, our studies not only reveal an essential H+-dissipating pathway in lysosomes, but also provide a molecular target to regulate pH-dependent lysosomal functions and associated pathologies.KEYWORDS: Proton channellysosomeacidificationH+ leakTMEM175 Disclosure statementNo potential conflict of interest was reported by the authors.AcknowledgementThis work was supported by an NIH Grant (RO1DK115474) and funds from the Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals.

ATP-sensitive K+(KATP) channels may be regulated by protons in addition to ATP, phospholipids, and other nucleotides. Such regulation allows a control of cellular excitability in conditions when pH is low but ATP concentration is normal. However, whether the KATP changes its activity with pH alterations remains uncertain. In this study we showed that the reconstituted KATP was strongly activated during hypercapnia and intracellular acidosis using whole-cell recordings. Further characterizations in excised patches indicated that channel activity increased with a moderate drop in intracellular pH and decreased with strong acidification. The channel activation was produced by a direct action of protons on the Kir6 subunit and relied on a histidine residue that is conserved in all KATP. The inhibition appeared to be a result of channel rundown and was not seen in whole-cell recordings. The biphasic response may explain the contradictory pH sensitivity observed in cell-endogenous KATP in excised patches. Site-specific mutations of two residues showed that pH and ATP sensitivities were independent of each other. Thus, these results demonstrate that the proton is a potent activator of the KATP. The pH-dependent activation may enable the KATP to control vascular tones, insulin secretion, and neuronal excitability in several pathophysiologic conditions. ATP-sensitive K+(KATP) channels may be regulated by protons in addition to ATP, phospholipids, and other nucleotides. Such regulation allows a control of cellular excitability in conditions when pH is low but ATP concentration is normal. However, whether the KATP changes its activity with pH alterations remains uncertain. In this study we showed that the reconstituted KATP was strongly activated during hypercapnia and intracellular acidosis using whole-cell recordings. Further characterizations in excised patches indicated that channel activity increased with a moderate drop in intracellular pH and decreased with strong acidification. The channel activation was produced by a direct action of protons on the Kir6 subunit and relied on a histidine residue that is conserved in all KATP. The inhibition appeared to be a result of channel rundown and was not seen in whole-cell recordings. The biphasic response may explain the contradictory pH sensitivity observed in cell-endogenous KATP in excised patches. Site-specific mutations of two residues showed that pH and ATP sensitivities were independent of each other. Thus, these results demonstrate that the proton is a potent activator of the KATP. The pH-dependent activation may enable the KATP to control vascular tones, insulin secretion, and neuronal excitability in several pathophysiologic conditions. Hypercapnia and acidosis affect vascular tone, skeletal muscle contractility, insulin secretion, epithelial transport, and neuronal excitability, which may be mediated by KATP1 (1Aspinwall C.A. Brooks S.A. Kennedy R.T. Lakey J.R. J. Biol. Chem. 1997; 272: 31308-31314Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 2Dost R. Rundfeldt C. Epilepsy Res. 2000; 38: 53-66Crossref PubMed Scopus (55) Google Scholar, 3Gramolini A. Renaud J.M. Am. J. Physiol. 1997; 272: C1936-C1946Crossref PubMed Google Scholar, 4Wang W. Hebert S.C. Giebisch G. Annu. Rev. Physiol. 1997; 59: 413-436Crossref PubMed Scopus (176) Google Scholar, 5Wei E.P. Kontos H.A. Stroke. 1999; 30: 851-853Crossref PubMed Scopus (23) Google Scholar). However, previous studies on the pH sensitivity of these K+channels were controversial and even contradictory. In the absence of ATP, acidic pH was shown to stimulate cell-endogenous KATP(6Koyano T. Kakei M. Nakashima H. Yoshinaga M. Matsuoka T. Tanaka H. J. Physiol. ( Lond. ). 1993; 463: 747-766Crossref PubMed Scopus (55) Google Scholar, 7Vivaudou M. Forestier C. J. Physiol. ( Lond .). 1995; 486: 629-645Crossref PubMed Scopus (35) Google Scholar), inhibit it (8Allard B. Lazdunski M. Rougier O. J. Physiol. ( Lond .). 1995; 485: 283-296Crossref PubMed Scopus (44) Google Scholar, 9Proks P. Takano M. Ashcroft F.M. J. Physiol. ( Lond .). 1994; 475: 33-44Crossref PubMed Scopus (26) Google Scholar), and have little or no effect (10Cook D.L. Hales C.N. Nature. 1984; 311: 271-273Crossref PubMed Scopus (969) Google Scholar, 11Davies N.W. Standen N.B. Stanfield P.R. J. Physiol. ( Lond .). 1992; 445: 549-568Crossref PubMed Scopus (92) Google Scholar). This inconsistency is further complicated by the indirect effect of ATP or Mg2+ and tissue-specific KATP species (8Allard B. Lazdunski M. Rougier O. J. Physiol. ( Lond .). 1995; 485: 283-296Crossref PubMed Scopus (44) Google Scholar, 9Proks P. Takano M. Ashcroft F.M. J. Physiol. ( Lond .). 1994; 475: 33-44Crossref PubMed Scopus (26) Google Scholar, 10Cook D.L. Hales C.N. Nature. 1984; 311: 271-273Crossref PubMed Scopus (969) Google Scholar, 11Davies N.W. Standen N.B. Stanfield P.R. J. Physiol. ( Lond .). 1992; 445: 549-568Crossref PubMed Scopus (92) Google Scholar, 12Fan Z. Tokuyama Y. Makielski J.C. Am. J. Physiol. 1994; 267: C1036-C1044Crossref PubMed Google Scholar). Consequently, it is unclear whether KATP is modulated during hypercapnia and acidosis and what molecular mechanisms are underlying the modulations. The cloned KATP channels are ideal for addressing these questions because they allow for fine dissection of the modulatory mechanisms and elaborate manipulation of PCO2 and pH in an expression system (13Baukrowitz T. Tucker S.J. Schulte U. Benndorf K. Ruppersberg J.P. Fakler B. EMBO J. 1999; 18: 847-853Crossref PubMed Scopus (78) Google Scholar, 14Zhu G.Y. Chanchevalap S. Liu C. Xu H. Jiang C. J. Cell. Physiol. 2000; 183: 53-64Crossref PubMed Scopus (38) Google Scholar). Therefore, we studied the modulation of the cloned KATP(Kir6 with SUR, Ref. 15Ashcroft F.M. Gribble F.M. Trends Neurosci. 1998; 21: 288-294Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar) by CO2 and acidic pH. To locate the pH sensors, we also studied Kir6.2 with a truncation of 36 amino acids at the C terminus (Kir6.2ΔC36) because it expresses functional channel without the SUR subunit and retains fair ATP sensitivity (16Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (675) Google Scholar). Oocytes from Xenopus laevis were used in the present studies. Frogs were anesthetized by bathing them in 0.3% 3-aminobenzoic acid ethyl ester. A few lobes of ovaries were removed after a small abdominal incision (∼5 mm). Then, the surgical incision was closed and the frogs were allowed to recover from the anesthesia.Xenopus oocytes were treated with 2 mg/ml collagenase (Type I, Sigma) in an OR2 solution consisting of (in mm) NaCl 82, KCl 2, MgCl2 1, and HEPES 5 (pH 7.4) for 90 min at room temperature. After three washes (10 min each) of the oocytes with the OR2 solution, cDNAs (25–50 ng in 50 nl of water) were injected into the oocytes. For coexpression, Kir6.x and SUR1 were injected in a 1:2 ratio. The oocytes were then incubated at 18 °C in the ND-96 solution containing (in mm) NaCl 96, KCl 2, MgCl2 1, CaCl2 1.8, HEPES 5, and sodium pyruvate 2.5 with 100 mg/liter geneticin added (pH 7.4). Rat Kir6.1 (uKATP, GenBank™/EBI accession number D42145) and mouse Kir6.2 (mBIR, GenBank™/EBI accession number D50581) cDNAs were generously provided by Dr. S. Seino. Hamster SUR1 (GenBankTM accession number L40623) was a gift from Dr. L. Bryan. The cDNAs were subcloned to a eukaryotic expression vector (pcDNA3.1, Invitrogen Inc., Carlsbad, CA) and used forXenopus oocyte expression without cRNA synthesis. Site-specific mutations were produced using a site-directed mutagenesis kit (Stratagene, La Jolla, CA). The orientation of the constructs and correct mutations were confirmed with DNA sequencing. Whole-cell currents were studied on the oocytes 2–4 days after injection using a two-electrode voltage clamp with an amplifier (Geneclamp 500, Axon Instruments Inc., Foster City, CA) at room temperature (∼24 °C). The extracellular solution contained (in mm) KCl 90, MgCl2 3, and HEPES 5 (pH 7.4). Extracellular acidification was done by titrating the extracellular solution to desired pH levels. The HEPES buffer was chosen because of its buffering range and membrane impermeability, as shown in our previous studies (17Qu Z. Zhu G.Y. Yang Z. Cui N. Li Y. Chanchevalap S. Sulaiman S. Haynie H. Jiang C. J. Biol. Chem. 1999; 274: 13783-13789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 18Xu H. Cui N. Yang Z. Qu Z. Jiang C. J. Physiol. ( Lond .). 2000; 524: 725-735Crossref PubMed Scopus (105) Google Scholar). In intracellular acidification experiments, 90 mm KCl was replaced with the same concentration of KHCO3 (pH titrated to 7.4), so that the K+concentration remained the same in these experiments (19Fakler B. Schultz J.H. Yang J. Schulte U. Brandle U. Zenner H.P. Jan L.Y. Ruppersberg J.P. EMBO J. 1996; 15: 4093-4099Crossref PubMed Scopus (162) Google Scholar). When oocytes were exposed to these perfusates, intracellular pH (pHi) and extracellular pH (pHo) were measured using ion-selective microelectrodes as described previously (14Zhu G.Y. Chanchevalap S. Liu C. Xu H. Jiang C. J. Cell. Physiol. 2000; 183: 53-64Crossref PubMed Scopus (38) Google Scholar). Whole-cell currents were also studied with nigericin (10 μm) at various bath pH levels. This protonophore forms cation channels permeable primarily to protons (20Grinstein S. Cohen S. Rothstein A. J. Gen. Physiol. 1984; 83: 341-369Crossref PubMed Scopus (312) Google Scholar, 21Wilding T.J. Cheng B. Roos A. J. Gen. Physiol. 1992; 100: 593-608Crossref PubMed Scopus (44) Google Scholar). Using 90 mm K+ in the bath solution as permeable cation, pHi becomes the same as pHo in the presence of nigericin (20Grinstein S. Cohen S. Rothstein A. J. Gen. Physiol. 1984; 83: 341-369Crossref PubMed Scopus (312) Google Scholar). Exposure to nigericin increased oocyte-endogenous currents at each pH point. The current changes in these pH points were measured in oocytes without any injection, averaged (n = 4), and subtracted from current records of the Kir6-expressing cells because the alterations were due to nigericin rather than changes of pHi in these cells. Experiments were performed in a semiclosed recording chamber (BSC-HT, Medical Systems Corp., Greenvale, NY) in which oocytes were placed on a supporting nylon mesh; the perfusion solution bathed both the top and bottom surface of the oocytes. The perfusate and the superfusion gas entered the chamber from two inlets at one end and flowed out at the other end. There was a 3 × 15-mm gap on the top cover of the chamber, which served as the gas outlet and an access to the oocytes for recording microelectrodes. At baseline the chamber was ventilated with atmospheric air. Exposure of the oocytes to CO2 was carried out by switching a perfusate that had been bubbled for at least 30 min with a gas mixture containing CO2 at various concentrations balanced with 21% O2 and N2 and superfused with the same gas (14Zhu G.Y. Chanchevalap S. Liu C. Xu H. Jiang C. J. Cell. Physiol. 2000; 183: 53-64Crossref PubMed Scopus (38) Google Scholar, 17Qu Z. Zhu G.Y. Yang Z. Cui N. Li Y. Chanchevalap S. Sulaiman S. Haynie H. Jiang C. J. Biol. Chem. 1999; 274: 13783-13789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 18Xu H. Cui N. Yang Z. Qu Z. Jiang C. J. Physiol. ( Lond .). 2000; 524: 725-735Crossref PubMed Scopus (105) Google Scholar). The high dissolvability of CO2 resulted in a detectable change in intra- or extracellular acidification as fast as 10 s in these oocytes. Macroscopic and single-channel currents were recorded in excised patches at room temperature (∼24 °C) as described previously (22Zhu G.Y. Chanchevalap S. Cui N. Jiang C. J. Physiol. ( Lond .). 1999; 516: 699-710Crossref PubMed Scopus (69) Google Scholar,23Yang Z. Xu H. Cui N. Qu Z. Chanchevalap S. Shen W. Jiang C. J. Gen. Physiol. 2000; 116: 33-46Crossref PubMed Scopus (95) Google Scholar). In brief, the oocyte vitelline membranes were mechanically removed after being exposed to hypertonic solution (400 mosmol) for 5 min. Recordings were performed on the stripped oocytes using the same solution applied to bath and recording pipettes. The solution contained (in mm) KCl 10, potassium gluconate 130, potassium fluoride 5, EGTA 1, and HEPES 10 (pH 7.4). A parallel perfusion system was used to deliver low pH perfusates at a rate of ∼1 ml/min with no dead space (22Zhu G.Y. Chanchevalap S. Cui N. Jiang C. J. Physiol. ( Lond .). 1999; 516: 699-710Crossref PubMed Scopus (69) Google Scholar, 23Yang Z. Xu H. Cui N. Qu Z. Chanchevalap S. Shen W. Jiang C. J. Gen. Physiol. 2000; 116: 33-46Crossref PubMed Scopus (95) Google Scholar). Macroscopic and single-channel currents were analyzed using the pClamp 6 software as detailed previously (22Zhu G.Y. Chanchevalap S. Cui N. Jiang C. J. Physiol. ( Lond .). 1999; 516: 699-710Crossref PubMed Scopus (69) Google Scholar, 23Yang Z. Xu H. Cui N. Qu Z. Chanchevalap S. Shen W. Jiang C. J. Gen. Physiol. 2000; 116: 33-46Crossref PubMed Scopus (95) Google Scholar). Data are presented as means ± S.E. analysis of variance or Student's t test was used. Differences of CO2and pH effects before versus during exposures were considered to be statistically significant if p ≤ 0.05. Expressions of Kir6.2 together with SUR1 (Kir6.2+SUR1) inXenopus oocytes produced K+ currents with clear inward rectification in whole-cell recordings and ATP-dependent inhibition in inside-out patches. When oocytes expressing Kir6.2+SUR1 were exposed to 15% CO2(14Zhu G.Y. Chanchevalap S. Liu C. Xu H. Jiang C. J. Cell. Physiol. 2000; 183: 53-64Crossref PubMed Scopus (38) Google Scholar, 18Xu H. Cui N. Yang Z. Qu Z. Jiang C. J. Physiol. ( Lond .). 2000; 524: 725-735Crossref PubMed Scopus (105) Google Scholar), massive activation of the whole-cell inward rectifying currents occurred (129 ± 31%, n = 5). Similar channel activation was observed in Kir6.2ΔC36 (143 ± 15%,n = 11), whose CO2 sensitivity did not show any significant difference from the Kir6.2+SUR1 and Kir6.2ΔC36+SUR1 (p > 0.05), indicating that the CO2-sensing mechanism is located on the Kir6.2 subunit (Fig. 1, A and B). The effect was reversible and dependent on CO2concentrations. An apparent increase in the current amplitude was seen with PCO2 as low as 7.6 torr (1%), and higherPCO2 resulted in stronger activation (Fig.1 C). Interestingly, the Kir6.1+SUR1 showed a similar CO2 sensitivity (141 ± 28%, n = 6; Fig. 1 B), suggesting that various KATP channels consisting of Kir6.1 or Kir6.2 are likely to be activated by CO2. In contrast, Kir2.1 had no response to 15% CO2, whereas Kir1.1, Kir2.3, and Kir4.1 were inhibited (Fig. 1 B). To understand whether the channel activation was produced by pH changes, currents were studied with a selective decrease in intracellular pH (pHi) to 6.6 or extracellular pH (pHo) to 6.2 as we have previously measured during CO2 (15%) exposure (14Zhu G.Y. Chanchevalap S. Liu C. Xu H. Jiang C. J. Cell. Physiol. 2000; 183: 53-64Crossref PubMed Scopus (38) Google Scholar). Selective intracellular acidification using bicarbonate (14Zhu G.Y. Chanchevalap S. Liu C. Xu H. Jiang C. J. Cell. Physiol. 2000; 183: 53-64Crossref PubMed Scopus (38) Google Scholar, 18Xu H. Cui N. Yang Z. Qu Z. Jiang C. J. Physiol. ( Lond .). 2000; 524: 725-735Crossref PubMed Scopus (105) Google Scholar, 19Fakler B. Schultz J.H. Yang J. Schulte U. Brandle U. Zenner H.P. Jan L.Y. Ruppersberg J.P. EMBO J. 1996; 15: 4093-4099Crossref PubMed Scopus (162) Google Scholar) without changing pHo activated the Kir6.2ΔC36 currents by 162 ± 31% (n = 4), which showed no significant difference from the hypercapnic effect (p> 0.05, Fig. 1, D and E). Lowering pHoto 6.2 without changing pHi, however, increased the currents only modestly (8 ± 2%, n = 4; Fig.1 E), suggesting that the channels are stimulated predominantly by intracellular protons. The pH sensitivity of whole-cell Kir6.2ΔC36 currents was examined by permeabilization of the plasma membranes using protonophore nigericin (10 μm) at various pHo levels (20Grinstein S. Cohen S. Rothstein A. J. Gen. Physiol. 1984; 83: 341-369Crossref PubMed Scopus (312) Google Scholar, 21Wilding T.J. Cheng B. Roos A. J. Gen. Physiol. 1992; 100: 593-608Crossref PubMed Scopus (44) Google Scholar). Graded activation of Kir6.2ΔC36 currents was seen with graded acidification (Fig.2 B). When the maximal activation was reached at pH ∼6.6, the current amplitude increased by 150 ± 11% (n = 6), which should mainly be produced by a drop in pH because acidic pHo had little effect on the currents. Thus, a consistent enhancement of the channel activity was demonstrated at pHi 6.6 in the presence or absence of CO2. To determine whether the channel activation is carried out by the inherent mechanism of Kir6.2 or mediated by cytosolic factors, Kir6.2ΔC36 and Kir6.2+SUR1 currents were studied in excised inside-out patches in the absence of ATP, ADP, and other cytosolic soluble factors (22Zhu G.Y. Chanchevalap S. Cui N. Jiang C. J. Physiol. ( Lond .). 1999; 516: 699-710Crossref PubMed Scopus (69) Google Scholar, 23Yang Z. Xu H. Cui N. Qu Z. Chanchevalap S. Shen W. Jiang C. J. Gen. Physiol. 2000; 116: 33-46Crossref PubMed Scopus (95) Google Scholar). Exposure of the internal surface of patch membranes to acidic pH augmented the macroscopic inward rectifying currents (Fig. 2 A). The peak activation occurred at pHi 6.6–6.8 (Fig. 2 B). A further decrease in pHi caused rapid inhibition of the Kir currents (Fig. 2,A and B). The inhibition appeared to be channel rundown because it was not seen in whole-cell recordings and channel activity showed little or no recovery during washout at pH 7.4, particularly after a long period of exposure in patches (>30 s). Thus, extremely acidic pH may accelerate KATP rundown as described previously (7Vivaudou M. Forestier C. J. Physiol. ( Lond .). 1995; 486: 629-645Crossref PubMed Scopus (35) Google Scholar, 11Davies N.W. Standen N.B. Stanfield P.R. J. Physiol. ( Lond .). 1992; 445: 549-568Crossref PubMed Scopus (92) Google Scholar, 12Fan Z. Tokuyama Y. Makielski J.C. Am. J. Physiol. 1994; 267: C1036-C1044Crossref PubMed Google Scholar, 13Baukrowitz T. Tucker S.J. Schulte U. Benndorf K. Ruppersberg J.P. Fakler B. EMBO J. 1999; 18: 847-853Crossref PubMed Scopus (78) Google Scholar). A similar bell-shaped current-pHi relationship was observed in Kir6.2+SUR1 (Fig.2 B), further supporting the observation that pHi sensitivity is independent of the SUR subunit. Single-channel recordings showed that the current stimulation was mainly caused by augmentation of the open state probability (NPo) with concurrent moderate suppression (by 10.7 ± 0.2% measured at pH 6.2, n = 4) of the single-channel conductance. When the NPo was plotted as a function of pHi a bell-shapedNPo-pH relationship was also obtained, which was almost identical to the response of macroscopic currents to acidic pHi (Figs. 2 B and 3). The relationship of channel activity (i.e. macroscopic currents and NPo) versus pHican be described using a sum of two Hill equations with one for channel activation (pKa 7.15, h 2.0) and the other for channel rundown (pKa 6.48, h6.0) (Fig. 2 B). The pHi-dependent channel activation was virtually superimposed with that of whole-cell currents, consistent with the idea that the pH sensing mechanism exists inherently in the channel protein. If the pHi sensing is indeed an intrinsic property of the channel protein, there should be specific protein domains or amino acid residues responsible for the proton detection. To test this hypothesis, we performed site-directed mutagenesis on potentially titratable residues of histidine, an amino acid with its side chain pK (6.04) closest to the channel activation pKa (7.15). Among nine histidine residues studied in the intracellular N- and C-terminal domains, we found that His-175 was critical for the pHi sensitivity of Kir6.2ΔC36. Mutation of this residue to lysine (residue found in other Kir channels, H175K) or alanine (H175A) completely eliminated the proton-induced channel activation (Fig. 4). These mutants were even inhibited during hypercapnia in whole-cell recordings (Fig. 4,A and C) and by acidic pHi in excised patches (Fig. 4 B), which were also observed in mutants expressed with SUR1. Single-channel conductance of the H175K was significantly smaller than its wild-type counterpart (64.4 ± 0.9 picosiemens, n = 4; p < 0.05). Interestingly, a relief of channel rundown was seen in some of the His-175 mutations. In the H175K, NPo remained above 0.1 after 200 s of perfusion with a solution of pH 6.2 (n = 3). In contrast, NPodropped to below 0.01 within 40 s in the wild-type channel (n = 12) and H175A mutant under the same condition (n = 3). Mutations of other histidine residues had no significant effect on the CO2 sensitivity (p > 0.05, Fig. 4 C). Figure 4Dependence of the pH sensitivity on histidine residues. A, whole-cell currents were recorded from an oocyte expressing the H175A mutant Kir6.2ΔC36 under the same conditions as described in Fig. 1 A. The currents were no longer stimulated by hypercapnia, although current inhibition was seen. B, in an inside-out patch, the H175A mutation completely abolished channel activation by low pHi, whereas the currents were suppressed at acidic pHi. Moderate recovery occurred after washout. C, comparison of the CO2sensitivity of Kir6.2ΔC36 currents with histidine mutations studied in the same experimental condition. Channel stimulation disappeared in the H175A and H175K, whereas other mutants showed no significant changes. D, alignment of amino acid sequences of several members of the Kir family around the His-175 in Kir6.2 (bold). This histidine residue is found exclusively in the Kir6 subfamily. WS, washout.View Large Image Figure ViewerDownload (PPT) Finally, we examined if ATP and pH sensitivities depended on each other. It is known that the K185E mutation greatly reduces the ATP sensitivity of Kir6.2ΔC36 (24Reimann F. Ryder T.J. Tucker S.J. Ashcroft F.M. J. Physiol. ( Lond .). 1999; 520: 661-669Crossref PubMed Scopus (41) Google Scholar). We found that the K185E had identical pH sensitivity to the wild-type Kir6.2ΔC36, whereas the H175K mutation did not affect the ATP sensitivity (Fig.5). KATP channels are regulated by ATP, ADP, and phospholipids (11Davies N.W. Standen N.B. Stanfield P.R. J. Physiol. ( Lond .). 1992; 445: 549-568Crossref PubMed Scopus (92) Google Scholar, 24Reimann F. Ryder T.J. Tucker S.J. Ashcroft F.M. J. Physiol. ( Lond .). 1999; 520: 661-669Crossref PubMed Scopus (41) Google Scholar, 25Larsson O. Ammala C. Bokvist K. Fredholm B. Rorsman P. J. Physiol. ( Lond .). 1993; 463: 349-365Crossref PubMed Scopus (77) Google Scholar, 26Fan Z. Makielski J.C. J. Biol. Chem. 1997; 272: 5388-5395Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 27Baukrowitz T. Schulte U. Oliver D. Herlitze S. Krauter T. Tucker S.J. Ruppersberg J.P. Fakler B. Science. 1998; 282: 1141-1144Crossref PubMed Scopus (438) Google Scholar, 28Shyng S.L. Nichols C.G. Science. 1998; 282: 1138-1141Crossref PubMed Scopus (483) Google Scholar). Such modulations allow them to control cellular activity during energy insufficiency. In the present studies, we have demonstrated the proton as another KATPregulator. We have found that hypercapnia and acidic pH at near physiological levels augment KATP activity in striking contrast to other members of the Kir family that are either inhibited by acidic pH or lack response (Fig. 1 B) (29Nichols C.G. Lopatin A.N. Annu. Rev. Physiol. 1997; 59: 171-191Crossref PubMed Scopus (659) Google Scholar). The effect of hypercapnia is likely to be mediated by a decrease in intracellular pH, inasmuch as similar channel activation occurs with a drop in pHi but not pHo. In cell-free excised patches we have found a moderate decrease in pHi augments KATP activity consistent with our whole-cell recordings in which pHi drops to 6.6 with 15% CO2 (14Zhu G.Y. Chanchevalap S. Liu C. Xu H. Jiang C. J. Cell. Physiol. 2000; 183: 53-64Crossref PubMed Scopus (38) Google Scholar). A further decrease in pHi causes channel rundown in excised patches consistent with previous observations (7Vivaudou M. Forestier C. J. Physiol. ( Lond .). 1995; 486: 629-645Crossref PubMed Scopus (35) Google Scholar, 11Davies N.W. Standen N.B. Stanfield P.R. J. Physiol. ( Lond .). 1992; 445: 549-568Crossref PubMed Scopus (92) Google Scholar, 12Fan Z. Tokuyama Y. Makielski J.C. Am. J. Physiol. 1994; 267: C1036-C1044Crossref PubMed Google Scholar, 13Baukrowitz T. Tucker S.J. Schulte U. Benndorf K. Ruppersberg J.P. Fakler B. EMBO J. 1999; 18: 847-853Crossref PubMed Scopus (78) Google Scholar). Such channel stimulation followed by inhibition may explain by and large the contradictory results of the pH sensitivity obtained previously in the cell-endogenous KATP using excised patches,i.e. when the KATP is studied with moderate acidification in a short period of exposure, channel activity increases. On the other hand, lower pH levels with a longer period of exposure produces channel inhibition with poor reversibility (8Allard B. Lazdunski M. Rougier O. J. Physiol. ( Lond .). 1995; 485: 283-296Crossref PubMed Scopus (44) Google Scholar, 9Proks P. Takano M. Ashcroft F.M. J. Physiol. ( Lond .). 1994; 475: 33-44Crossref PubMed Scopus (26) Google Scholar). In the presence of ATP, however, channel rundown is largely diminished so that only activation remains (6Koyano T. Kakei M. Nakashima H. Yoshinaga M. Matsuoka T. Tanaka H. J. Physiol. ( Lond. ). 1993; 463: 747-766Crossref PubMed Scopus (55) Google Scholar, 7Vivaudou M. Forestier C. J. Physiol. ( Lond .). 1995; 486: 629-645Crossref PubMed Scopus (35) Google Scholar, 8Allard B. Lazdunski M. Rougier O. J. Physiol. ( Lond .). 1995; 485: 283-296Crossref PubMed Scopus (44) Google Scholar, 9Proks P. Takano M. Ashcroft F.M. J. Physiol. ( Lond .). 1994; 475: 33-44Crossref PubMed Scopus (26) Google Scholar, 10Cook D.L. Hales C.N. Nature. 1984; 311: 271-273Crossref PubMed Scopus (969) Google Scholar, 11Davies N.W. Standen N.B. Stanfield P.R. J. Physiol. ( Lond .). 1992; 445: 549-568Crossref PubMed Scopus (92) Google Scholar, 12Fan Z. Tokuyama Y. Makielski J.C. Am. J. Physiol. 1994; 267: C1036-C1044Crossref PubMed Google Scholar). Because the biphasic response to acidic pH is not seen in our whole-cell recordings with severe hypercapnia (15% CO2) and acidification (pH 5.8), the channel inhibition or rundown may not occur in intact cells. Our current studies have also begun to shed insight into the molecular mechanisms underlying KATP activation during acidosis. We have shown that the pH-sensing mechanisms are located on the Kir6 subunit rather than in the SUR. Indeed, we have demonstrated that mutation of a single histidine residue (H175A or H175K) is sufficient to eliminate completely the acid-induced channel activation. Instead of stimulation, the His-175 mutants show a significant inhibition by hypercapnic acidosis. The inhibition appears to suggest that the pH-dependent channel rundown may include a proportion of channel inhibition that is not seen in the wild-type channels under whole-cell recordings when the channel activation is dominant. The channel inhibition manifests itself when the channel stimulation is eliminated, as for His-175 mutations. When this inhibition is considered, the activation phase of the pH-current relationship curve shown in Figs. 2 B and 5A can be even steeper. The inhibition, however, cannot totally account for the pH-dependent rundown, because rundown, though relieved in the H175K, is still seen in the H175A mutation. The histidine-dependent pH sensitivity thus is consistent with the proton-mediated regulation of a large number of proteins. Because the histidine is conserved in both Kir6.1 and Kir6.2 in all known species (Fig. 4 D), it is very likely that the pH-sensing mechanism exists in KATP channels in various tissues. This unique site may offer an approach to control the KATPselectively without interference of other Kir channels. Our data indicate that pH sensitivity is independent of ATP for the following reasons: 1) acid-induced channel activation is seen in the absence of ATP; 2) channel pH sensitivity in excised patches resembles that in whole-cell recordings; 3) the K185E mutation greatly reduces ATP sensitivity without affecting the pH sensitivity; and 4) the H175K mutation does not compromise the ATP sensitivity. Therefore, proton sensing in the KATP is very unlikely to be mediated by ATP, although it may be modulated by ATP. Because a drop in pH often accompanies metabolic stresses and is more frequently seen than sole energy depletion, pH sensitivity enables the KATP channels to play a role in a wide variety of pathophysiological conditions. Pharmacological manipulation of the KATP in coronary arteries and cerebral vasculature has been shown to affect vascular tones during hypercapnia and acidosis (5Wei E.P. Kontos H.A. Stroke. 1999; 30: 851-853Crossref PubMed Scopus (23) Google Scholar, 30Ishizaka H. Gudi S.R. Frangos J.A. Kuo L. Circulation. 1999; 99: 558-563Crossref PubMed Scopus (53) Google Scholar). KATP may be activated by lactoacidosis during skeletal muscle fatigue (31Light P.E. Comtois A.S. Renaud J.M. J. Physiol. ( Lond .). 1994; 475: 495-507Crossref PubMed Scopus (54) Google Scholar), contributing to the decrease in tetanic force and the protection against injury (3Gramolini A. Renaud J.M. Am. J. Physiol. 1997; 272: C1936-C1946Crossref PubMed Google Scholar). Excessive neuronal activity also reduces pHi (32Trapp S. Luckermann M. Brooks P.A. Ballanyi K. J. Physiol. ( Lond .). 1996; 496: 695-710Crossref PubMed Scopus (68) Google Scholar), which may activate the KATP, leading to a suppression of hyperexcitability and a cessation of seizure activity (2Dost R. Rundfeldt C. Epilepsy Res. 2000; 38: 53-66Crossref PubMed Scopus (55) Google Scholar). Therefore, the demonstration of KATPmodulation by pHi has a profound impact on understanding cellular functions during metabolic stress and offers a potential intervention to control the cellular activity by manipulating the inherent pH sensing mechanism of the KATP channels in the treatment and prevention of stroke, epilepsy, diabetes mellitus, and coronary heart diseases. We thank Dr. S. Seino for providing Kir6.1 and Kir6.2 cNDAs and Dr. L. Bryan for the SUR1 cDNA. ATP-sensitive K+ sulfonylurea receptor intracellular pH extracellular pH open state probability

Abstract Although numerous fluorescent Zn 2+ sensors have been reported, it is unclear whether and how Zn 2+ can be released from the intracellular compartments into the cytosol due to a lack of probes that can detect physiological dynamics of cytosolic Zn 2+ . Here, we create a genetically encoded sensor, GZnP3, which demonstrates unprecedented sensitivity for Zn 2+ at sub-nanomolar concentrations. Using GZnP3 as well as GZnP3-derived vesicular targeted probes, we provide the first direct evidence that Zn 2+ can be released from endolysosomal vesicles to the cytosol in primary hippocampal neurons through the TRPML1 channel. Such TRPML1-mediated Zn 2+ signals are distinct from Ca 2+ in that they are selectively present in neurons, sustain longer, and are significantly higher in neurites as compared to the soma. Together, our work not only creates highly sensitive probes for investigating sub-nanomolar Zn 2+ dynamics, but also reveals new pools of Zn 2+ signals that can play critical roles in neuronal function.

The lysosome is an essential organelle to recycle cellular materials and maintain nutrient homeostasis, but the mechanism to down-regulate its membrane proteins is poorly understood. In this study, we performed a cycloheximide (CHX) chase assay to measure the half-lives of approximately 30 human lysosomal membrane proteins (LMPs) and identified RNF152 and LAPTM4A as short-lived membrane proteins. The degradation of both proteins is ubiquitin dependent. RNF152 is a transmembrane E3 ligase that ubiquitinates itself, whereas LAPTM4A uses its carboxyl-terminal PY motifs to recruit NEDD4-1 for ubiquitination. After ubiquitination, they are internalized into the lysosome lumen by the endosomal sorting complexes required for transport (ESCRT) machinery for degradation. Strikingly, when ectopically expressed in budding yeast, human RNF152 is still degraded by the vacuole (yeast lysosome) in an ESCRT-dependent manner. Thus, our study uncovered a conserved mechanism to down-regulate lysosome membrane proteins.

Lysosomes mediate hydrolase-catalyzed macromolecule degradation to produce building block catabolites for reuse. Lysosome function requires an osmo-sensing machinery that regulates osmolytes (ions and organic solutes) and water flux. During hypoosmotic stress or when undigested materials accumulate, lysosomes become swollen and hypo-functional. As a membranous organelle filled with cargo macromolecules, catabolites, ions, and hydrolases, the lysosome must have mechanisms that regulate its shape and size while coordinating content exchange. In this review, we discussed the mechanisms that regulate lysosomal fusion and fission as well as swelling and condensation, with a focus on solute and water transport mechanisms across lysosomal membranes. Lysosomal H+, Na+, K+, Ca2+, and Cl− channels and transporters sense trafficking and osmotic cues to regulate both solute flux and membrane trafficking. We also provide perspectives on how lysosomes may adjust the volume of themselves, the cytosol, and the cytoplasm through the control of lysosomal solute and water transport.

Divalent metal transporter-1 (DMT1/DCT1/Nramp2) is the major Fe(2+) transporter mediating cellular iron uptake in mammals. Phenotypic analyses of animals with spontaneous mutations in DMT1 indicate that it functions at two distinct sites, transporting dietary iron across the apical membrane of intestinal absorptive cells, and transporting endosomal iron released from transferrin into the cytoplasm of erythroid precursors. DMT1 also acts as a proton-dependent transporter for other heavy metal ions including Mn(2+), Co(2+), and Cu(2), but not for Mg(2+) or Ca(2+). A unique mutation in DMT1, G185R, has occurred spontaneously on two occasions in microcytic (mk) mice and once in Belgrade (b) rats. This mutation severely impairs the iron transport capability of DMT1, leading to systemic iron deficiency and anemia. The repeated occurrence of the G185R mutation cannot readily be explained by hypermutability of the gene. Here we show that G185R mutant DMT1 exhibits a new, constitutive Ca(2+) permeability, suggesting a gain of function that contributes to remutation and the mk and b phenotypes.

Niemann-Pick type C disease is a lysosomal storage disorder most often caused by loss-of-function mutations in the NPC1 gene. The encoded multipass transmembrane protein is required for cholesterol efflux from late endosomes and lysosomes. Numerous missense mutations in the NPC1 gene cause disease, including the prevalent I1061T mutation that leads to protein misfolding and degradation. Here, we sought to modulate the cellular proteostasis machinery to achieve functional recovery in primary patient fibroblasts. We demonstrate that targeting endoplasmic reticulum (ER) calcium levels using ryanodine receptor (RyR) antagonists increased steady-state levels of the NPC1 I1061T protein. These compounds also promoted trafficking of mutant NPC1 to late endosomes and lysosomes and rescued the aberrant storage of cholesterol and sphingolipids that is characteristic of disease. Similar rescue was obtained using three distinct RyR antagonists in cells with missense alleles, but not with null alleles, or by over-expressing calnexin, a calcium-dependent ER chaperone. Our work highlights the utility of proteostasis regulators to remodel the protein-folding environment in the ER to recover function in the setting of disease-causing missense alleles.

Background & AimsThe multiple endocrine neoplasia, type 1 (MEN1) locus encodes the nuclear protein and tumor suppressor menin. MEN1 mutations frequently cause neuroendocrine tumors such as gastrinomas, characterized by their predominant duodenal location and local metastasis at time of diagnosis. Diffuse gastrin cell hyperplasia precedes the appearance of MEN1 gastrinomas, which develop within submucosal Brunner’s glands. We investigated how menin regulates expression of the gastrin gene and induces generation of submucosal gastrin-expressing cell hyperplasia.MethodsPrimary enteric glial cultures were generated from the VillinCre:Men1FL/FL:Sst–/– mice or C57BL/6 mice (controls), with or without inhibition of gastric acid by omeprazole. Primary enteric glial cells from C57BL/6 mice were incubated with gastrin and separated into nuclear and cytoplasmic fractions. Cells were incubated with forskolin and H89 to activate or inhibit protein kinase A (a family of enzymes whose activity depends on cellular levels of cyclic AMP). Gastrin was measured in blood, tissue, and cell cultures using an ELISA. Immunoprecipitation with menin or ubiquitin was used to demonstrate post-translational modification of menin. Primary glial cells were incubated with leptomycin b and MG132 to block nuclear export and proteasome activity, respectively. We obtained human duodenal, lymph node, and pancreatic gastrinoma samples, collected from patients who underwent surgery from 1996 through 2007 in the United States or the United Kingdom.ResultsEnteric glial cells that stained positive for glial fibrillary acidic protein (GFAP+) expressed gastrin de novo through a mechanism that required PKA. Gastrin-induced nuclear export of menin via cholecystokinin B receptor (CCKBR)-mediated activation of PKA. Once exported from the nucleus, menin was ubiquitinated and degraded by the proteasome. GFAP and other markers of enteric glial cells (eg, p75 and S100B), colocalized with gastrin in human duodenal gastrinomas.ConclusionsMEN1-associated gastrinomas, which develop in the submucosa, might arise from enteric glial cells through hormone-dependent PKA signaling. This pathway disrupts nuclear menin function, leading to hypergastrinemia and associated sequelae. The multiple endocrine neoplasia, type 1 (MEN1) locus encodes the nuclear protein and tumor suppressor menin. MEN1 mutations frequently cause neuroendocrine tumors such as gastrinomas, characterized by their predominant duodenal location and local metastasis at time of diagnosis. Diffuse gastrin cell hyperplasia precedes the appearance of MEN1 gastrinomas, which develop within submucosal Brunner’s glands. We investigated how menin regulates expression of the gastrin gene and induces generation of submucosal gastrin-expressing cell hyperplasia. Primary enteric glial cultures were generated from the VillinCre:Men1FL/FL:Sst–/– mice or C57BL/6 mice (controls), with or without inhibition of gastric acid by omeprazole. Primary enteric glial cells from C57BL/6 mice were incubated with gastrin and separated into nuclear and cytoplasmic fractions. Cells were incubated with forskolin and H89 to activate or inhibit protein kinase A (a family of enzymes whose activity depends on cellular levels of cyclic AMP). Gastrin was measured in blood, tissue, and cell cultures using an ELISA. Immunoprecipitation with menin or ubiquitin was used to demonstrate post-translational modification of menin. Primary glial cells were incubated with leptomycin b and MG132 to block nuclear export and proteasome activity, respectively. We obtained human duodenal, lymph node, and pancreatic gastrinoma samples, collected from patients who underwent surgery from 1996 through 2007 in the United States or the United Kingdom. Enteric glial cells that stained positive for glial fibrillary acidic protein (GFAP+) expressed gastrin de novo through a mechanism that required PKA. Gastrin-induced nuclear export of menin via cholecystokinin B receptor (CCKBR)-mediated activation of PKA. Once exported from the nucleus, menin was ubiquitinated and degraded by the proteasome. GFAP and other markers of enteric glial cells (eg, p75 and S100B), colocalized with gastrin in human duodenal gastrinomas. MEN1-associated gastrinomas, which develop in the submucosa, might arise from enteric glial cells through hormone-dependent PKA signaling. This pathway disrupts nuclear menin function, leading to hypergastrinemia and associated sequelae.

The transient receptor potential (TRP) channel superfamily consists of a large group of non-selective cation channels that serve as cellular sensors for a wide spectrum of physical and environmental stimuli. The 28 mammalian TRPs, categorized into six subfamilies, including TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPML (mucolipin) and TRPP (polycystin), are widely expressed in different cells and tissues. TRPs exhibit a variety of unique features that not only distinguish them from other superfamilies of ion channels, but also confer diverse physiological functions. Located at the plasma membrane or in the membranes of intracellular organelles, TRPs are the cellular safeguards that sense various cell stresses and environmental stimuli and translate this information into responses at the organismal level. Loss- or gain-of-function mutations of TRPs cause inherited diseases and pathologies in different physiological systems, whereas up- or down-regulation of TRPs is associated with acquired human disorders. In this Cell Science at a Glance article and the accompanying poster, we briefly summarize the history of the discovery of TRPs, their unique features, recent advances in the understanding of TRP activation mechanisms, the structural basis of TRP Ca2+ selectivity and ligand binding, as well as potential roles in mammalian physiology and pathology.

Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] is a low-abundance phospholipid known to be associated with a wide variety of physiological functions in plants. However, the localization and dynamics of PI(3,5)P2 in plant cells remain largely unknown, partially due to the lack of an effective fluorescent probe. Using Arabidopsis transgenic plant expressing the PI(3,5)P2-labeling fluorescent probe (tagRFP–ML1N*2) developed based on a tandem repeat of the cytosolic phosphoinositide-interacting domain (ML1N) of the mammalian lysosomal transient receptor potential cation channel, Mucolipin 1 (TRPML1), here we show that PI(3,5)P2 is predominantly localized on the limited membranes of the FAB1- and SNX1-positive late endosomes, but rarely localized on the membranes of plant vacuoles or trans-Golgi network/early endosomes of cortical cells of the root differentiation zone. The late endosomal localization of tagRFP–ML1N*2 is reduced or abolished by pharmacological inhibition or genetic knockdown of expression of genes encoding PI(3,5)P2-synthesizing enzymes, FAB1A/B, but markedly increased with FAB1A overexpression. Notably, reactive oxygen species (ROS) significantly increase late endosomal levels of PI(3,5)P2. Thus, tandem ML1N-based PI(3,5)P2 probes can reliably monitor intracellular dynamics of PI(3,5)P2 in Arabidopsis cells with less binding activity to other endomembrane organelles.

Epithelial homeostasis and regeneration require a pool of quiescent cells. How the quiescent cells are established and maintained is poorly understood. Here, we report that Trpv6, a cation channel responsible for epithelial Ca2+ absorption, functions as a key regulator of cellular quiescence. Genetic deletion and pharmacological blockade of Trpv6 promoted zebrafish epithelial cells to exit from quiescence and re-enter the cell cycle. Reintroducing Trpv6, but not its channel dead mutant, restored the quiescent state. Ca2+ imaging showed that Trpv6 is constitutively open in vivo. Mechanistically, Trpv6-mediated Ca2+ influx maintained the quiescent state by suppressing insulin-like growth factor (IGF)-mediated Akt-Tor and Erk signaling. In zebrafish epithelia and human colon carcinoma cells, Trpv6/TRPV6 elevated intracellular Ca2+ levels and activated PP2A, which down-regulated IGF signaling and promoted the quiescent state. Our findings suggest that Trpv6 mediates constitutive Ca2+ influx into epithelial cells to continuously suppress growth factor signaling and maintain the quiescent state.

Lysosomal hydrolases require an acidic lumen for their optimal activities. In this issue, two independent groups (Wu et al. 2023. J. Cell Biol.https://doi.org/10.1083/jcb.202208155; Zhang et al. 2023. J. Cell. Biol.https://doi.org/10.1083/jcb.202210063) report that hydrolase activation also requires high intralysosomal Cl-, which is established by the lysosomal Cl-/H+ exchanger ClC-7.

The modulation of KATP channels during acidosis has an impact on vascular tone, myocardial rhythmicity, insulin secretion, and neuronal excitability. Our previous studies have shown that the cloned Kir6.2 is activated with mild acidification but inhibited with high acidity. The activation relies on His-175, whereas the molecular basis for the inhibition remains unclear. To elucidate whether the His-175 is indeed the protonation site and what other structures are responsible for the pH-induced inhibition, we performed these studies. Our data showed that the His-175 is the only proton sensor whose protonation is required for the channel activation by acidic pH. In contrast, the channel inhibition at extremely low pH depended on several other histidine residues including His-186, His-193, and His-216. Thus, proton has both stimulatory and inhibitory effects on the Kir6.2 channels, which attribute to two sets of histidine residues in the C terminus. The modulation of KATP channels during acidosis has an impact on vascular tone, myocardial rhythmicity, insulin secretion, and neuronal excitability. Our previous studies have shown that the cloned Kir6.2 is activated with mild acidification but inhibited with high acidity. The activation relies on His-175, whereas the molecular basis for the inhibition remains unclear. To elucidate whether the His-175 is indeed the protonation site and what other structures are responsible for the pH-induced inhibition, we performed these studies. Our data showed that the His-175 is the only proton sensor whose protonation is required for the channel activation by acidic pH. In contrast, the channel inhibition at extremely low pH depended on several other histidine residues including His-186, His-193, and His-216. Thus, proton has both stimulatory and inhibitory effects on the Kir6.2 channels, which attribute to two sets of histidine residues in the C terminus. sulfonylurea receptor phosphatidylinositol 4,5-biphosphate wild type ATP-sensitive K+ (KATP) channels couple the intermediary metabolism to cellular activity and play important roles in numerous cellular functions such as insulin secretion, neuronal excitability, vascular tones, and muscle contractability (1Aspinwall C.A. Brooks S.A. Kennedy R.T. Lakey J.R. J. Biol. Chem. 1997; 272: 31308-31314Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 2Dost R. Rundfeldt C. Epilepsy Res. 2000; 38: 53-66Crossref PubMed Scopus (54) Google Scholar, 3Gramolini A. Renaud J.M. Am. J. Physiol. 1997; 272: C1936-C1946Crossref PubMed Google Scholar, 4Kontos H.A. Wei E.P. Am. J. Physiol. 1998; 274: H974-H981Crossref PubMed Google Scholar, 5Light P.E. Comtois A.S. Renaud J.M. J. Physiol. (Lond.). 1994; 475: 495-507Crossref Scopus (56) Google Scholar, 6Wang W. Hebert S.C. Giebisch G. Annu. Rev. Physiol. 1997; 59: 413-436Crossref PubMed Scopus (176) Google Scholar). The KATP channels consist of the pore-forming Kir6 and sulfonylurea receptor (SUR)1 subunits. Without SUR, the Kir6.2 alone can express functional channels with essential ATP sensitivity when the last 26 or 36 amino acids are deleted (i.e. Kir6.2ΔC26 or Kir6.2ΔC36) (7Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (683) Google Scholar). The KATP channels are modulated by multiple cytosolic factors, likely through allosteric mechanisms. Their hallmark feature is the sensitivity to intracellular ATP that inhibits channel activity. ADP and phosphatidylinositol 4,5-bisphosphate (PIP2) are another two regulators with opposite effects to ATP (8Allard B. Lazdunski M. Pfluegers Arch. 1992; 422: 185-192Crossref PubMed Scopus (43) Google Scholar, 9Fan Z. Makielski J.C. J. Biol. Chem. 1997; 272: 5388-5395Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 10Larsson O. Ammala C. Bokvist K. Fredholm B. Rorsman P. J. Physiol. (Lond.). 1993; 463: 349-365Crossref Scopus (77) Google Scholar). In the presence of PIP2, the IC50 of ATP concentration increases by at least 100-fold, allowing these channels to be activated at near-physiological ATP levels (11Baukrowitz T. Schulte U. Oliver D. Herlitze S. Krauter T. Tucker S.J. Ruppersberg J.P. Fakler B. Science. 1998; 282: 1141-1144Crossref PubMed Scopus (442) Google Scholar, 12Shyng S.L. Nichols C.G. Science. 1998; 282: 1138-1141Crossref PubMed Scopus (487) Google Scholar). Similar to ATP, ADP, and PIP2, proton is a potent KATP regulator in a number of tissues (13Allard B. Lazdunski M. Rougier O. J. Physiol. (Lond.). 1995; 485: 283-296Crossref Scopus (44) Google Scholar, 14Davies N.W. Standen N.B. Stanfield P.R. J. Physiol. 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Some studies have suggested that proton rather than ATP or ADP is the ultimate signal reflecting the metabolic status in the muscle cells (9Fan Z. Makielski J.C. J. Biol. Chem. 1997; 272: 5388-5395Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). The pH sensitivity is more complex in KATP than in all other Kir channels. In excised patches, the KATP first undergoes activation with mild acidosis and then is strongly inhibited with further acidification (15Fan Z. Tokuyama Y. Makielski J.C. Am. J. Physiol. 1994; 267: C1036-C1044Crossref PubMed Google Scholar, 16Koyano T. Kakei M. Nakashima H. Yoshinaga M. Matsuoka T. Tanaka H. J. Physiol. (Lond.). 1993; 463: 747-766Crossref Scopus (55) Google Scholar, 20Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The activation is a reversible process relying on inherent properties of the channel protein, whereas the inhibition manifests itself when the activation is removed and shows rather poor reversibility (20Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). We have shown previously that His-175 is crucial for the pH-dependent channel activation (20Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). This histidine residue is conserved in Kir6 but not seen in any other Kir channels. Therefore, detailed studies of such a critical residue may lead to a discovery of molecular intervention to KATP channels by manipulating this histidine residue. Although histidine is the ultimate proton sensor according to its side-chain pK value, it can be involved in channel gating via other mechanisms. Thus, the demonstration of the real proton sensor becomes crucial in the understanding of the pH-dependent KATP activation. To determine whether titration of the side-chain amine group of His-175 indeed occurs at acidic pH, we performed systematic mutagenesis experiments on the His-175 using whole-cell voltage clamp and single-channel recordings. We reasoned that if the channel activation requires protonation of the His-175, a replacement of it with a positively charged residue should lead to an enhancement of the base-line currents. On the other hand, its substitution with a neutral or negative residue should have no effect or even suppress the base-line currents if the channel expression in the plasma membranes remains unchanged. Furthermore, mutations to any other amino acids should abolish the acid-induced channel activation, because of their titratability in an environment that favors the histidine titration at pH 6–7. The pH-dependent channel inhibition is another important characteristic of the KATP, which is shown in excised patches at low pH. Since such an inhibition is also pH-dependent, it should have its own pH-sensing mechanisms in the Kir6.2 protein, which may depend on histidine residues as well. To understand the proton-sensing mechanisms for the Kir6.2 activation and inhibition, we performed these experiments in which all histidine residues in the Kir6.2 protein were studied either alone or in combination with other histidines. Our results indicate that there are two sets of histidine residues in the Kir6.2 channels critical for the pH-dependent activation and inhibition, respectively. Oocytes from Xenopus laevis were used in the present studies. Frogs were anesthetized by bathing them in 0.3% 3-aminobenzoic acid ethyl ester. A few lobes of ovaries were removed after a small abdominal incision (∼5 mm). The surgical incision was then closed, and the frogs were allowed to recover from the anesthesia.Xenopus oocytes were treated with 2 mg/ml collagenase (type I, Sigma) in the OR2 solution (in mm): NaCl 82, KCl 2, MgCl2 1, and HEPES 5, pH 7.4, for 90 min at room temperature. After 3 washes (10 min each) of the oocytes with the OR2 solution, cDNAs (25–50 ng in 50 nl of water) were injected into the oocytes. The oocytes were then incubated at 18 °C in the ND-96 solution containing (in mm) NaCl 96, KCl 2, MgCl2 1, CaCl2 1.8, HEPES 5, and sodium pyruvate 2.5 with 100 mg/liter geneticin added, pH 7.4. Mouse Kir6.2 (GenBankTM accession number D50581) cDNA was generously provided by Dr. S. Seino and subcloned into a eukaryotic expression vector (pcDNA3.1, Invitrogen Inc., Carlsbad, CA). Site-specific mutations were produced using a site-directed mutagenesis kit (Stratagene, La Jolla, CA). The correct mutations were confirmed with DNA sequencing. Whole-cell currents were studied on the oocytes 2–4 days after injection. Two-electrode voltage clamp was performed using an amplifier (Geneclamp 500, Axon Instruments Inc., Foster City, CA) at room temperature (∼24 °C). The extracellular solution contained (in mm): KCl 90, MgCl2 3, and HEPES 5, pH 7.4. The HEPES buffer was chosen because of its buffering range and membrane impermeability, as shown in our previous studies (20Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 23Xu H. Cui N. Yang Z. Qu Z. Jiang C. J. Physiol. (Lond.). 2000; 524: 725-735Crossref Scopus (108) Google Scholar). Experiments were performed in a semi-closed recording chamber (BSC-HT, Medical System, Greenvale, NY), in which oocytes were placed on a supporting nylon mesh, and the perfusion solution bathed both the top and bottom surface of the oocytes. The perfusate and the superfusion gas entered the chamber from two inlets at one end and flowed out at the other end. There was a 3 × 15-mm gap on the top cover of the chamber, which served as the gas outlet and an access to the oocytes for recording microelectrodes. At base line, the chamber was ventilated with atmospheric air. Exposure of the oocytes to CO2 was carried out by switching a perfusate that had been bubbled for at least 30 min with a gas mixture containing CO2 at various concentrations balanced with 21% O2 and N2 and superfused with the same gas (23Xu H. Cui N. Yang Z. Qu Z. Jiang C. J. Physiol. (Lond.). 2000; 524: 725-735Crossref Scopus (108) Google Scholar, 24Yang Z. Xu H. Cui N. Qu Z. Chanchevalap S. Shen W. Jiang C. J. Gen. Physiol. 2000; 116: 33-45Crossref PubMed Scopus (96) Google Scholar, 25Zhu G. Chanchevalap S. Cui N. Jiang C. J. Physiol. (Lond.). 1999; 516: 699-710Crossref Scopus (70) Google Scholar). The high dissolvability of CO2 resulted in a detectable change in intra- or extracellular pH values as fast as 10 s in these oocytes. Macroscopic and single-channel currents were recorded in excised patches at room temperature (∼24 °C) as described previously (24Yang Z. Xu H. Cui N. Qu Z. Chanchevalap S. Shen W. Jiang C. J. Gen. Physiol. 2000; 116: 33-45Crossref PubMed Scopus (96) Google Scholar,25Zhu G. Chanchevalap S. Cui N. Jiang C. J. Physiol. (Lond.). 1999; 516: 699-710Crossref Scopus (70) Google Scholar). In brief, the oocyte vitelline membranes were mechanically removed after exposing to hypertonic solution (400 mOsm) for 5 min. Recordings were performed on the stripped oocytes using the same solution applied to bath and recording pipettes. The solution contained (in mm) KCl 10, potassium gluconate 130, potassium fluoride 5, EGTA 1, and HEPES 10, pH 7.4. A parallel perfusion system was used to deliver low pH perfusates at a rate of ∼1 ml/min with no dead space (24Yang Z. Xu H. Cui N. Qu Z. Chanchevalap S. Shen W. Jiang C. J. Gen. Physiol. 2000; 116: 33-45Crossref PubMed Scopus (96) Google Scholar, 25Zhu G. Chanchevalap S. Cui N. Jiang C. J. Physiol. (Lond.). 1999; 516: 699-710Crossref Scopus (70) Google Scholar). Macroscopic and single-channel currents were analyzed using the pClamp 6 software as detailed previously (24Yang Z. Xu H. Cui N. Qu Z. Chanchevalap S. Shen W. Jiang C. J. Gen. Physiol. 2000; 116: 33-45Crossref PubMed Scopus (96) Google Scholar, 25Zhu G. Chanchevalap S. Cui N. Jiang C. J. Physiol. (Lond.). 1999; 516: 699-710Crossref Scopus (70) Google Scholar). Data were further filtered (1 kHz) with a Gaussian filter. This filtering causes events shorter than 200 µs to be ignored. No correction was attempted for the missed events. Single-channel conductance was measured as a slope conductance with at least two voltage points. The open-state probability (Po) was calculated by first measuring the time, tj, spent at current levels corresponding to j = 0,1,2,···N channels open (24Yang Z. Xu H. Cui N. Qu Z. Chanchevalap S. Shen W. Jiang C. J. Gen. Physiol. 2000; 116: 33-45Crossref PubMed Scopus (96) Google Scholar, 25Zhu G. Chanchevalap S. Cui N. Jiang C. J. Physiol. (Lond.). 1999; 516: 699-710Crossref Scopus (70) Google Scholar). ThePo was then obtained asPo = ( Σ jN = 1tjj)/TN, where Nis the number of channels active in the patch, and T is the duration of recordings. Po values were calculated from stretches of data having a total duration of 20–60 s. The current amplitude was described using Gaussian distributions, and the difference between two adjacent fitted peaks was taken as unitary current amplitude. The charge density of the side chain of an amino acid was calculated using the classical Henderson-Hasselbach equation pH = pK + log ([base]/[acid]). The ratio of [base]/[acid] was calculated using the side-chain pK value of a titratable amino acid in free base at different pH levels and was used as an index for the ratio of the protonated versus non-protonated state of the amino acid. The pH-current relationship was described using a sum of a double Hill equation: y = {m/(1 + (pH/pK1)h1)} + {m/(1 + (pK2/pH)h2)} −m, where pK1 is the midpoint channel activation; h1 is the Hill coefficient for channel activation; pK2 is the midpoint channel inhibition; h2 is the Hill coefficient for channel inhibition; and m = 1.2 (assuming 80% peak activation is reach before rundown starts). Data are presented as means ± S.E. Student's t or analysis of variance test was used. Differences of CO2 and pH effects beforeversus during exposures were considered to be statistically significant if p ≤ 0.05. Whole-cell currents were studied in the two-electrode voltage clamp mode using an extracellular solution containing 90 mm K+. Depolarizing and hyperpolarizing command pulses were given to the cell in a range from −160 (−120 mV in some cells) to 100 mV with a 20-mV increment at a holding potential of 0 mV. Under such a condition, the inward rectifying currents were observed 2–4 days after cDNA injection. Consistent with our previous observations, currents produced by Kir6.2 and SUR1 or a single Kir6.2ΔC36 were equally stimulated when the cell was exposed to 15% CO2 (Fig.1A). Therefore, to simplify the studies, the Kir6.2ΔC36 was used. This effect resulted from intracellular acidification and was independent of ATP, as it was seen in the excised patch in the absence of ATP (20Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). In excised patches, the channel displayed a rapid inhibition at extremely acidic pH (20Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), which showed a poor reversibility and appeared similar to channel rundown (Fig. 1B). As we reported previously (20Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), mutation of His-175 to lysine (a residue found in several other Kir channels, H175K) or alanine (H175A) completely eliminated the acid-induced channel activation. Instead, these mutants were inhibited during hypercapnia in whole-cell recordings (Fig.2A) and by acidic pH in excised patches (Fig. 1B). To determine how this histidine residue is involved in pH-dependent Kir6.2 activation, systematic mutation analysis was carried out. If certain side-chain properties of this residue such as the size, charged state, or hydrophobicity but not the titratability were critical for the proton sensitivity, a substitution of the histidine with another amino acid with similar side-chain properties would retain, to certain degree, the pH-dependent activation. To test this hypothesis, the His-175 was mutated to positively charged (H175K and H175R), neutral polar (H175N and H175C), neutral non-polar (H175A), or negatively charged (H175E and H175D) amino acids. When these mutants were tested, we found that the pH-dependent activation was totally lost in all these mutants (Fig. 2B). Among these substitutes are arginine and alanine. Arginine is highly hydrophilic and capable of forming hydrogen bonds with adjacent residues, whereas alanine is not. Arginine but not alanine has a large side chain and is positively charged at physiological pH. Despite these distinct properties, replacement of the His-175 with arginine affected the CO2 sensitivity almost identically to that with alanine. We noticed that the amplitude of base-line currents before CO2 exposure were closely associated with the charge characteristics of the amino acid at this location. Mutations to positively charged amino acids yielded channels with large base-line currents (H175K, 8.1 ± 1.1 µA, n = 8; H175R, 9.2 ± 1.1 µA, n = 5); mutations to neutral amino acid gave rise to moderate currents (H175A, 2.4 ± 0.4 µA,n = 11) similar to that of the wt Kir6.2ΔC36 (H175H, 2.1 ± 0.3 µA, n = 12); and no detectable current was seen when His-175 was mutated to negatively charged residues (H175D and H175E). The relationship of base-line currents with charge density was calculated according to the Henderson-Hasselbach equation (see “Materials and Methods”). When the base-line currents were plotted against the charge density, a strong correlation was revealed with r > 0.99 andp < 0.001 (Fig. 3), indicating that the current amplitude is a function of the positive charge at this location. There are two possible explanations for the difference in the base-line currents among these mutants. First, mutations to acidic or neutral amino acids are nonfunctional, owing to either misfolding, defective endoplasmic reticulum trafficking, or poor membrane insertion of the channel proteins. Second, the substitution with negative or neutral residues may lead most of expressed channels to the closed state, whereas mutations to the positive ones favor channel openings. If the mutant channels are expressed in the plasma membranes and stay in the closed state, their membrane expression can be demonstrated by activating these channels. Because the KATP channels are normally inhibited by ATP, lowering the ATP sensitivity can enhance the open-state probability, increasing the likelihood to see their expression. Therefore, another mutation on Cys-166 was introduced, as the C166S mutant has been reported previously to stabilize the channel to the open conformation (21Piao H. Cui N. Xu H. Mao J. Rojas A. Wang R. Abdulkadir L. Li L. Wu J. Jiang C. J. Biol. Chem. 2001; 276: 36673-36680Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 26Trapp S. Proks P. Tucker S.J. Ashcroft F.M. J. Gen. Physiol. 1998; 112: 333-349Crossref PubMed Scopus (151) Google Scholar). By adding the C166S mutation, several His-175 mutants (H175D, H175C, H175E, and H175Q) that failed to show channel activity were rescued (TableI). All of them were inhibited during CO2 exposure, even though the side-chain properties of the amino acids vary greatly (Table I). These results therefore suggest that the limited currents seen with negative charge or neutrality at locus 175 are likely to be due to the inadequate channel openings rather than the lack of functional expression.Table IMutation analyses of histidines involved in pH-dependent activation and inhibition of Kir6.2 channelNameBL currentCO2 effectnµA%Kir6.2ΔC362.1 ± 0.3148.0 ± 14.912His-175H175A2.4 ± 0.4−28.7 ± 6.911H175K8.1 ± 1.1−21.4 ± 2.18*H175K8.8 ± 2.6−15.8 ± 3.84*H175D15.8 ± 4.8−67.6 ± 6.05*H175E5.5 ± 0.5−38.1 ± 2.76*H175N18.7 ± 5.7−35.7 ± 4.74*H175C12.7 ± 1.8−23.7 ± 7.14H175R9.2 ± 1.1−7.8 ± 2.95Multiple histidine mutants*H175D15.8 ± 4.8−67.6 ± 6.05*H175D/H46A11.7 ± 1.1−67.6 ± 2.64*H175D/H70A26.4 ± 2.4−76.6 ± 2.64*H175D/H186A2.4 ± 0.2−5.4 ± 3.14*H175D/H193N32.7 ± 6.7−4.7 ± 2.85*H175D/H216Q15.7 ± 6.0−6.9 ± 4.14*H175D/H234A10.5 ± 3.4−66.7 ± 5.44*H175D/H259A11.1 ± 2.5−67.1 ± 3.35*H175D/H276A13.1 ± 4.0−54.5 ± 3.64*H175D/H277Q/278NNFH175K8.1 ± 1.1−21.4 ± 2.18H175K/H186A17.0 ± 2.46.3 ± 3.45H175K/H193N8.7 ± 0.4−0.8 ± 7.67H175K/H216Q8.4 ± 1.72.6 ± 7.94H175K/H186A/H913N15.5 ± 2.93.6 ± 0.95H175K/H70A15.1 ± 3.2−11.9 ± 1.97H175K/H259A5.0 ± 1.0−18.4 ± 0.75H186A2.0 ± 0.7169.1 ± 29.46H193N2.0 ± 0.3179.3 ± 29.34H216Q2.1 ± 0.4139.7 ± 14.64H186A/H193N2.7 ± 0.4190.6 ± 37.34H186A/H193N/H216Q3.5 ± 0.5213.7 ± 23.15Other mutantsE179Q15.4 ± 2.728.6 ± 10.04C166S14.7 ± 3.310.0 ± 6.97The mutants with * were constructed using the C166S mutant as a template. The abbreviations used are: BL, base line; n, number of observations; NF, nonfunctional. Open table in a new tab The mutants with * were constructed using the C166S mutant as a template. The abbreviations used are: BL, base line; n, number of observations; NF, nonfunctional. To understand further the relationship of the open-state probability (Po) with the number of expressed channels, we studied single-channel currents in excised inside-out patches. The H175K had higher base-line activity (Po = 0.123 ± 0.016, n = 4, p < 0.001) than the wt channel (Po = 0.021 ± 0.040,n = 5) and H175A mutant (Po = 0.029 ± 0.006, n = 5) (Fig.4A). Based on the relationshipG =g·N·Po (whereG is conductance of the macroscopic currents; gis single-channel conductance, and N is number of functional channels), the number of functional channels expressed in the plasma membranes can be estimated. We thereby measured base-line macroscopic currents and the single-channel conductance under the identical conditions. The base-line macroscopic currents were 26.1 ± 2.3 pA (n = 12) in the Kir6.2ΔC36, 30.3 ± 4.4 pA (n = 7) in the H175A, and 93.6 ± 17.4 pA (n = 8) in the H175K. The single-channel conductance was 74.1 ± 1.0 pS (n = 15) in the Kir6.2ΔC36, 74.0 ± 2.0 pS (n = 4) in the H175A, and 67.0 ± 1.3 pS (n = 5) in the H175K. We found that the large base-line H175K currents were due to a higher Poinstead of a greater expression of functional channels in the plasma membranes, as theG/(Po·g) ratio, or theN value, for the H175K is not greater than those for H175A and Kir6.2ΔC36 (Fig. 4B). The whole-cell base-line currents of the H175K were also about 3–4 times larger than those of wt Kir6.2ΔC36 and H175A (Table I). The change in the base-line currents was not caused by a decrease in ATP sensitivity either, since H175K and H175A had a similar ATP sensitivity as wt Kir6.2ΔC36 (20Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). These results thus indicate that protonation of the His-175 occurs at acidic pH leading to the channel activation. The H175K mutant showed high channel activity at pH 7.4–9.0. The channel activity decreased when the pH levels became higher or lower. The pK value for channel activation was pH 10.5 (Fig.5A), suggesting that the lysine residue is also protonated at near its pK value for free-base amino acids in the microenvironment of this residue. The PHDsec and Chou-Fasman analyses show that the secondary structure around the His-175 is α-helical. Thus, we studied two residues in the immediate vicinity of the His-175, which are aligned onto the same surface of His-175. Interestingly, when we neutralized Glu-179, the E179Q mutant showed CO2 sensitivity significantly smaller than that of the wt channel (Fig. 5B), indicating that the titratability of His-175 is strongly influenced by its microenvironment. We also studied Thr-171 by mutating it to a lysine (T171K), but we failed to obtain functional expression. Although the His-175 mutations eliminate the pH-dependent activation, the acid-induced channel inhibition remained, which indeed was more obvious in these mutants. It is possible that there is another mechanism responsible for the channel inhibition, which manifests itself only when the dominant activation is removed. To understand the molecular basis of this inhibition and to see whether this inhibition has a similar origin as the channel rundown that is usually seen in excised patch, further mutation analysis was performed. Mutations of one of several histidines were constructed on the H175D, H175K, or C166S/H175D mutants. The rationale for choosing the C166S/H175D as a template is that this mutant is strongly inhibited by CO2, raising the resolution of the CO2/pH-induced channel inhibition (Fig.6A). Note that the C166S mutant itself is not inhibited by 15% CO2 (Table I), similar to what we have shown recently (21Piao H. Cui N. Xu H. Mao J. Rojas A. Wang R. Abdulkadir L. Li L. Wu J. Jiang C. J. Biol. Chem. 2001; 276: 36673-36680Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Consistent with the CO2 sensitivity of whole-cell currents, the C166S/H175D mutant was inhibited by intracellular acidosis in inside-out patches with pK = 6.68 (h = 2.1,n = 4, Fig. 6, B and C). Concerning the CO2/pH sensitivities, the mutant channel behaved just like Kir1.1 and Kir2.3 channels where histidines are critical for the acid-induced channel inhibition (23Xu H. Cui N. Yang Z. Qu Z. Jiang C. J. Physiol. (Lond.). 2000; 524: 725-735Crossref Scopus (108) Google Scholar, 27Coulter K.L. Perier F. Radeke C.M. Vandenberg C.A. Neuron. 1995; 15: 1157-1168Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 28Qu Z. Yang Z. Cui N. Zhu G. Liu C. Xu H. Chanchevalap S. Shen W. Wu J. Li Y. Jiang C. J. Biol. Chem. 2000; 275: 31573-31580Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 29Tsai T.D. Shuck M.E. Thompson D.P. Bienkowski M.J. Lee K.S. Am. J. Physiol. 1995; 268: C1173-C1178Crossref PubMed Google Scholar). Therefore, we systematically studied all histidine residues in the channel protein. We found that His-193 was a player. The acid-induced inhibition was almost completely removed by mutation of His-193 to glutamine (Fig. 7, A andB). Mutations of His-186 and His-216 had a similar effect, whereas replacements of other histidines did not (Fig.8A and Table I). Mutation of these histidines did not compromise the channel activation. Indeed, the magnitude of the activation was significantly larger in the H186A/H193N/H216Q triple mutant than in the wt Kir6.2ΔC36 (Fig.8, B and C, Table I). Interestingly, mutations of these histidine residues markedly diminished the channel rundown, as shown in the H186A/H193N/H216Q and C166S/H175D/H193N (Fig.9, A and B). These results thus indicate that another set of histidine residues are involved in the pH-dependent inhibition of Kir6.2 channels.Figure 7Elimination of the acid-dependent inhibition by mutation of His-193. The H193N was constructed using the H175D/C166S as a template, and a triple mutation H175D/H193N/C166S was obtained. A, when the H175D/H193N/C166S was studied in whole-cell recording, this mutant showed large base-line (BL) currents and became insensitive to CO2(15%, 6 min). B, in an inside-out patch, the H175D/H193N/C166S mutant shows only a modest inhibition. C,sequence alignment of a cytoplasmic domain containing a few critical histidine residues including the His-193.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8Involvement of other histidine residues in the acid-induced channel inhibition.A, the CO2-induced channel inhibition was studied in several histidine mutants constructed on the basis of the C166S/H175D that shows a strong inhibition by CO2 (see Fig. 6A). The channel inhibition is greatly diminished when His-186, His-193, or His-216 is mutated, whereas mutations of other histidine residues have no significant effect. B, mutation of these histidines alone or combined does not reduce the channel activation during CO2 (15%) exposure. Indeed, the CO2sensitivity of the H186A/H193N/H216Q triple mutant is significantly enhanced, which is detailed in C. *, p < 0.05; **, p < 0.001; dashed line, control level of channel response to 15% CO2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 9A, pH sensitivity of the H186A/H193N/H216Q mutant. Currents were studied in an inside-out patch. Moderate decrease in pH still activates the H186A/H193N/H216Q currents. Although the current amplitude drops with further decrease in pH, such an inhibition is much less severe in comparison to the wt Kir6.2ΔC36. There are substantial currents remaining at pH 6.2, at which the wt Kir6.2ΔC36 currents are almost completely inhibited. B,the pH-current relationship of the wt Kir6.2ΔC36, C166S/H175D/H193N, and H186A/H193N/H216Q triple histidine mutant channels. The H186A/H193N/H216Q currents at pH 6.2 remain above the base-line level, pH 7.4. The C166C/H175D/H193N responds to acid similarly.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Proton is an important regulator of the KATPchannels (30Quayle J.M. Nelson M.T. Standen N.B. Physiol. Rev. 1997; 77: 1165-1232Crossref PubMed Scopus (722) Google Scholar). However, previous studies on the pH sensitivity of the KATP in several tissues and cells were controversial and even contradictory. Channel response varies from activation (16Koyano T. Kakei M. Nakashima H. Yoshinaga M. Matsuoka T. Tanaka H. J. Physiol. (Lond.). 1993; 463: 747-766Crossref Scopus (55) Google Scholar, 19Vivaudou M. Forestier C. J. Physiol. (Lond.). 1995; 486: 629-645Crossref Scopus (35) Google Scholar), no effect (22Davies N.W. Nature. 1990; 343: 375-377Crossref PubMed Scopus (190) Google Scholar, 31Cook D.L. Hales C.N. Nature. 1984; 311: 271-273Crossref PubMed Scopus (976) Google Scholar), to inhibition (13Allard B. Lazdunski M. Rougier O. J. Physiol. (Lond.). 1995; 485: 283-296Crossref Scopus (44) Google Scholar, 18Proks P. Takano M. Ashcroft F.M. J. Physiol. (Lond.). 1994; 475: 33-44Crossref Scopus (26) Google Scholar). By using the cloned KATP channels, we have shown recently that acidic pH has dual effects depending on the pH range. The channels are activated by a moderate decrease in intracellular pH and inhibited at high acidity. Such two-phase effects, which have been observed in insulin-secreting cells and cardiac myocytes (15Fan Z. Tokuyama Y. Makielski J.C. Am. J. Physiol. 1994; 267: C1036-C1044Crossref PubMed Google Scholar, 16Koyano T. Kakei M. Nakashima H. Yoshinaga M. Matsuoka T. Tanaka H. J. Physiol. (Lond.). 1993; 463: 747-766Crossref Scopus (55) Google Scholar), may underlie the controversy in the pH sensitivity of the cell-endogenous KATP. Our previous studies have shown that the channel activation relies on His-175 (20Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Mutation of this histidine residue completely eliminates the acid-induced channel activation (20Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The molecular basis of the His-175-dependent channel activation was examined in the present study. Our data have shown that protonation of His-175 is essential for the channel activation. First, histidine is absolutely necessary for the pH-dependent activation that is lost with a single mutation of this residue. Second, other side-chain properties of amino acid such as size, hydrophobicity, charge status, and hydrogen bond do not play a role, as mutations to other amino acids that have some of these properties cannot produce the pH-dependent activation. Third, the base-line channel activity is a function of the charge density. The largest currents are seen when residue 175 has a positive charge by protonation of the His-175 or by mutation to lysine or arginine. Fourth, the pKvalue of residue 175 is close to that of amino acid in its free base, which shifts to 10.5 when the His-175 is mutated to a lysine. These as well as the fact that the histidine side chain has a pKvalue most close to the physiological pH levels indicate that His-175 is the only proton sensor responsible for the pH-dependent Kir6.2 activation. We believe that finding this proton sensor is important since this histidine residue is exclusively seen in the Kir6 subfamily but not in any other Kir channels. The demonstration of such a site critical for controlling channel activity opens a major avenue to the understanding of the KATP regulation and the control of cellular activity in a number of physiologic and pathophysiologic conditions. At pH levels below 6.5, the Kir6.2 channel activity drops rapidly. In the present study, we have looked at the molecular basis of the inhibition and found three histidine residues that are likely to play a role. Mutations of these residues markedly attenuate the pH-dependent channel inhibition with no sacrifice of the activation. The inhibition becomes more obvious when the activation is removed. Considering only partial recovery following acid exposure in Kir1.1 (32Schulte U. Hahn H. Wiesinger H. Ruppersberg J.P. Fakler B. J. Biol. Chem. 1998; 273: 34575-34579Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), the H175D/C166S mutant responds to acidic pH almost identically to the typical pH-sensitive Kir channels (23Xu H. Cui N. Yang Z. Qu Z. Jiang C. J. Physiol. (Lond.). 2000; 524: 725-735Crossref Scopus (108) Google Scholar, 24Yang Z. Xu H. Cui N. Qu Z. Chanchevalap S. Shen W. Jiang C. J. Gen. Physiol. 2000; 116: 33-45Crossref PubMed Scopus (96) Google Scholar, 25Zhu G. Chanchevalap S. Cui N. Jiang C. J. Physiol. (Lond.). 1999; 516: 699-710Crossref Scopus (70) Google Scholar, 27Coulter K.L. Perier F. Radeke C.M. Vandenberg C.A. Neuron. 1995; 15: 1157-1168Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 28Qu Z. Yang Z. Cui N. Zhu G. Liu C. Xu H. Chanchevalap S. Shen W. Wu J. Li Y. Jiang C. J. Biol. Chem. 2000; 275: 31573-31580Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 29Tsai T.D. Shuck M.E. Thompson D.P. Bienkowski M.J. Lee K.S. Am. J. Physiol. 1995; 268: C1173-C1178Crossref PubMed Google Scholar). Thus, it is possible that the Kir6.2 shares the same molecular basis for the pHdependent inhibition. At this stage, the physiological significance for the pHdependent inhibition of Kir6.2 is still unclear. Since such an inhibition shows very poor recovery and is clearly seen in excised patches but not in whole-cell recordings, it may be related to the channel rundown. Previous studies on the cell-endogenous KATP have shown that channel rundown can be greatly attenuated when the cytosolic side of patch membranes is exposed to protease (33Fan Z. Makielski J.C. Circ. Res. 1993; 72: 715-722Crossref PubMed Google Scholar, 34Lee K. Ozanne S.E. Rowe I.C. Hales C.N. Ashford M.L. Mol. Pharmacol. 1994; 46: 176-185PubMed Google Scholar). Therefore, it is possible that the KATPchannel rundown involves a large scale of movement of intracellular protein domains as demonstrated in the Kir1.1 (32Schulte U. Hahn H. Wiesinger H. Ruppersberg J.P. Fakler B. J. Biol. Chem. 1998; 273: 34575-34579Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), whereas these histidine residues may play a part in the movement or even possibly initiate the movement. The KATP channels are gated by several intracellular regulators including ATP, ADP, PIP2, and proton. More complicated are the channel gating mechanisms, as proton itself has biphasic effects on channel activity showing how sophisticated the KATP gating can be. Although the precise mechanisms for channel gating are still not clear, a better understanding of the KATP regulation can be achieved with the availability of the information about all its key regulators. Therefore, the demonstration of the molecular basis for the pH-dependent channel activation and inhibition in the present study provides important information in this aspect. In conclusion, the Kir6.2 channel shows a biphasic response to acidic pH. The channel activation requires a histidine residue (His-175) near the M2 region in the C terminus. The His-175 is very likely to be the proton sensor that couples the pH change to channel activation. The channel inhibition, on the other hand, is related to another three histidine residues, i.e. His-186, His-193, and His-216. Mutations of these residues greatly diminish the pH-dependent inhibition. Thus, proton has both stimulatory and inhibitory effects on the KATP channels by acting on two distinct sets of histidine residues. We thank Dr. S. Seino for providing Kir6.2 cDNA.

A continuous-flow photocatalytic synthesis of the industrially important thiuram disulfides has been developed, utilizing O₂ as the oxidant and Eosin Y as the photoredox catalyst.

Thermosensitive transient receptor potential V3 (TRPV3) is a polymodal receptor implicated in nociceptive, thermoceptive, pruritoceptive, and inflammatory pathways. Reports focused on understanding the role of TRPV3 in thermoception or nociception are not conclusive. Previous studies also show that aberrant hyperactivity of TRPV3 channels results in spontaneous itch and dermatitis-like symptoms, but the resultant behavior is highly dependent on the background of the animal and the skin microbiome. To determine the function of hyperactive TRPV3 channels in somatosensory sensations, we tested different somatosensory behaviors using a genetic mouse model that carries a gain-of-function point mutation G573S in the Trpv3 gene (Trpv3G573S ). Here we report that Trpv3G573S mutants show reduced perception of cold, acetone-induced cooling, punctate, and sharp mechanical pain. By contrast, locomotion, noxious heat, touch, and mechanical itch are unaffected in Trpv3G573S mice. We fail to observe any spontaneous itch responses and/or dermatitis in Trpv3G573S mutants under specific pathogen (Staphylococcus aureus)-free conditions. However, we find that the scratching events in response to various pruritogens are dramatically decreased in Trpv3G573S mice in comparison to wild-type littermates. Interestingly, we observe sensory hypoinnervation of the epidermis in Trpv3G573S mutants, which might contribute to the deficits in acute mechanical pain, cool, cold, and itch sensations.

Hypercapnia has been shown to affect cellular excitability by modulating K+ channels. To understand the mechanisms for this modulation, four cloned K+ channels were studied by expressing them in Xenopus oocytes. Exposures of the oocytes to CO2 for 4–6 min produced reversible and concentration-dependent inhibitions of Kir1.1 and Kir2.3 currents, but had no effect on Kir2.1 and Kir6.1 currents. Intra- and extracellular pH (pHi, pHo) dropped during CO2 exposures. The inhibition of Kir2.3 currents was mediated by reductions in both intra- and extracellular pH, whereas the suppression of Kir1.1 resulted from intracellular acidification. In cell-free excised inside-out patches with cytosolic-soluble factors washed out, a decrease in pHi produced a fast and reversible inhibition of macroscopic Kir2.3 currents. The degree of this inhibition was similar to that produced by hypercapnia when compared at the same pHi level. Exposure of cytosolic surface of patch membranes to a perfusate bubbled with 15% CO2 without changing pH failed to inhibit the Kir2.3 currents. These results therefore indicate that (1) hypercapnia inhibits specific K+ channels, (2) these inhibitions are caused by intra- and extracellular protons rather than molecular CO2, and (3) these effects are independent of cytosol-soluble factors. J. Cell. Physiol. 183:53–64, 2000. © 2000 Wiley-Liss, Inc.

Ion channels play an important role in cellular functions, and specific cellular activity can be produced by gating them. One important gating mechanism is produced by intra- or extracellular ligands. Although the ligand-mediated channel gating is an important cellular process, the relationship between ligand binding and channel gating is not well understood. It is possible that ligands are involved in the interactions of different protein domains of the channel leading to opening or closing. To test this hypothesis, we studied the gating of Kir2.3 (HIR) by intracellular protons. Our results showed that hypercapnia or intracellular acidification strongly inhibited these channels. This effect relied on both the N and C termini. The CO2/pH sensitivities were abolished or compromised when one of the intracellular termini was replaced. Using purified N- and C-terminal peptides, we found that the N and C termini bound to each other in vitro. Although their binding was weak at pH 7.4, stronger binding was seen at pH 6.6. Two short sequences in the N and C termini were found to be critical for the N/C-terminal interaction. Interestingly, there was no titratable residue in these motifs. To identify the potential protonation sites, we systematically mutated most histidine residues in the intracellular N and C termini. We found that mutations of several histidine residues in the C but not the N terminus had a major effect on channel sensitivities to CO2 and pHi. These results suggest that at acidic pH, protons appear to interact with the C-terminal histidine residues and present the C terminus to the N terminus. Consequentially, these two intracellular termini bound to each other through two short motifs and closed the channel. Thus, a novel mechanism for K+ channel gating is demonstrated, which involves the N- and C-terminal interaction with protons as the mediator. Ion channels play an important role in cellular functions, and specific cellular activity can be produced by gating them. One important gating mechanism is produced by intra- or extracellular ligands. Although the ligand-mediated channel gating is an important cellular process, the relationship between ligand binding and channel gating is not well understood. It is possible that ligands are involved in the interactions of different protein domains of the channel leading to opening or closing. To test this hypothesis, we studied the gating of Kir2.3 (HIR) by intracellular protons. Our results showed that hypercapnia or intracellular acidification strongly inhibited these channels. This effect relied on both the N and C termini. The CO2/pH sensitivities were abolished or compromised when one of the intracellular termini was replaced. Using purified N- and C-terminal peptides, we found that the N and C termini bound to each other in vitro. Although their binding was weak at pH 7.4, stronger binding was seen at pH 6.6. Two short sequences in the N and C termini were found to be critical for the N/C-terminal interaction. Interestingly, there was no titratable residue in these motifs. To identify the potential protonation sites, we systematically mutated most histidine residues in the intracellular N and C termini. We found that mutations of several histidine residues in the C but not the N terminus had a major effect on channel sensitivities to CO2 and pHi. These results suggest that at acidic pH, protons appear to interact with the C-terminal histidine residues and present the C terminus to the N terminus. Consequentially, these two intracellular termini bound to each other through two short motifs and closed the channel. Thus, a novel mechanism for K+ channel gating is demonstrated, which involves the N- and C-terminal interaction with protons as the mediator. inward rectifier K+ 1,4-piperazinediethanesulfonic acid glutathione S-transferase amino acid(s) Ion channels are a group of membrane proteins characterized by ion-selective permeation and event-specific gating. By controlling or gating the transition between their open and closed states, specific cellular functions can be produced (1Hille B. Ionic Channels of Excitable Membranes. Sinauer Associates, Sunderland, MA1992: 472-503Google Scholar). Several membrane and cytosolic mechanisms are involved in the channel gating. For instance, a voltage-gated ion channel can be activated by depolarization; intra- and extracellular ligands can retain a ligand-gated ion channel to the open or closed state. The gating of inward rectifier K+(Kir)1 channels is carried out by membrane-bound and cytosolic molecules including G proteins, nucleotides, and protons (2Nichols C.G. Lopatin A.N. Annu. Rev. Physiol. 1997; 59: 171-191Crossref PubMed Scopus (657) Google Scholar). The gating of Kir channels by protons may allow cells to produce corresponding responses to a change in intra- and extracellular pH seen under a number of pathophysiological conditions. Several members in the Kir family are regulated by intra- and extracellular protons (3Tsai T.D. Shuck M.E. Thompson D.P. Bienkowski M.J. Lee K.S. Am. J. Physiol. 1995; 268: C1173-C1178Crossref PubMed Google Scholar, 4Coulter K.L. Perier F. Radeke C.M. Vandenberg C.A. Neuron. 1995; 15: 1157-1168Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 5Fakler B. Schultz J.H. Yang J. Schulte U. Brandle U. Zenner H.P. Jan L.Y. Ruppersberg J.P. EMBO J. 1996; 15: 4093-4099Crossref PubMed Scopus (162) Google Scholar, 6Choe H. Zhou H. Palmer L.G. Sackin H. Am. J. Physiol. 1997; 273: F516-F529PubMed Google Scholar, 7Shuck M.E. Piser T.M. Bock J.H. Slightom J.L. Lee K.S. Bienkowski M.J. J. Biol. Chem. 1997; 272: 586-593Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 8Zhu G.Y. Chanchevalap S. Cui N.R. Jiang C. J. Physiol. (Lond. ). 1999; 516: 699-710Crossref Scopus (69) Google Scholar). Channels activity is completely shut off by acidification, whereas full channel openings are achieved with an increase in the pH level. In Kir1.1, Lys80 is a critical player in channel gating by intracellular protons, although several other residues are also involved (5Fakler B. Schultz J.H. Yang J. Schulte U. Brandle U. Zenner H.P. Jan L.Y. Ruppersberg J.P. EMBO J. 1996; 15: 4093-4099Crossref PubMed Scopus (162) Google Scholar, 6Choe H. Zhou H. Palmer L.G. Sackin H. Am. J. Physiol. 1997; 273: F516-F529PubMed Google Scholar, 9Schulte U. Hahn H. Konrad M. Jeck N. Derst C. Wild K. Weidemann S. Ruppersberg J.P. Fakler B. Ludwig J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15298-15303Crossref PubMed Scopus (132) Google Scholar, 10Chanchevalap, S., Yang, Z. J., Cui, N. R., Qu, Z., Zhu, G. Y., Liu, C. X., Giwa, L. R., Abdulkadir, L., and Jiang, C. J. Biol. Chem. 275, 7811–7817.Google Scholar). Mutation of this lysine residue totally abolishes the pH sensitivity (5Fakler B. Schultz J.H. Yang J. Schulte U. Brandle U. Zenner H.P. Jan L.Y. Ruppersberg J.P. EMBO J. 1996; 15: 4093-4099Crossref PubMed Scopus (162) Google Scholar). The Lys80 may become protonated at acidic pH by its interactions with other protein domains, leading to a closure of the channel (9Schulte U. Hahn H. Konrad M. Jeck N. Derst C. Wild K. Weidemann S. Ruppersberg J.P. Fakler B. Ludwig J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15298-15303Crossref PubMed Scopus (132) Google Scholar). In Kir2.3 (HIR), we have previously identified a short motif in the N terminus that is crucial in the gating of this channel by intracellular protons (11Qu Z.Q. Zhu G.Y. Yang J. Cui N.R. Li Y. Chanchevalap S. Sulaiman S. Haynie H. Jiang C. J. Biol. Chem. 1999; 274: 13783-13789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Unlike Kir1.1, there is no proton-binding site within or nearby to this motif in HIR. Thus, it is possible that protons interact with other intermediate sites that subsequently act on this short motif and close the channel. Recent studies have shown that a large number of residues in the channel proteins are involved in the gating process by pH (5Fakler B. Schultz J.H. Yang J. Schulte U. Brandle U. Zenner H.P. Jan L.Y. Ruppersberg J.P. EMBO J. 1996; 15: 4093-4099Crossref PubMed Scopus (162) Google Scholar, 6Choe H. Zhou H. Palmer L.G. Sackin H. Am. J. Physiol. 1997; 273: F516-F529PubMed Google Scholar, 9Schulte U. Hahn H. Konrad M. Jeck N. Derst C. Wild K. Weidemann S. Ruppersberg J.P. Fakler B. Ludwig J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15298-15303Crossref PubMed Scopus (132) Google Scholar,10Chanchevalap, S., Yang, Z. J., Cui, N. R., Qu, Z., Zhu, G. Y., Liu, C. X., Giwa, L. R., Abdulkadir, L., and Jiang, C. J. Biol. Chem. 275, 7811–7817.Google Scholar). However, it is unknown how these multiple sites are harmonized in opening or closing a channel. A potential mechanism is that proton binding initiates a cascade of events involving consequential interactions of the proton-binding sites with other protein domains, leading to a rearrangement of protein conformation and a switch of channel activity. To test this hypothesis, we studied the gating of Kir by intracellular protons (8Zhu G.Y. Chanchevalap S. Cui N.R. Jiang C. J. Physiol. (Lond. ). 1999; 516: 699-710Crossref Scopus (69) Google Scholar, 11Qu Z.Q. Zhu G.Y. Yang J. Cui N.R. Li Y. Chanchevalap S. Sulaiman S. Haynie H. Jiang C. J. Biol. Chem. 1999; 274: 13783-13789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 12Zhu G Chanchevalap S Liu C Xu H. Jiang C. J. Cell. Physiol. 2000; 183: 53-64Crossref PubMed Scopus (38) Google Scholar). We chose to use HIR because there is no titratable residue in the identified N-terminal motif. Thus, the gating site seems to be separate from the proton-sensing sites in this channel. Our results indicate that interactions of different parts of channel proteins occur during the gating process, demonstrating a novel channel-gating mechanism of K+ channels by intracellular protons. Kir2.3 (HIR) and Kir2.1 (IRK1) cDNAs were generously provided by Carol A. Vandenberg and Lily L. Jan, respectively. These cDNAs were inserted into pcDNA3.1, a eukaryotic expression vector that allows expression of its insert gene in eukaryotic cells without injecting it into the nucleus (Invitrogen, Carlsbad, NM). Chimerical constructs were prepared by the overlap extension at the junction of the interested domains using polymerase chain reaction (Pfu DNA polymerase, Stratagene, La Jolla, CA). Junction sites of all chimeras were chosen where there were conserved amino acid residues. Thus, none of the chimeras had missing or additional residues at the junction sites. Site-specific mutations were made using a site-directed mutagenesis kit (Stratagene). Orientation of the constructs and correct mutations were confirmed with DNA sequencing. Oocytes were surgically removed from adult frog (Xenopus laevis) and treated with 2 mg/ml collagenase (Type I, Sigma) in the OR2 solution (82 mm NaCl, 2 mm KCl, 1 mm MgCl2, and HEPES, pH 7.4) for 90 min at room temperature (∼25 °C). After three washes with OR2 solution, cDNA in the pcDNA3.1 vector (40–50 ng in 50 nl of double-distilled water) was injected into the oocytes. The oocytes were then incubated at 18 °C in ND-96 solution containing (in mm) NaCl 96, KCl 2, MgCl2 1, CaCl21.8, HEPES 5, and sodium pyruvate 2.5, plus 100 mg/liter geneticin (pH 7.4). Xenopus oocytes were placed in a semi-closed recording chamber (Medical System, Greenvale, NY), where perfusion solution bathed both the top and bottom surfaces of the oocytes. The perfusate and the superfused gas entered the chamber from the inlet at one end and flowed out at the other end. There was a 3 × 15-mm gap on the top cover of the chamber, which served as the gas outlet and as access to the oocytes for recording microelectrodes. The perfusate (KD 90) contained 90 mm KCl, 3 mm MgCl2, and 5 mm HEPES (pH 7.4). At baseline, the chamber was ventilated with atmospheric air. Exposure of the oocytes to CO2 was carried out by switching the superfused air to a gas mixture containing CO2 (15%) balanced with 21% O2 and N2. The high dissolvability of CO2 resulted in a detectable change in intra- or extracellular acidification in as fast as 10 s in these oocytes (11Qu Z.Q. Zhu G.Y. Yang J. Cui N.R. Li Y. Chanchevalap S. Sulaiman S. Haynie H. Jiang C. J. Biol. Chem. 1999; 274: 13783-13789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Whole-cell currents were studied on the oocytes 2–4 days after injection. Two-electrode voltage clamp was performed using an amplifier (Geneclamp 500, Axon Instruments Inc., Foster City, CA) at room temperature (∼25 °C). The extracellular solution contained 90 mm KCl, 3 mmMgCl2, and 5 mm HEPES (pH 7.4). Cells were impaled using electrodes filled with 3 m KCl. One of the electrodes (1.0–2.0 megaohms) served as voltage and the other electrode (0.3–0.6 megaohms) was used for current recording. Current records were low-pass-filtered (Bessel, 4-pole filter, 3 dB at 5 kHz), digitized at 5 kHz (12-bit resolution), and stored on computer disc for later analysis (pClamp 6, Axon Instruments). Patch clamp experiments were performed at room temperature as described (8Zhu G.Y. Chanchevalap S. Cui N.R. Jiang C. J. Physiol. (Lond. ). 1999; 516: 699-710Crossref Scopus (69) Google Scholar, 13Yang Z. Jiang C. J. Physiol. (Lond. ). 1999; 520: 921-927Crossref Scopus (48) Google Scholar). In brief, fire-polished patch pipettes (0.5–2.0 megaohms) were made from 1.2-mm borosilicate capillary glass. The oocyte vitelline membranes were mechanically removed after exposing hypertonic solution (400 mosm) for 5 min. The stripped oocytes were placed in FVPP solution (in mm: 40 KCl, 75 potassium gluconate, 5 potassium fluoride, 0.1 sodium vanadate, 10 potassium pyrophosphate, 1 EGTA, 0.2 adenosine diphosphate, 10 PIPES, pH 7.4, 10 glucose, and 0.1 spermine) for giant inside-out patch preparation. The same solution was applied to the pipette. Macroscopic current recordings were performed using FVPP solutions containing equal concentrations of K+applied to both sides of the patch membranes. In a control experiment, we found that macroscopic currents recorded from giant inside-out patches showed less than 10% reduction over a 17-min period of recordings in FVPP solution. Current records were low-pass filtered (2000 Hz, Bessel 4-pole filter, −3 dB), digitized (10 kHz, 12-bit resolution) and stored on computer disc for later analysis (PCLAMP 6, Axon Instruments). Junction potentials between bath and pipette solutions were appropriately nulled before seal formation. A parallel perfusion system was used to administer agents to patches at a rate of ∼1 ml/min with no dead space (8Zhu G.Y. Chanchevalap S. Cui N.R. Jiang C. J. Physiol. (Lond. ). 1999; 516: 699-710Crossref Scopus (69) Google Scholar, 13Yang Z. Jiang C. J. Physiol. (Lond. ). 1999; 520: 921-927Crossref Scopus (48) Google Scholar). Low pH exposures were carried out using FVPP solutions that had been titrated to various pH levels as required. DNA fragments encoding the sequences of N- or C-terminal regions of HIR (HIRn, AA 1–50; HIRc, AA 171–445) were synthesized using polymerase chain reaction. The N-terminal region was fused to glutathionineS-transferase (GST) in pGEX-4T-3 (Amersham Pharmacia Biotech) by insertion of a polymerase chain reaction fragment of the HIRn at BamHI and XhoI restriction sites created with primers. Immediately following the BamHI site was the first methionine residue of the HIRn, so that there was no additional residue introduced. At the C-terminal end of the GST-HIRn, however, there were seven additional residues (LERPHRD) coming along with the vector before the stop codon in the plasmid. The HIRn was expressed inEscherichia coli BL21 cells (Amersham Pharmacia Biotech) by transformation using 0.25 mmisopropyl-β-d-thiogalactoside (IPTG, Amersham Pharmacia Biotech) at 25–30 °C for 3–5 h. Bacteria were lysed with a French pressure cell in lysis buffer (50 mm Tris-HCl, pH 7.4, or PIPES, pH 6.6, 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1 mm dithiothreitol, 0.1 mm EDTA) containing protease inhibitors. Soluble proteins were isolated by centrifugation at 17,000 × g. The fusion proteins were purified with glutathionine-Sepharose beads (Amersham Pharmacia Biotech). The FLAG epitope was fused to HIRc using polymerase chain reaction. At the N-terminal end, a methonine residue was added with the Kozak sequence, followed by Ile171 of HIR. The FLAG sequence was introduced to the C-terminal end by removing the stop codon. Thus, there was no other residue missing in the HIRc peptide. The construct was then cloned into pcDNA3.1 and expressed in the human embryonic kidney cell line (HEK-293, ATCC, Manassas, VA). 48 h after transfection, proteins were extracted with lysis buffer (pH 7.4 or 6.6). Glutathionine-Sepharose beads that had been pre-linked to GST fusion proteins were mixed with HEK cell extracts in lysis buffer (pH 7.4 or 6.6) rotated at 4 °C for 2 h. Similar amounts of GST fusion proteins were used for each assay. Beads were centrifuged at 2000 × g for 2 min and washed three times with 0.7 ml of lysis buffer (pH 7.5 or 6.6). After a final wash, beads were heated in sample buffers for SDS-polyacrylamide gel electrophoresis, and bound proteins were separated with 15% SDS-polyacrylamide gel (Bio-Rad). After transferring the protein to a polyvinylidene difluoride membrane, binding to GST fusion proteins were detected by an anti-FLAG antibody (Sigma) followed by a secondary antibody conjugated with alkaline phosphatase (Bio-Rad). Color development was completed with an alkaline phosphatase conjugate substrate kit (Bio-Rad). Interaction assays were repeated two to four times to determine their reproducibility. Mutation analyses were performed on HIR and IRK1. Chimerical recombinations of intracellular N and C termini were presented using three letters, with the first and last representing the originations of N and C termini and the middle letter for the source of the rest sequence, e.g. HIH, C and N termini of the mutant were from HIR and the rest from IRK1; HII, the N terminus was from HIR and rest from IRK1. Chimerical mutants within the C or N terminus were named with two pieces of information: 1) the channel in which mutations were constructed, such XXXHIR, XXXIRK, and HIHXXX (mutants were made based on HIR, IRK1, and HIH, respectively); 2) the location of the mutations, such as N1–50HIH, amino acids from 1 to 50 in HIH were mutated to a corresponding sequence in IRK1; IRK179–238C, residues from 179 to 238 were mutated to those in HIR. Site-specific mutations were described using single-letter amino acids symbols, e.g. H191A, histidine at position 191 was mutated to alanine. Data are presented as means ± S.E. (n ≥ 4). Differences in means were examined with the Student's ttest or an analysis-of-variance test and were accepted as significant if p ≤ 0.05. Whole-cell currents were studied inXenopus oocytes that had received an injection of HIR cDNA or one of its mutants. In the two-electrode voltage clamp mode, inward rectifying currents as large as 20 μA were seen in most oocytes. These currents were sensitive to micromolar concentrations of Ba2+ and Cs+ (8Zhu G.Y. Chanchevalap S. Cui N.R. Jiang C. J. Physiol. (Lond. ). 1999; 516: 699-710Crossref Scopus (69) Google Scholar, 11Qu Z.Q. Zhu G.Y. Yang J. Cui N.R. Li Y. Chanchevalap S. Sulaiman S. Haynie H. Jiang C. J. Biol. Chem. 1999; 274: 13783-13789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Exposure of the oocytes to 15% CO2 for 4–5 min produced a fast and reversible inhibition of these inward rectifying currents (Fig.1 A). The inhibition of HIR currents by CO2 was mediated by decreases in pH, since selectively lowering intra- and extracellular pH (pHi, pHo) to the corresponding levels (pHi 6.6, pHo6.2) seen during 15% CO2 exposure inhibited the HIR currents to the same degree as hypercapnia (8Zhu G.Y. Chanchevalap S. Cui N.R. Jiang C. J. Physiol. (Lond. ). 1999; 516: 699-710Crossref Scopus (69) Google Scholar, 11Qu Z.Q. Zhu G.Y. Yang J. Cui N.R. Li Y. Chanchevalap S. Sulaiman S. Haynie H. Jiang C. J. Biol. Chem. 1999; 274: 13783-13789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Macroscopic currents recorded in excised patches were similarly inhibited (Fig.1 B). The consistent pH sensitivity seen in whole-cell recordings and excised patches suggests that proton-mediated channel inhibition is independent of other cytosolic factors. The IRK1 channel, however, did not respond to 15% CO2 and pHi6.0–7.4 (Fig. 2).Figure 2Dependence of the CO2/pH sensitivity on certain parts of the HIR channel protein. A, because HIR responds to hypercapnia (15% CO2) but Kir2.1 (IRK1) does not, chimeras were constructed between these two channels to identify the intracellular pH-sensing domain. Whole-cell currents were studied in two-electrode clamp. Their sensitivity to 15% CO2 was examined as described for Fig. 1 A and are presented as percentage inhibition of the currents. The HIH carrying both the N (AA 1–50) and C termini (AA 171–445) from HIR and the rest of its structures from IRK1 (AA 87–178) showed CO2 sensitivity almost identical to the wild-type HIR. In IHH, the entire HIR N-terminal region (AA 1–50) was substituted with its counterpart in IRK1 (AA 1–86). The IHH lost sensitivity to CO2. When IRK1 N-terminal domain (AA 1–86) was replaced with the corresponding sequence in HIR (AA 1–50), the mutant channel (HII) became CO2-sensitive. Extension of the mutation to include the N terminus through the M2 region of HIR (HHI; AA 171–445 in HIR were replaced with AA 179–428 in IRK1) did not significantly change the CO2sensitivity. B, concentration-dependent inhibition of K+ currents by acidic pH in inside-out patches. Currents were studied in inside-out patches under conditions described in Fig. 1 B. The current amplitude can be expressed as a function of pHi using the Hill equation:y = 1/[1 + (pK a/x) h ], where pK a (apparent pK) is the midpoint pH value for channel inhibition, and h is the Hill coefficient. Although HIR currents showed strong pHi sensitivity with pK a 6.76 (h = 3.1), IRK1 and IHH had almost no response to a pHi change from 7.4 to 5.8 (pK a 4.95, h 0.9, and pK a5.25, h 0.9, respectively). Although its pHi sensitivity decreased (pK a 6.45,h 2.2), HII responded to pHi changes more like HIR than IRK1. With both N and C termini from HIR and the rest of the sequences from IRK1, HIH had almost identical pH sensitivity (pK a6.77, h 3.1) to HIR. Data are presented as means ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The effect of CO2 and pH on HIR currents depended on certain portions of the channel protein. A substitution of the N terminus of HIR (HIRn) with that of IRK1 (IHH, see “Materials and Methods” for nomenclature) completely abolished the CO2/ pHi sensitivities (Fig. 2), whereas a replacement of the N terminus of IRK1 (IRKn) with HIRn rendered the mutant channel (HII) substantial CO2/ pHi sensitivities, indicating that the pH-dependent gating mechanism is related to the N terminus. Although the recombinant HIR-IRK channels with the N terminus from HIR remained pH-sensitive, the apparent pK (pH value for 50% channel inhibition, pK a) value of the HII was ∼0.3 pH units lower than that of the wild-type HIR (Fig. 2 B). Thus, it is possible that other parts of the HIR protein are also involved in the pH sensing. To test this possibility, we extended the recombinant channel to include the N terminus as well as the M1, H5, and M2 regions of HIR (HHI). When this construct was expressed, we did not see any significant increase in CO2 sensitivity over the HII (Fig.2 A). A chimera (HIH) was then constructed which contained both HIRn and the C terminus of HIR (HIRc) and other sequences (M1 through M2) from IRK1. This mutant expressed inward rectifying currents with the amplitude and rectification comparable with those of HIR. When the HIH was exposed to 15% CO2 or acidic pH, we found that this channel was inhibited to exactly the same degree as the wild-type HIR (Fig. 2). The function of the HIRc in pH sensing was not simply contributive, because introducing the HIRc alone to the IRK1 (IIH, IHH) failed to produce any CO2/ pHi sensitivity (Fig.2). Thus, the HIRc is also required for pH sensing, although its role strictly depends on the presence of HIRn. The requirement of both the N and C termini for pHi sensing suggests that the channel gating may result from interactions of both the C and N termini. If the N and C termini can interact in pH sensing, there should be amino acid sequences critical for the interaction. To locate the sequences, several N-terminal mutants were constructed based on HIH and IRK1. We chose to use HIH rather than HIR, because the extracellular pH sensor was removed in this mutant (4Coulter K.L. Perier F. Radeke C.M. Vandenberg C.A. Neuron. 1995; 15: 1157-1168Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 11Qu Z.Q. Zhu G.Y. Yang J. Cui N.R. Li Y. Chanchevalap S. Sulaiman S. Haynie H. Jiang C. J. Biol. Chem. 1999; 274: 13783-13789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) without affecting baseline currents or the pHi sensitivity, as mentioned above. HIRn and IRKn were divided into three segments at two conserved areas. Each chimera carried one or two segments from HIR and the rest from IRK1. The pHi sensitivity was then studied (Fig.3). If an area is not involved in interactions with the HIRc, mutation of this sequence should not affect the distinct pH sensitivity of channels carrying HIRc or IRKc as shown in Fig. 2 B (HIH versus HII); if it is involved, on the other hand, substitution of this sequence with that in IRK1 should produce a channel with identical pH sensitivity regardless of the presence of HIRc or IRKc. When a short sequence near the M1 domain was mutated to that in IRK1, the pH sensitivity of this mutant channel could no longer be enhanced by HIRc with a marked reduction in pH sensitivity (N51–60HIH versus N1–76IRK, Fig.3 A). This short motif contained about 10 amino acids with only three residues (TYM) different from those in IRK1. In contrast, mutations of other N-terminal domains did not eliminate the effect of HIRc in augmenting pH sensitivity although decreases in pH sensitivity were also observed (Fig. 3, B–D). These results suggest that the TYM motif that we described previously (11Qu Z.Q. Zhu G.Y. Yang J. Cui N.R. Li Y. Chanchevalap S. Sulaiman S. Haynie H. Jiang C. J. Biol. Chem. 1999; 274: 13783-13789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) is indeed the N-terminal interaction site. The C terminus of HIR has 275 amino acids with a number of feature structures. At its C-terminal end, there is a potential PDZ-binding sequence. Immediately before this sequence, there are a cluster of negatively charged residues and a proline-rich motif. To determine whether these structures are involved in CO2 and pH sensing, several deletions of the C-terminal sequences were created by introducing a stop codon at positions 361, 381, or 393 (Fig.4 A). However, the CO2 sensitivity of these C-truncated HIR channels did not show any significant difference from the wild-type HIR (Fig.4 B), indicating that these structures are not necessary for the CO2 sensing. Several chimeras were constructed using C-terminal segments of HIR and IRK1, shown in Fig. 4 A. CO2 and pH sensitivities of these chimeras were then compared with those of HIH and HII. The substitution of a sequence from position 231 or 261 to the end of the C terminus (HIH231–445C, HIH261–445C) had no effect on the CO2 and pHi sensitivities of the mutant channels, which remained identical to HIH (Fig. 4, B andC). A chimera with sequence 196–445 replaced by that in IRK1 showed CO2 and pHi sensitivities similar to the HII, suggesting that the region between 196 and 230 is critical in CO2/ pHi sensing (Fig. 4, B andC). In region 196–230, there are only six residues that differ between HIR and IRK1, with four of them (PYMQ) clearly in contrast with their counterparts. Mutations of all four of these residues to those in IRK1 (HIH-SRIS) completely eliminated the C-terminal effect on enhancing pH sensitivity (Fig. 4, B andC). Introducing these four residues to HII significantly increased the CO2/ pHi sensitivity of the mutant channel over the HII (HII-PYMQ, Fig. 4, B and C). These results suggest, therefore, that the C-terminal domain for the N/C-terminal interaction is located at the PYMQ motif. Simultaneous mutations of both the N-terminal TYM and C-terminal PYMQ sequences (TYM-PYMQ) rendered the mutant channel the pHisensitivity identical to that of N51–60HIH (Fig. 4 C), suggesting the mutual dependence of these two motifs. Although the TYM and PYMQ motifs are critical in channel gating, there is no titratable residue within them. Apparently, the proton-binding sites are located in areas other than these two motifs. If protons mediate the N/C-terminal interaction, there should be proton-binding sites in the channel protein. Histidine has a pK value 6.04 in its side chain, which is close to the physiological pH level. This property makes the histidine residue the highly promising proton-binding site (10Chanchevalap, S., Yang, Z. J., Cui, N. R., Qu, Z., Zhu, G. Y., Liu, C. X., Giwa, L. R., Abdulkadir, L., and Jiang, C. J. Biol. 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The endometrium microvessel system, responsible for supplying oxygen and nutrients to the embryo, holds significant importance in evaluating endometrial receptivity (ER). Visualizing this system directly can significantly enhance ER evaluation. Currently, clinical methods like Narrow-band hysteroscopy and Color Doppler ultrasound are commonly used for uterine blood vessel examination, but they have limitations in depth or resolution. Endoscopic Photoacoustic Imaging (PAE) has proven effective in visualizing microvessels in the digestive tract, while its adaptation to uterine imaging faces challenges due to the uterus's unique physiological characteristics. This paper for the first time that uses high-resolution PAE in vivo to capture a comprehensive network of endometrial microvessels non-invasively. Followed by continuous observation and quantitative analysis in the endometrial injury model, we further corroborated that PAE detection of endometrial microvessels stands as a valuable indicator for evaluating ER. The PAE system showcases its promising potential for integration into reproductive health assessments.

Insect antennae facilitate the nuanced detection of vibrations and deflections, and the non-contact perception of magnetic or chemical stimuli, capabilities not found in mammalian skin. Here, we report a neuromorphic antennal sensory system that emulates the structural, functional, and neuronal characteristics of ant antennae. Our system comprises electronic antennae sensor with three-dimensional flexible structures that detects tactile and magnetic stimuli. The integration of artificial synaptic devices adsorbed with solution-processable MoS2 nanoflakes enables synaptic processing of sensory information. By emulating the architecture of receptor-neuron pathway, our system realizes hardware-level, spatiotemporal perception of tactile contact, surface pattern, and magnetic field (detection limits: 1.3 mN, 50 μm, 9.4 mT). Vibrotactile-perception tasks involving profile and texture classifications were accomplished with high accuracy (> 90%), surpassing human performance in "blind" tactile explorations. Magneto-perception tasks including magnetic navigation and touchless interaction were successfully completed. Our work represents a milestone for neuromorphic sensory systems and biomimetic perceptual intelligence.

The endomembrane system consists of organellar membranes in the biosynthetic pathway: endoplasmic reticulum (ER), Golgi apparatus, and secretory vesicles, as well as those in the degradative pathway: early endosomes, macropinosomes, phagosomes, autophagosomes, late endosomes, and lysosomes. These endomembrane organelles/vesicles work together to synthesize, modify, package, transport, and degrade proteins, carbohydrates, and lipids, regulating the balance between cellular anabolism and catabolism. Large ion concentration gradients exist across endomembranes - Ca2+ gradients for most endomembrane organelles and H+ gradients for the acidic compartments. Ion (Na+, K+, H+, Ca2+, and Cl-) channels on the organellar membranes control ion flux in response to cellular cues, allowing rapid informational exchange between the cytosol and organelle lumen. Recent advances in organelle proteomics, organellar electrophysiology, luminal and juxta-organellar ion imaging have led to molecular identification and functional characterization of about two dozen endomembrane ion channels. For example, whereas IP3R1-3 channels mediate Ca2+ release from the ER in response to neurotransmitter and hormone stimulation, TRPML1-3 and TMEM175 channels mediate lysosomal Ca2+ and H+ release, respectively, in response to nutritional and trafficking cues. This review aims to summarize the current understandings of these endomembrane channels, with a focus on their subcellular localizations, ion permeation properties, gating mechanisms, cell biological functions, and disease relevance.

Photoacoustic molecular imaging technology has a wide range of applications in biomedical research. In practical scenarios, both the probes and blood generate signals, resulting in the saliency of the probes in the blood environment being diminished, impacting imaging quality. Although several methods have been proposed for saliency enhancement, they inevitably suffer from moderate generality and detection speed. The Grüneisen relaxation (GR) nonlinear effect offers an alternative for enhancing saliency and can improve generality and speed. In this article, the excitation and detection efficiencies are optimized to enhance the GR signal amplitude. Experimental studies show that the saliency of the probe is enhanced. Moreover, the issue of signal aliasing is studied to ensure the accuracy of enhancement results in the tissues. In a word, the feasibility of the GR-based imaging method in saliency enhancement is successfully demonstrated in the study, showing the superiorities of good generality and detection speed.

Abstract The superior recognition ability and excitatory–inhibitory balance of the olfactory system has important applications in the efficient recognition, analysis, and processing of data. In this study, transistor synaptic devices are prepared utilizing poly‐diketo‐pyrrolopyrrole‐selenophene polymer (PTDPPSe‐5Si) with excellent electrical properties as the active layer, and dual‐gas pulses are applied for the first time to simulate excitatory and inhibitory behaviors in the olfactory system. Basic synaptic properties are successfully simulated, such as excitatory/inhibitory postsynaptic currents (EPSC/IPSC), and long‐term potentiation/depression (LTP/LTD). The regulation of excitatory–inhibitory balance in biomimetic olfaction is successfully simulated. This working mechanism is attributed to the capture and release of carriers in the channel induced by the gas's electron‐donating and electron‐withdrawing characteristics. The neuromotor pathway is constructed using synaptic devices as the key processing unit, which enables the integration of information from neurons and the output of information from motor neurons. A convolutional neural network is constructed to achieve recognition of eight common laboratory gas types and concentrations with a recognition accuracy of over 97%. The simulated excitatory and inhibitory behaviors exhibited by this device hold significant importance for the development of artificial neural networks, intelligent frameworks, and neural robots.

Central chemoreceptors (CCRs) play a crucial role in autonomic respiration. Although a variety of brainstem neurons are CO(2) sensitive, it remains to know which of them are the CCRs. In this article, we discuss a potential alternative approach that may allow an access to the CCRs. This approach is based on identification of specific molecules that are CO(2) or pH sensitive, exist in brainstem neurons, and regulate cellular excitability. Their molecular identity may provide another measure in addition to the electrophysiologic criteria to indicate the CCRs. The inward rectifier K(+) channels (Kir) seem to be some of the CO(2) sensing molecules, as they regulate membrane potential and cell excitability and are pH sensitive. Among homomeric Kirs, we have found that even the most sensitive Kir1.1 and Kir2.3 have pK approximately 6.8, suggesting that they may not be capable of detecting hypocapnia. We have studied their biophysical properties, and identified a number of amino acid residues and molecular motifs critical for the CO(2) sensing. By comparing all Kirs using the motifs, we found the same amino acid sequence in Kir5.1, and demonstrated the pH sensitivity in heteromeric Kir4.1 and Kir5.1 channels to be pK approximately 7.4. In current clamp, we show evidence that the Kir4.1-Kir5.1 can detect P(CO(2)) changes in either hypercapnic or hypocapnic direction. Our in-situ hybridization studies have indicated that they are coexpressed in brainstem cardio-respiratory nuclei. Thus, it is likely that the heteromeric Kir4.1-Kir5.1 contributes to the CO(2)/pH sensitivity in these neurons. We believe that this line of research intended to identify CO(2) sensing molecules is an important addition to current studies on the CCRs.

The ATP‐sensitive K + (K ATP ) channels are regulated by intracellular H + in addition to ATP, ADP, and phospholipids. Here we show evidence for the interaction of H + with ATP in regulating a cloned K ATP channel, i.e. Kir6.2 expressed with and without the SUR1 subunit. Channel sensitivity to ATP decreases at acidic pH, while the pH sensitivity also drops in the presence of ATP. These effects are more evident in the presence of the SUR1 subunit. In the Kir6.2 + SUR1, the pH sensitivity is reduced by about 0.4 pH units with 100 μM ATP and 0.6 pH units with 1 m m ATP, while a decrease in pH from 7.4 to 6.8 lowers the ATP sensitivity by about fourfold. The Kir6.2 + SUR1 currents are strongly activated at pH 5.9‐6.5 even in the presence of 1 m m ATP. The modulations appear to take place at His175 and Lys185 that are involved in proton and ATP sensing, respectively. Mutation of His175 completely eliminates the pH effect on the ATP sensitivity. Similarly, the K185E mutant‐channel loses the ATP‐dependent modulation of the pH sensitivity. Thus, allosteric modulations of the cloned K ATP channel by ATP and H + are demonstrated. Such a regulation allows protons to activate directly the K ATP channels and release channel inhibition by intracellular ATP; the pH effect is further enhanced with a decrease in ATP concentration as seen in several pathophysiological conditions.

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina propria. EGCs play important roles in gut homeostasis through the release of various trophic factors and contribute to the integrity of the epithelial barrier. Most studies of primary enteric glial cultures use cells isolated from the myenteric plexus after enzymatic dissociation. Here, a non-enzymatic method to isolate and culture EGCs from the intestinal submucosa and lamina propria is described. After manual removal of the longitudinal muscle layer, EGCs were liberated from the lamina propria and submucosa using sequential HEPES-buffered EDTA incubations followed by incubation in commercially available non-enzymatic cell recovery solution. The EDTA incubations were sufficient to strip most of the epithelial mucosa from the lamina propria, allowing the cell recovery solution to liberate the submucosal EGCs. Any residual lamina propria and smooth muscle was discarded along with the myenteric glia. EGCs were easily identified by their ability to express glial fibrillary acidic protein (GFAP). Only about 50% of the cell suspension contained GFAP+ cells after completing tissue incubations and prior to plating on the poly-D-lysine/laminin substrate. However, after 3 days of culturing the cells in glial cell-derived neurotrophic factor (GDNF)-containing culture media, the cell population adhering to the substrate-coated plates comprised of >95% enteric glia. We created a hybrid mouse line by breeding a hGFAP-Cre mouse to the ROSA-tdTomato reporter line to track the percentage of GFAP+ cells using endogenous cell fluorescence. Thus, non-myenteric enteric glia can be isolated by non-enzymatic methods and cultured for at least 5 days.

ATP-sensitive K+ channels (KATP) are regulated by pH in addition to ATP, ADP, and phospholipids. In the study we found evidence for the molecular basis of gating the cloned KATP by intracellular protons. Systematic constructions of chimerical Kir6.2-Kir1.1 channels indicated that full pH sensitivity required the N terminus, C terminus, and M2 region. Three amino acid residues were identified in these protein domains, which are Thr-71 in the N terminus, Cys-166 in the M2 region, and His-175 in the C terminus. Mutation of any of them to their counterpart residues in Kir1.1 was sufficient to completely eliminate the pH sensitivity. Creation of these residues rendered the mutant channels clear pH-dependent activation. Thus, critical players in gating KATP by protons are demonstrated. The pH sensitivity enables the KATP to regulate cell excitability in a number of physiological and pathophysiological conditions when pH is low but ATP concentration is normal. ATP-sensitive K+ channels (KATP) are regulated by pH in addition to ATP, ADP, and phospholipids. In the study we found evidence for the molecular basis of gating the cloned KATP by intracellular protons. Systematic constructions of chimerical Kir6.2-Kir1.1 channels indicated that full pH sensitivity required the N terminus, C terminus, and M2 region. Three amino acid residues were identified in these protein domains, which are Thr-71 in the N terminus, Cys-166 in the M2 region, and His-175 in the C terminus. Mutation of any of them to their counterpart residues in Kir1.1 was sufficient to completely eliminate the pH sensitivity. Creation of these residues rendered the mutant channels clear pH-dependent activation. Thus, critical players in gating KATP by protons are demonstrated. The pH sensitivity enables the KATP to regulate cell excitability in a number of physiological and pathophysiological conditions when pH is low but ATP concentration is normal. ATP-sensitive K+ channels Kir6.2ΔC36 wild type sulfonylurea receptor ATP-sensitive K+ channel (KATP)1is a unique member in the K+ channel family, which directly couples the intermediary metabolism to cellular excitability (1Ashcroft F.M. Gribble F.M. Trends Neurosci. 1998; 21: 288-294Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 2Quayle J.M. Nelson M.T. Standen N.B. Physiol. Rev. 1997; 77: 1165-1232Crossref PubMed Scopus (718) Google Scholar). Such a property enables the KATP to play an important role in regulating vascular tone, skeletal muscle contractility, insulin secretion, epithelial transport, and neuronal excitability under a variety of physiological and pathophysiological conditions (3Dost R. Rundfeldt C. Epilepsy Res. 2000; 38: 53-66Crossref PubMed Scopus (55) Google Scholar, 4Gramolini A. Renaud J.M. Am. J. Physiol. 1997; 272: C1936-C1946Crossref PubMed Google Scholar, 5Light P.E. Comtois A.S. Renaud J.M. J. Physiol. (Lond.). 1994; 475: 495-507Crossref Scopus (54) Google Scholar, 6Wang W. Hebert S.C. Giebisch G. Annu. Rev. Physiol. 1997; 59: 413-436Crossref PubMed Scopus (176) Google Scholar, 7Wei E.P. Kontos H.A. Beckman J.S. Stroke. 1998; 29: 817-822Crossref PubMed Scopus (31) Google Scholar). Although ATP is the primary regulator of theKATP, several other cytosolic factors are also involved in the control of channel activity, including ADP, phospholipids, and hydrogen ion (8–20). Our recent studies have shown that the cloned KATP also responds to acidic pH (21Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). These channels are strongly stimulated by hypercapnia and intracellular acidosis. The pH sensitivity is independent of the SUR subunit and other cytosolic factors, suggesting that the pH sensing mechanisms are located in the Kir (inward rectifier K+ channel) subunit (21Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). If the pH sensitivity is an inherent property of Kir6 proteins, there should be special structures responsible for channel gating by protons. These structures are likely to be located in the Kir6 subunit, since the SUR subunit is not required for the pH sensitivity. To identify these structures, we performed these experiments in which we used the Kir6.2 with a truncation of 36 amino acids at the C-terminal end,i.e. Kir6.2ΔC36 that expresses functional channels without the SUR subunit and retains fair ATP sensitivity (22Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (680) Google Scholar). Several chimerical channels were generated based on peptide sequences of Kir6.2 and Kir1.1, a Kir channel that is inhibited by intracellular acidosis (23Chanchevalap S. Yang Z. Cui N. Qu Z. Zhu G. Liu C. Giwa L.R. Abdulkadir L. Jiang C. J. Biol. Chem. 2000; 275: 7811-7817Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 24Fakler B. Schultz J.H. Yang J. Schulte U. Brandle U. Zenner H.P. Jan L.Y. Ruppersberg J.P. EMBO J. 1996; 15: 4093-4099Crossref PubMed Scopus (163) Google Scholar, 25Tsai T.D. Shuck M.E. Thompson D.P. Bienkowski M.J. Lee K.S. Am. J. Physiol. 1995; 268: C1173-C1178Crossref PubMed Google Scholar) and has been shown to express functional channels in its chimeras with Kir6.2 (26Drain P. Li L. Wang J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13953-13958Crossref PubMed Scopus (174) Google Scholar). These chimeras were studied in whole-cell recording using hypercapnia, a condition that does not cause channel rundown (21Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Our results indicate that there are three separate protein domains in the Kir6.2 protein that are crucial for the pH sensitivity. We have identified critical amino acid residues within each of these protein domains. Replacement of any of them with their counterpart residues in Kir1.1 interrupts the channel sensitivity to CO2/pH. Frog oocytes were obtained from Xenopus laevis. The frogs were anesthetized by bathing them in 0.3% 3-aminobenzoic acid ethyl ester. A few lobes of ovaries were removed after a small abdominal incision (∼5 mm). Then the surgical incision was closed, and the frogs were allowed to recover from the anesthesia.Xenopus oocytes were treated with 2 mg/ml collagenase (Type I, Sigma) in OR2 solution (NaCl 82 mm, KCl 2 mm, MgCl2 1 mm, and HEPES 5 mm, pH 7.4) for 90 min at room temperature. After 3 washes (10 min each) of the oocytes with the OR2 solution, cDNAs (25–50 ng in 50 nl of water) were injected into the oocytes. The oocytes were then incubated at 18 °C in the ND-96 solution containing 96 mm NaCl, 2 mm KCl, 1 mmMgCl2, 1.8 mm CaCl2, 5 mm HEPES, and 2.5 mm sodium pyruvate with 100 mg/liter Geneticin added, pH 7.4. Rat Kir1.1 (ROMK1, GenBankTM accession number X72341) and mouse Kir6.2 (mBIR, GenBankTM accession number D50581) cDNAs were generously provided by Dr. S. Hebert at Yale University and Dr. S. Seino at Chiba University in Japan, respectively. The cDNAs were subcloned to a eukaryotic expression vector (pcDNA3.1, Invitrogen Inc., Carlsbad, CA) and used forXenopus oocyte expression without cRNA synthesis. Chimerical constructs were prepared by the overlap extension at the junction of the interested domains using the polymerase chain reaction (Pfu DNA polymerase, Stratagene, La Jolla, CA). Site-specific mutations were made using a site-directed mutagenesis kit (Stratagene). The orientation of the constructs and correct mutations were confirmed with DNA sequencing. Whole-cell currents were studied on the oocytes 2–4 days after injection. Two-electrode voltage clamp was performed using an amplifier (Geneclamp 500, Axon Instruments Inc., Foster City, CA) at room temperature (∼24 °C). The microelectrodes were filled with 3m KCl. One of the electrodes (1.0–2.0 megaohms) served for voltage measurements, and the other electrode (0.3–0.6 megaohms) was used for current recording. Current records were low-pass filtered (Bessel, 4-pole filter, 3 dB at 5 kHz), digitized at 5 kHz (12-bit resolution), and stored on computer disc for later analysis (pClamp 6, Axon Instruments) (21Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 27Xu H. Yang Z. Cui N. Giwa L.R. Abdulkadir L. Patel M. Sharma P. Shan G. Shen W. Jiang C. Am. J. Physiol. Cell Physiol. 2000; 279: C1464-C1471Crossref PubMed Google Scholar, 28Yang Z. Jiang C. J. Physiol. (Lond.). 1999; 520: 921-927Crossref Scopus (48) Google Scholar, 29Yang Z. Xu H. Cui N. Qu Z. Chanchevalap S. Shen W. Jiang C. J. Gen. Physiol. 2000; 116: 33-45Crossref PubMed Scopus (96) Google Scholar, 30Zhu G. Chanchevalap S. Cui N. Jiang C. J. Physiol. (Lond.). 1999; 516: 699-710Crossref Scopus (70) Google Scholar). The extracellular solution contained 90 mm KCl, 3 mm MgCl2, and 5 mm HEPES, pH 7.4. Under this condition, cells showed a membrane potential of ∼0 mV, so that we studied the currents with a holding potential of 0 mV. Expressions of functional Kir channels were confirmed using one or two of the following methods. 1) The amplitude of the inward rectifying currents was significantly larger than that recorded from the pcDNA3-injected oocytes, 2) the currents were strongly activated with the exposure to 3 mm azide, and 3) 100 μm Ba2+ inhibited the currents. When a mutant failed to express functional channels in ∼60 oocytes tested, another two injections of the same mutant from different colonies were followed in ∼60 cells each. If there was still a lack of expression, we believed that the mutation was too severe to produce any functional channels, and further experimentation was not attempted. Experiments were performed in a semi-closed recording chamber (BSC-HT, Medical System, Greenvale, NY) in which oocytes were placed on a supporting nylon mesh, and the perfusion solution bathed both the top and bottom surface of the oocytes. The perfusate and the superfusion gas entered the chamber from two inlets at one end and flowed out at the other end. There was a 3 × 15-mm gap on the top cover of the chamber that served as the gas outlet and an access to the oocytes for recording microelectrodes. At base line, the chamber was ventilated with atmospheric air. Exposure of the oocytes to CO2 was carried out by switching to a perfusate that had been bubbled for at least 30 min with a gas mixture containing CO2 at various concentrations balanced with 21% O2 and N2 and superfused with the same gas (21Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 27Xu H. Yang Z. Cui N. Giwa L.R. Abdulkadir L. Patel M. Sharma P. Shan G. Shen W. Jiang C. Am. J. Physiol. Cell Physiol. 2000; 279: C1464-C1471Crossref PubMed Google Scholar, 28Yang Z. Jiang C. J. Physiol. (Lond.). 1999; 520: 921-927Crossref Scopus (48) Google Scholar, 29Yang Z. Xu H. Cui N. Qu Z. Chanchevalap S. Shen W. Jiang C. J. Gen. Physiol. 2000; 116: 33-45Crossref PubMed Scopus (96) Google Scholar, 30Zhu G. Chanchevalap S. Cui N. Jiang C. J. Physiol. (Lond.). 1999; 516: 699-710Crossref Scopus (70) Google Scholar). The high solubility of CO2 resulted in a detectable change in intra- or extracellular acidification as fast as 10 s in these oocytes. The following nomenclatures were used in the present study. Kir6.2 and Kir1.1 were divided into three segments in their peptide chains,i.e. 1) the N terminus, 2) C terminus, and 3) all the rest M1 through M2 regions. A chimera with both N and C termini from Kir6.2 and the rest from Kir1.1 was named SOS. If only the N terminus was from Kir6.2, it was called SOO. If the SOS carried the M1 region from Kir6.2, it was referred to SOSm1. To facilitate the comparison of amino acid residues between the SO chimeras and the wt channels, they were numbered by their original locations in the wt Kir1.1 or Kir6.2 protein rather than by their new positions in the sequences of the chimeras. Data are presented as means ± S.E. Analysis of variance or Student's t test was used. Differences of CO2and pH effects before versus during exposures were considered to be statistically significant if p ≤ 0.05. The Kir6.2ΔC36 was expressed in Xenopus oocytes. As reported previously (21Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 22Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (680) Google Scholar), inward rectifying currents were recorded in the whole-cell configuration after Kir6.2ΔC36 cDNA injection. Exposure to CO2 produced reversible and concentration-dependent activation of the inward rectifying currents (Fig. 1A). The effect was mediated by pH rather than molecular CO2, as intracellular, but not extracellular, acidification to the same levels seen during CO2 exposures produced the same degrees of channel activation (22Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (680) Google Scholar). In excised inside-out patches, the Kir6.2ΔC36 currents were also stimulated by modest acidification on the cytosolic side of membranes in the absence of ATP and other cytosolic soluble factors. With strong acidification, however, the channel activation was followed by marked inhibition that appeared to be a result of channel rundown as reported previously (8Baukrowitz T. Schulte U. Oliver D. Herlitze S. Krauter T. Tucker S.J. Ruppersberg J.P. Fakler B. Science. 1998; 282: 1141-1144Crossref PubMed Scopus (441) Google Scholar, 11Davies N.W. Nature. 1990; 343: 375-377Crossref PubMed Scopus (189) Google Scholar, 13Koyano T. Kakei M. Nakashima H. Yoshinaga M. Matsuoka T. Tanaka H. J. Physiol. (Lond.). 1993; 463: 747-766Crossref Scopus (55) Google Scholar, 20Vivaudou M. Forestier C. J. Physiol. (Lond.). 1995; 486: 629-645Crossref Scopus (35) Google Scholar,31Fan Z. Tokuyama Y. Makielski J.C. Am. J. Physiol. 1994; 267: C1036-C1044Crossref PubMed Google Scholar). Similar effects were observed in the presence of 1 mmATP in the internal solution, 2J. Wu and C. Jiang, unpublished observations. indicating that the pH sensitivity is independent of ATP. Since the channel rundown occurs in excised patches, further studies were done using whole-cell recording and hypercapnic acidosis with 15% CO2, an experimental condition that we have well documented previously (21Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar,27Xu H. Yang Z. Cui N. Giwa L.R. Abdulkadir L. Patel M. Sharma P. Shan G. Shen W. Jiang C. Am. J. Physiol. Cell Physiol. 2000; 279: C1464-C1471Crossref PubMed Google Scholar, 28Yang Z. Jiang C. J. Physiol. (Lond.). 1999; 520: 921-927Crossref Scopus (48) Google Scholar, 29Yang Z. Xu H. Cui N. Qu Z. Chanchevalap S. Shen W. Jiang C. J. Gen. Physiol. 2000; 116: 33-45Crossref PubMed Scopus (96) Google Scholar, 30Zhu G. Chanchevalap S. Cui N. Jiang C. J. Physiol. (Lond.). 1999; 516: 699-710Crossref Scopus (70) Google Scholar). In contrast to the Kir6.2, Kir1.1 was inhibited during hypercapnia and intracellular acidification (Fig. 1B), as demonstrated previously (23Chanchevalap S. Yang Z. Cui N. Qu Z. Zhu G. Liu C. Giwa L.R. Abdulkadir L. Jiang C. J. Biol. Chem. 2000; 275: 7811-7817Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 24Fakler B. Schultz J.H. Yang J. Schulte U. Brandle U. Zenner H.P. Jan L.Y. Ruppersberg J.P. EMBO J. 1996; 15: 4093-4099Crossref PubMed Scopus (163) Google Scholar, 25Tsai T.D. Shuck M.E. Thompson D.P. Bienkowski M.J. Lee K.S. Am. J. Physiol. 1995; 268: C1173-C1178Crossref PubMed Google Scholar, 27Xu H. Yang Z. Cui N. Giwa L.R. Abdulkadir L. Patel M. Sharma P. Shan G. Shen W. Jiang C. Am. J. Physiol. Cell Physiol. 2000; 279: C1464-C1471Crossref PubMed Google Scholar, 32Doi T. Fakler B. Schultz J.H. Schulte U. Brandle U. Weidemann S. Zenner H.P. Lang F. Ruppersberg J.P. J. Biol. Chem. 1996; 271: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Our results suggest that the pH sensitivity in the Kir6.2 is an inherent property of the channel protein, similar to those described in several other Kir channels (23Chanchevalap S. Yang Z. Cui N. Qu Z. Zhu G. Liu C. Giwa L.R. Abdulkadir L. Jiang C. J. Biol. Chem. 2000; 275: 7811-7817Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 24Fakler B. Schultz J.H. Yang J. Schulte U. Brandle U. Zenner H.P. Jan L.Y. Ruppersberg J.P. EMBO J. 1996; 15: 4093-4099Crossref PubMed Scopus (163) Google Scholar, 25Tsai T.D. Shuck M.E. Thompson D.P. Bienkowski M.J. Lee K.S. Am. J. Physiol. 1995; 268: C1173-C1178Crossref PubMed Google Scholar, 27Xu H. Yang Z. Cui N. Giwa L.R. Abdulkadir L. Patel M. Sharma P. Shan G. Shen W. Jiang C. Am. J. Physiol. Cell Physiol. 2000; 279: C1464-C1471Crossref PubMed Google Scholar, 30Zhu G. Chanchevalap S. Cui N. Jiang C. J. Physiol. (Lond.). 1999; 516: 699-710Crossref Scopus (70) Google Scholar, 32Doi T. Fakler B. Schultz J.H. Schulte U. Brandle U. Weidemann S. Zenner H.P. Lang F. Ruppersberg J.P. J. Biol. Chem. 1996; 271: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 33Coulter K.L. Perier F. Radeke C.M. Vandenberg C.A. Neuron. 1995; 15: 1157-1168Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 34Qu Z. Yang Z. Cui N. Zhu G. Liu C. Xu H. Chanchevalap S. Shen W. Wu J. Li Y. Jiang C. J. Biol. Chem. 2000; 275: 31573-31580Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). To identify the specific structures in the Kir6.2 protein that enable the channel to respond to acidic pH, chimerical Kir channels were constructed by recombination of protein domains of Kir6.2 and Kir1.1. We reasoned that the pH sensitivity relied on the integrity of the proton-sensing and channel-gating mechanisms in the Kir6.2 protein. Interruption of the integrity will cause a loss of the pH sensitivity. Thus, we divided Kir6.2 and Kir1.1 into three segments in their peptide chains, i.e. 1) the N terminus, 2) C terminus, and 3) all the rest M1 through M2 regions. The chimera with both N and C termini from Kir6.2 and the rest from Kir1.1 is named SOS (S refers to six, and O to one). If the N terminus is the only part from Kir6.2, it is called SOO. Accordingly, the wild-type (wt) Kir6.2ΔC36 refers to SSS, and the wt Kir1.1 to OOO (Fig.2A). Six chimerical channels were systematically constructed using these Kir6.2 and Kir1.1 protein domains (Fig. 2A). Most of the recombinant channels showed inward rectifying currents of 1–3 μA, which were significantly larger than those recorded from the vector-injected cells (0.5 ± 0.1 μA, n = 9). The SSO and OSO were the only two that did not show evident channel expression. Azide (3 mm) treatment of the cells injected with SSO or OSO did not reveal any additional increase in the current amplitude. Thus, how SSO and OSO respond to acidosis is unclear. Although all other chimeras expressed detectable inward rectifying currents, none of them displayed a hypercapnic response as large as the wt Kir6.2ΔC36 (Fig. 2A, TableI). Clearly, these chimeras had caused interruptions of the pH-dependent gating mechanisms.Table IKir6.2, Kir6.2ΔC36. See Fig. 2 for nomenclature of chimeras. Data are presented as means ± S.E.NameBase-line current% CO2effectnExpressionμA Kir6.2 (SSS)2.1 ± 0.4130.3 ± 12.214Yes Kir1.1 (OOO)19.3 ± 5.5−69.0 ± 5.412YesChimeras OOS0.8 ± 0.345.7 ± 11.29Yes OSONo OSS1.0 ± 0.423.5 ± 10.57Yes SOO2.4 ± 0.517.8 ± 6.85Yes SOS6.8 ± 1.143.8 ± 7.94Yes SSONo OOSm2No SOOm2No SOSm11.9 ± 0.519.2 ± 5.84Yes SOSm24.9 ± 3.0123.9 ± 15.17YesN terminus Kir6.2-T71DNo Kir6.2-T71K2.4 ± 0.5−11.7 ± 6.48Yes Kir6.2-T71M1.6 ± 0.447.1 ± 14.94Yes Kir6.2-T71N4.6 ± 1.1160.3 ± 19.45Yes Kir6.2-T71S4.2 ± 1.1112.7 ± 7.65Yes OOSm2-K80TNo OSS-K80T1.9 ± 0.472.0 ± 18.25Yes SOSm2-T71K3.3 ± 0.6−1.9 ± 1.55YesM2 region Kir1.1-F173I5.2 ± 1.0−27.2 ± 3.84Yes Kir1.1-A177C1.7 ± 0.9−29.2 ± 9.014Yes Kir1.1-C175L/A177CNo Kir1.1-L179FNo Kir6.2-C166A12.5 ± 4.6−1.9 ± 1.74Yes Kir6.2-C166E19.6 ± 5.85.1 ± 1.54Yes Kir6.2-C166H5.6 ± 0.563.4 ± 2.74Yes Kir6.2-C166KNo Kir6.2-C166S18.7 ± 5.0−0.5 ± 1.86Yes Kir6.2-C166V1.5 ± 0.3114.7 ± 26.15Yes Kir6.2-F168LNo Kir6.2-I162FNo SOS-A177C6.7 ± 2.5−4.5 ± 4.54Yes SOS-F173I31.2 ± 9.5−3.9 ± 1.85Yes SOSm2-C166A1.7 ± 2.0−13.4 ± 8.08Yes SOSm2-C166E19.5 ± 0.81.5 ± 1.84Yes SOSm2-C166KNo SOSm2-C166S1.7 ± 0.64.7 ± 0.54Yes SOSm2-F168LNo SOSm2-I162F1.1 ± 0.274.4 ± 14.68YesC terminus Kir6.2-H175K5.6 ± 1.2−18.6 ± 2.75Yes SOSm2-H175K11.6 ± 3.16.5 ± 0.74Yes SOOm2-K186HNo Kir1.1-K186H12.0 ± 3.4−63.1 ± 4.15Yes SOO-K186H3.4 ± 0.544.4 ± 12.96YesMultiple domains Kir1.1-K80T/C175L/A177C/K186HNo OOS-K80T/C175L/A177C2.1 ± 0.555.1 ± 12.84Yes SOO-C175L/A177C/K186HNo SOS-C175L/A177C4.7 ± 1.7−20.5 ± 3.54Yes SOS-ALC1.1 ± 0.2142.5 ± 15.05Yes SOS-ILC3.9 ± 0.77.0 ± 6.56Yes SOS-AILC1.3 ± 0.2118.3 ± 23.05Yes Open table in a new tab Because none of the chimeras had a full CO2/pH sensitivity as did the wt Kir6.2ΔC36, it is possible that the essential structures for the pH sensitivity are not contained in these chimeras. To include them, we extended the C terminus to the entire M2 region of Kir6.2. This chimera SOSm2 expressed large inward rectifying currents (4.9 ± 3.0 μA, n = 7), with its inward rectification more like Kir6.2 than Kir1.1 (Fig. 2B). Exposure of the SOSm2 to 15% CO2 enhanced the inward rectifying currents by 123.9 ± 15.1% (n = 7), which were slightly smaller but not significantly different from the wt Kir6.2ΔC36 (p > 0.05) (Figs. 2B). Inclusion of the M1 sequence to the N terminus (SOSm1), however, did not produce any significant additional effect on the CO2sensitivity over the SOS (Fig. 2A). To determine if two of the three protein domains are sufficient for the pH sensitivity, chimeras containing two of the M2, N terminus and C terminus from Kir6.2 and the remaining sequences from Kir1.1 were constructed. The OOSm2 and SOOm2 failed to yield any functional channels, whereas the SOS had pH sensitivity of only about one-third that of the SOSm2 (Fig. 2A). These results thus indicate that protein domains necessary for the full CO2/pH sensitivity consist of at least the N terminus, C terminus, and M2 region in Kir6.2. The N terminus has been previously shown to play an important part in pH sensitivity in several Kir channels. A lysine residue at near the M1 region (Lys-80 in Kir1.1, Lys67 in Kir4.1) is a critical player (24Fakler B. Schultz J.H. Yang J. Schulte U. Brandle U. Zenner H.P. Jan L.Y. Ruppersberg J.P. EMBO J. 1996; 15: 4093-4099Crossref PubMed Scopus (163) Google Scholar, 27Xu H. Yang Z. Cui N. Giwa L.R. Abdulkadir L. Patel M. Sharma P. Shan G. Shen W. Jiang C. Am. J. Physiol. Cell Physiol. 2000; 279: C1464-C1471Crossref PubMed Google Scholar). This lysine residue is not found in Kir6.2. At the same location, the Kir6.2 has a threonine instead (Thr-71). Since Kir1.1 and Kir4.1 channels with this lysine residue are inhibited by acidic pH (25Tsai T.D. Shuck M.E. Thompson D.P. Bienkowski M.J. Lee K.S. Am. J. Physiol. 1995; 268: C1173-C1178Crossref PubMed Google Scholar, 28Yang Z. Jiang C. J. Physiol. (Lond.). 1999; 520: 921-927Crossref Scopus (48) Google Scholar), it is possible that the lack of the positively charged residues renders the Kir6.2 an opposite pH sensitivity. To test this hypothesis, we performed site-directed mutagenesis experiments at this site. Mutation of the Thr-71 to lysine yielded fair inward rectifying currents in whole-cell recordings. When the T71K was exposed to 15% CO2, we found that the mutant channels became insensitive to hypercapnia (Fig.3A). The same result was obtained in the SOSm2 carrying the T71K mutation (Fig. 3D). Systematic mutagenesis was subsequently carried out on the Thr-71 by replacing it with acidic, nonpolar, and other polar-neutral residues. There is a methionine at this site in Kir2.1, Kir2.3, Kir5.1, and Kir7.1. Thus we constructed the T71M mutant. The substitution of the Thr-71 with such a nonpolar residue greatly reduced the pH sensitivity (Fig. 3D). Replacement of the Thr-71 with an aspartate did not produce a functional channel. When it was mutated to serine, the mutant T71S showed an increase in the current amplitude by >100% with hypercapnia (Fig. 3D). Since both threonine and serine are the potential substrate of phosphorylation, we mutated the Thr-71 to another polar-neutral residue, asparagine. Currents of the T71N mutant were enhanced by 160.3 ± 19.4% (n = 5) by 15% CO2 (Fig. 3, B and D), suggesting that a polar-neutral residue at this site is necessary for maintaining the pH sensitivity in Kir6.2. Creation of the threonine residue in the OSS produced clear inward rectifying currents that increased reversibly by 72.0 ± 18.2% (n = 5) in response to 15% CO2 (Fig. 3,C and D), an increase that was significantly larger than the OSS (p < 0.01). Construction of this threonine in OOSm2 failed to produce functional expression. These results therefore suggest that the Thr-71 is the determinant residue in the N terminus for the pH sensitivity of Kir6.2. The M2 region of several Kir channels was aligned in Fig.4A. Amino acid residues in this region are highly homologous between Kir1.1 and Kir6.2. It is known that the area on the cytosolic side of the M2 domain is involved in lining the conductive pore. There are five residues in this area that are clearly different between Kir6.2 and Kir1.1. Among them are two phenylalanines with one found in Kir1.1 (Phe-173) and the other in Kir6.2 (Phe-168). Phenylalanine has a side chain much larger than that of leucine and valine, seen at its counterpart positions. To elucidate whether the size of residues controls the pH sensitivity, we site-specifically mutated these residues in Kir6.2 to those found in Kir1.1. The F168L and I162F mutants were constructed using both Kir6.2ΔC36 and SOSm2 as the template. Of the four mutants studied, the SOSm2-I162F was the only one expressing inward rectifying currents (Table I), which remained, although smaller, to be stimulated during hypercapnia by 74.4 ± 14.6% (n = 8) (Fig.4B). Subsequently, we studied two other sites in which a cysteine residue exists in each of Kir6.2 and Kir1.1. Mutations of Cys-175 to leucine in Kir1.1 and SOS did not produce channels that were stimulated by CO2. The other cysteine mutation, however, was remarkable. The Kir6.2-C166A mutant showed large base-line currents (12.5 ± 4.6 μA, n = 4) and a much weaker inward rectification than the wt Kir6.2ΔC36 (Fig. 4C). At highly negative membrane potentials, the inward rectifying currents also became weaker, suggesting that this site contributes to the voltage-dependent rectification. More interestingly, substitution of this single residue with alanine (Kir6.2-C166A) completely abolished the CO2 sensitivity of Kir6.2 (−1.9 ± 1.1%, n = 4) (Fig. 4, B andC). The same effect was also observed in the SOSm2-based mutant SOSm2-C166A (−13.4 ± 8.0%, n = 8). Systematic mutations of the Cys-166 were thereafter performed using Kir6.2ΔC36 and SOSm2. The mutant channels became CO2-insensitive when this residue was replaced with a negative or a polar-neutral residue, i.e. Kir6.2-C166E, Kir6.2-C166S, SOSm2-C166E, and SOSm2-C166S (Fig. 4B). Mutation to a positive residue (Kir6.2-C166K and SOSm2-C166K) did not yield functional expression. In contrast, the mutant channels remained stimulated by hypercapnic acidosis, when a valine or histidine was the replacement (Table I). Indeed, the Kir6.2-C166V showed CO2sensitivity almost identical to the SOSm2 (Fig. 4B). Reversal mutations of this cysteine residue, however, did not show any dramatic effect. The Kir1.1-A177C was still inhibited by CO2, and the SOS-A177C remained pH-insensitive (Fig.4B). Therefore, it is possible that other adjacent residues within the M2 are also involved. To identify these residues, we performed further mutagenesis studies. We found that the formation of disulfide bond with Cys-175 was not a reason, because combined mutations of these two residues still showed an inhibited phenotype in SOS (SOS-C175L/A177C, -20.5 ± 3.5%, n = 4). Subsequently, we created mutants to include Ser-172 and/or Phe-173. The SOS-based mutants S172A/F173I/C175L/A177C (SOS-AILC) and S172A/C175L/A177C (SOS-ALC) expressed functional currents with clear inward rectification. Both of them were strongly stimulated by 15% CO2 to a degree that statistically was not different from the SOSm2 and the wt Kir6.2ΔC36 channels (Fig. 4D). In contrast, the F173I/C175L/A177C (SOS-ILC) had no significant effect. Thus, these results suggest that the molecular determinant for the pH-dependent activation is likely to be the short motif (∼ 6 amino acids) at the intracellular end of the M2 centered by the Cys-166. Our previous studies show that His-175 in the C terminus is critical for the pH sensitivity of Kir6.2 (21Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). This was confirmed in our current studies (Table I). In addition, we found that mutation of this histidine residue in SOSm2 greatly reduced the current response to 15% CO2 (6.5 ± 0.7%, n = 4) (Fig.5A). Although construction of a histidine residue at the same site in SOOm2 did not produce functional expression, creation of such a residue in the SOO (SOO-K186H) gave rise to funct

Kir1.1 and Kir4.1 channels may be involved in the maintenance of pH and K+ homeostasis in renal epithelial cells and CO2 chemoreception in brainstem neurons. To understand the molecular determinants for their characteristic differences, the structure-function relationship was studied using site-directed mutagenesis. According to previous studies, Glu158 in Kir4.1 is likely to be the major rectification controller. This was confirmed in both Kir1.1 and Kir4.1. Mutation of Gly210, the second potential rectification controller, to glutamate did not show any additional effect on the inward rectification. More interestingly, we found that Glu158 in Kir4.1 was also an important residue contributing to single channel conductance and pH sensitivity. The E158N Kir4.1 mutant had a unitary conductance of 35 pS and a midpoint pH for channel inhibition (pKa) value of 6.72, both of which were almost identical to those of the wild-type (WT) Kir1.1. Flickering channel activity was clearly seen in the E158N mutant at positive membrane potentials, which is typical in the WT Kir1.1 but absent in the WT Kir4.1. Reverse mutation in Kir1.1 (N171E) reduced the unitary conductance to 27 pS (23 pS in WT Kir4.1). However, the pH sensitivity of this mutant did not show a marked difference from the WT Kir1.1. Therefore, it is possible that a residue(s) in addition to Asn171 is also involved. Thus we studied several other residues in both M2 and H5 regions. We found that joint mutations of Val140 and Asn171 to residues seen in Kir4.1 greatly reduced the pH sensitivity (pKa 6. 08). The V140T mutation in Kir1.1 led to a unitary conductance of approximately 70 pS, and the G210E mutation in Kir4.1 caused a decrease in pH sensitivity of 0.4 pH units. These results indicate that the pore-forming sequences are targets for modulations of multiple channel-biophysical properties and demonstrate a site contributing to rectification, unitary conductance and proton sensitivity in these Kir channels.

Kir1.1 (ROMK1) is inhibited by hypercapnia and intracellular acidosis with midpoint pH for channel inhibition (pK(a)) of approximately 6.7. Another close relative, Kir4.1 (BIR10), is also pH sensitive with much lower pH sensitivity (pK(a) approximately 6. 0), although it shares a high sequence homology with Kir1.1. To find the molecular determinants for the distinct pH sensitivity, we studied the structure-functional relationship using site-directed mutagenesis. An NH(2)-terminal residue (Lys-53) was found to be responsible for the low pH sensitivity in Kir4.1. Mutation of this lysine to valine (K53V), a residue seen at the same position in Kir1. 1, markedly increased channel sensitivity to CO(2)/pH. Reverse mutation on Kir1.1 (V66K) decreased the CO(2)/pH sensitivities. Interestingly, mutation of these residues to glutamate greatly enhanced the pH sensitivity in both channels. Other contributors to the distinct pH sensitivity were histidine residues in the COOH terminus, whose numbers are fewer in Kir4.1 than Kir1.1. Mutation of two of these histidine residues in Kir1.1 (H342Q/H354N) reduced CO(2)/pH sensitivities, whereas the creation of two histidines (S328H/G340H) in Kir4.1 increased the CO(2)/pH sensitivities. Combined mutations of the lysine and histidine residues in Kir4.1 (K53V/S328H/G340H) gave rise to a channel that had CO(2)/pH sensitivities almost identical to those of the wild-type Kir1.1. Thus the residues demonstrated in our current studies are likely the molecular basis for the distinct pH sensitivity between Kir1.1 and Kir4.1.

Several inward rectifier K(+) (Kir) channels are pH-sensitive, making them potential candidates for CO(2) chemoreception in cells. However, there is no evidence showing that Kir channels change their activity at near physiological level of P(CO(2)), as most previous studies were done using high concentrations of CO(2). It is known that the heteromeric Kir4.1-Kir5.1 channels are highly sensitive to intracellular protons with pKa value right at the physiological pH level. Such a pKa value may allow these channels to regulate membrane potentials with modest changes in P(CO(2)). To test this hypothesis, we studied the Kir4.1-Kir5.1 currents expressed in Xenopus oocytes and membrane potentials in the presence and absence of bicarbonate. Evident inhibition of these currents (by approximately 5%) was seen with P(CO(2)) as low as 8 torr. Higher P(CO(2)) levels (23-60 torr) produced stronger inhibitions (by 30-40%). The inhibitions led to graded depolarizations (5-45 mV with P(CO(2)) 8-60 torr). Similar effects were observed in the presence of 24 mM bicarbonate and 5% CO(2). Indeed, the Kir4.1-Kir5.1 currents were enhanced with 3% CO(2) and suppressed with 8% CO(2) in voltage clamp, resulting in hyper- (-9 mV) and depolarization (16 mV) in current clamp, respectively. With physiological concentration of extracellular K(+), the Kir4.1-Kir5.1 channels conduct substantial outward currents that were similarly inhibited by CO(2) as their inward rectifying currents. These results therefore indicate that the heteromeric Kir4.1-Kir5.1 channels are modulated by a modest change in P(CO(2)) levels. Such a modulation alters cellular excitability, and enables the cell to detect hypercapnia and hypocapnia in the presence of bicarbonate.

The TRP superfamily of channels (nomenclature as agreed by NC-IUPHAR [145, 915]), whose founder member is the Drosophila Trp channel, exists in mammals as six families; TRPC, TRPM, TRPV, TRPA, TRPP and TRPML based on amino acid homologies. TRP subunits contain six putative transmembrane domains and assemble as homo- or hetero-tetramers to form cation selective channels with diverse modes of activation and varied permeation properties (reviewed by [630]). Established, or potential, physiological functions of the individual members of the TRP families are discussed in detail in the recommended reviews and in a number of books [344, 589, 979, 216]. The established, or potential, involvement of TRP channels in disease is reviewed in [384, 588] and [591], together with a special edition of Biochemica et Biophysica Acta on the subject [588]. Additional disease related reviews, for pain [542], stroke [967], sensation and inflammation [843], itch [109], and airway disease [261, 896], are available. The pharmacology of most TRP channels has been advanced in recent years. Broad spectrum agents are listed in the tables along with more selective, or recently recognised, ligands that are flagged by the inclusion of a primary reference. See Rubaiy (2019) for a review of pharmacological tools for TRPC1/C4/C5 channels [692]. Most TRP channels are regulated by phosphoinostides such as PtIns(4,5)P2 although the effects reported are often complex, occasionally contradictory, and likely to be dependent upon experimental conditions, such as intracellular ATP levels (reviewed by [862, 592, 689]). Such regulation is generally not included in the tables.When thermosensitivity is mentioned, it refers specifically to a high Q10 of gating, often in the range of 10-30, but does not necessarily imply that the channel's function is to act as a 'hot' or 'cold' sensor. In general, the search for TRP activators has led to many claims for temperature sensing, mechanosensation, and lipid sensing. All proteins are of course sensitive to energies of binding, mechanical force, and temperature, but the issue is whether the proposed input is within a physiologically relevant range resulting in a response. TRPA (ankyrin) familyTRPA1 is the sole mammalian member of this group (reviewed by [246]). TRPA1 activation of sensory neurons contribute to nociception [356, 763, 516]. Pungent chemicals such as mustard oil (AITC), allicin, and cinnamaldehyde activate TRPA1 by modification of free thiol groups of cysteine side chains, especially those located in its amino terminus [491, 47, 311, 493]. Alkenals with α, β-unsaturated bonds, such as propenal (acrolein), butenal (crotylaldehyde), and 2-pentenal can react with free thiols via Michael addition and can activate TRPA1. However, potency appears to weaken as carbon chain length increases [21, 47]. Covalent modification leads to sustained activation of TRPA1. Chemicals including carvacrol, menthol, and local anesthetics reversibly activate TRPA1 by non-covalent binding [364, 438, 923, 922]. TRPA1 is not mechanosensitive under physiological conditions, but can be activated by cold temperatures [365, 175]. The electron cryo-EM structure of TRPA1 [639] indicates that it is a 6-TM homotetramer. Each subunit of the channel contains two short ‘pore helices’ pointing into the ion selectivity filter, which is big enough to allow permeation of partially hydrated Ca2+ ions. TRPC (canonical) familyMembers of the TRPC subfamily (reviewed by [239, 673, 14, 4, 79, 382, 638, 55]) fall into the subgroups outlined below. TRPC2 is a pseudogene in humans. It is generally accepted that all TRPC channels are activated downstream of Gq/11-coupled receptors, or receptor tyrosine kinases (reviewed by [661, 814, 915]). A comprehensive listing of G-protein coupled receptors that activate TRPC channels is given in [4]. Hetero-oligomeric complexes of TRPC channels and their association with proteins to form signalling complexes are detailed in [14] and [383]. TRPC channels have frequently been proposed to act as store-operated channels (SOCs) (or compenents of mulimeric complexes that form SOCs), activated by depletion of intracellular calcium stores (reviewed by [640, 14, 665, 703, 954, 132, 626, 51, 133]). However, the weight of the evidence is that they are not directly gated by conventional store-operated mechanisms, as established for Stim-gated Orai channels. TRPC channels are not mechanically gated in physiologically relevant ranges of force. All members of the TRPC family are blocked by 2-APB and SKF96365 [295, 294]. Activation of TRPC channels by lipids is discussed by [55]. Important progress has been recently made in TRPC pharmacology [692, 529, 372, 87]. TRPC channels regulate a variety of physiological functions and are implicated in many human diseases [248, 56, 759, 879]. TRPC1/C4/C5 subgroup TRPC1 alone may not form a functional ion channel [191]. TRPC4/C5 may be distinguished from other TRP channels by their potentiation by micromolar concentrations of La3+. TRPC2 is a pseudogene in humans, but in other mammals appears to be an ion channel localized to microvilli of the vomeronasal organ. It is required for normal sexual behavior in response to pheromones in mice. It may also function in the main olfactory epithelia in mice [951, 625, 624, 952, 462, 988, 947].TRPC3/C6/C7 subgroup All members are activated by diacylglycerol independent of protein kinase C stimulation [295].TRPM (melastatin) familyMembers of the TRPM subfamily (reviewed by [230, 294, 640, 978]) fall into the five subgroups outlined below. TRPM1/M3 subgroupIn darkness, glutamate released by the photoreceptors and ON-bipolar cells binds to the metabotropic glutamate receptor 6 , leading to activation of Go . This results in the closure of TRPM1. When the photoreceptors are stimulated by light, glutamate release is reduced, and TRPM1 channels are more active, resulting in cell membrane depolarization. Human TRPM1 mutations are associated with congenital stationary night blindness (CSNB), whose patients lack rod function. TRPM1 is also found melanocytes. Isoforms of TRPM1 may present in melanocytes, melanoma, brain, and retina. In melanoma cells, TRPM1 is prevalent in highly dynamic intracellular vesicular structures [341, 609]. TRPM3 (reviewed by [615]) exists as multiple splice variants which differ significantly in their biophysical properties. TRPM3 is expressed in somatosensory neurons and may be important in development of heat hyperalgesia during inflammation (see review [803]). TRPM3 is frequently coexpressed with TRPA1 and TRPV1 in these neurons. TRPM3 is expressed in pancreatic beta cells as well as brain, pituitary gland, eye, kidney, and adipose tissue [614, 802]. TRPM3 may contribute to the detection of noxious heat [870].TRPM2TRPM2 is activated under conditions of oxidative stress (respiratory burst of phagocytic cells) and ischemic conditions. However, the direct activators are ADPR(P) and calcium. As for many ion channels, PIP2 must also be present (reviewed by [935]). Numerous splice variants of TRPM2 exist which differ in their activation mechanisms [200]. The C-terminal domain contains a TRP motif, a coiled-coil region, and an enzymatic NUDT9 homologous domain. TRPM2 appears not to be activated by NAD, NAAD, or NAADP, but is directly activated by ADPRP (adenosine-5'-O-disphosphoribose phosphate) [827]. TRPM2 is involved in warmth sensation [724], and contributes to neurological diseases [61]. Recent study shows that 2'-deoxy-ADPR is an endogenous TRPM2 superagonist [231]. TRPM4/5 subgroupTRPM4 and TRPM5 have the distinction within all TRP channels of being impermeable to Ca2+ [915]. A splice variant of TRPM4 (i.e.TRPM4b) and TRPM5 are molecular candidates for endogenous calcium-activated cation (CAN) channels [278]. TRPM4 is active in the late phase of repolarization of the cardiac ventricular action potential. TRPM4 deletion or knockout enhances beta adrenergic-mediated inotropy [507]. Mutations are associated with conduction defects [347, 507, 753]. TRPM4 has been shown to be an important regulator of Ca2+ entry in to mast cells [847] and dendritic cell migration [39]. TRPM5 in taste receptor cells of the tongue appears essential for the transduction of sweet, amino acid and bitter stimuli [460] TRPM5 contributes to the slow afterdepolarization of layer 5 neurons in mouse prefrontal cortex [439]. Both TRPM4 and TRPM5 are required transduction of taste stimuli [206].TRPM6/7 subgroupTRPM6 and 7 combine channel and enzymatic activities (‘chanzymes’). These channels have the unusual property of permeation by divalent (Ca2+, Mg2+, Zn2+) and monovalent cations, high single channel conductances, but overall extremely small inward conductance when expressed to the plasma membrane. They are inhibited by internal Mg2+ at ~0.6 mM, around the free level of Mg2+ in cells. Whether they contribute to Mg2+ homeostasis is a contentious issue. When either gene is deleted in mice, the result is embryonic lethality. The C-terminal kinase region is cleaved under unknown stimuli, and the kinase phosphorylates nuclear histones. TRPM7 is responsible for oxidant- induced Zn2+ release from intracellular vesicles [3] and contributes to intestinal mineral absorption essential for postnatal survival [532]. TRPM8Is a channel activated by cooling and pharmacological agents evoking a ‘cool’ sensation and participates in the thermosensation of cold temperatures [50, 147, 186] reviewed by [864, 481, 391, 556]. TRPML (mucolipin) familyThe TRPML family [676, 964, 670, 926, 156] consists of three mammalian members (TRPML1-3). TRPML channels are probably restricted to intracellular vesicles and mutations in the gene (MCOLN1) encoding TRPML1 (mucolipin-1) cause the neurodegenerative disorder mucolipidosis type IV (MLIV) in man. TRPML1 is a cation selective ion channel that is important for sorting/transport of endosomes in the late endocytotic pathway and specifically, fission from late endosome-lysosome hybrid vesicles and lysosomal exocytosis [704]. TRPML2 and TRPML3 show increased channel activity in low extracellular sodium and are activated by similar small molecules [270]. A naturally occurring gain of function mutation in TRPML3 (i.e. A419P) results in the varitint waddler (Va) mouse phenotype (reviewed by [676, 593]). TRPP (polycystin) familyThe TRPP family (reviewed by [179, 177, 252, 905, 320]) or PKD2 family is comprised of PKD2 (PC2), PKD2L1 (PC2L1), PKD2L2 (PC2L2), which have been renamed TRPP1, TRPP2 and TRPP3, respectively [915]. It should also be noted that the nomenclature of PC2 was TRPP2 in old literature. However, PC2 has been uniformed to be called TRPP2 [293]. PKD2 family channels are clearly distinct from the PKD1 family, whose function is unknown. PKD1 and PKD2 form a hetero-oligomeric complex with a 1:3 ratio. [775]. Although still being sorted out, TRPP family members appear to be 6TM spanning nonselective cation channels. TRPV (vanilloid) familyMembers of the TRPV family (reviewed by [849]) can broadly be divided into the non-selective cation channels, TRPV1-4 and the more calcium selective channels TRPV5 and TRPV6.TRPV1-V4 subfamilyTRPV1 is involved in the development of thermal hyperalgesia following inflammation and may contribute to the detection of noxius heat (reviewed by [660, 756, 786]). Numerous splice variants of TRPV1 have been described, some of which modulate the activity of TRPV1, or act in a dominant negative manner when co-expressed with TRPV1 [722]. The pharmacology of TRPV1 channels is discussed in detail in [280] and [868]. TRPV2 is probably not a thermosensor in man [635], but has recently been implicated in innate immunity [469]. TRPV3 and TRPV4 are both thermosensitive. There are claims that TRPV4 is also mechanosensitive, but this has not been established to be within a physiological range in a native environment [106, 454].TRPV5/V6 subfamily TRPV5 and TRPV6 are highly expressed in placenta, bone, and kidney. Under physiological conditions, TRPV5 and TRPV6 are calcium selective channels involved in the absorption and reabsorption of calcium across intestinal and kidney tubule epithelia (reviewed by [901, 168, 558, 227]).

Significance Many neurons release neuropeptides as well as classical neurotransmitters. Neuropeptides often regulate neural circuits controlling behaviors such as emotional behaviors. However, how neuropeptide release is regulated is not well-understood. We identified a Drosophila gene, Dstac , that is similar to a vertebrate gene that regulates voltage-dependent calcium channels in skeletal muscles and found that it is active in muscles and a subset of neurons, including motor neurons. Using live imaging, electrophysiology, and genetic manipulations, we found that Dstac localizes to motor synapses with muscles and regulates the voltage response of calcium channels and the release of neuropeptides. Since Stac proteins are found in neurons in the vertebrate CNS, they may also be regulators of neuropeptide release in the vertebrate brain.

For the RNA world hypothesis, the direct condensation between nucleobase and ribose to afford canonical nucleoside has been a formidable challenge in terms of regioselectivity. In this report, we demonstrate that cavitation, a frequently occurring energy-intensive phenomenon upon the collapse of bubbles, could be a plausible driving force to form the purine nucleosides directly from nucleobase and ribose with the desired regioselectivity. Under cavitation, the overall yield of N9 nucleosides increases compared with non-cavitation conditions, whereas the amount of the undesired ribosylamino isomers decreases. Plausible geochemical scenarios on primordial Earth are proposed that could facilitate enrichment of the desired N9 nucleosides. Under salt-free dry-wet conditions, much improved yield (7.2%) and selectivity (N9/ribosylamino = 0.45) of the N9 isomers are achieved compared with the classical routes.

Lysosome acidification is a dynamic equilibrium of H+ influx and efflux across the membrane, which is crucial for cell physiology. The vacuolar H+ ATPase (V-ATPase) is responsible for the H+ influx or refilling of lysosomes. TMEM175 was identified as a novel H+ permeable channel on lysosomal membranes, and it plays a critical role in lysosome acidification. However, how TMEM175 participates in lysosomal acidification remains unknown. Here, we present evidence that TMEM175 regulates lysosomal H+ influx and efflux in enlarged lysosomes isolated from COS1 treated with vacuolin-1. By utilizing the whole-endolysosome patch-clamp recording technique, a series of integrated lysosomal H+ influx and efflux signals in a ten-of-second time scale under the physiological pH gradient (luminal pH 4.60, and cytosolic pH 7.20) was recorded from this in vitro system. Lysosomal H+ fluxes constitute both the lysosomal H+ refilling and releasing, and they are asymmetrical processes with distinct featured kinetics for each of the H+ fluxes. Lysosomal H+ fluxes are entirely abolished when TMEM175 losses of function in the F39V mutant and is blocked by the antagonist (2-GBI). Meanwhile, lysosomal H+ fluxes are modulated by the pH-buffering capacity of the lumen and the lysosomal glycosylated membrane proteins, lysosome-associated membrane protein 1 (LAMP1). We propose that the TMEM175-mediated lysosomal H+ fluxes model would provide novel thoughts for studying the pathology of Parkinson's disease and lysosome storage disorders.

Intracellular organelles exchange their luminal contents with each other via both vesicular and non-vesicular mechanisms. By forming membrane contact sites (MCSs) with ER and mitochondria, lysosomes mediate bidirectional transport of metabolites and ions between lysosomes and organelles that regulate lysosomal physiology, movement, membrane remodeling, and membrane repair. In this chapter, we will first summarize the current knowledge of lysosomal ion channels and then discuss the molecular and physiological mechanisms that regulate lysosome-organelle MCS formation and dynamics. We will also discuss the roles of lysosome-ER and lysosome-mitochondria MCSs in signal transduction, lipid transport, Ca 2+ transfer, membrane trafficking, and membrane repair, as well as their roles in lysosome-related pathologies.

Abstract Three ionic liquids (ILs) of pyrazolium cations associated with hydrogen sulfate (HSO 4 − ) anions were synthesized and quinoline denitrification from simulated oil was investigated. 1‐Hexyl‐2,3,5‐trimethylpyzolium ([HexPZM]HSO 4 ) showed the highest extraction efficiency (98%) for denitrification of quinoline from simulated oil, and could be reused more than five times without obvious decrease of extraction efficiency. The denitrification of fluid catalytic cracking (FCC) gasoline and diesel with [HexPZM]HSO 4 were determined with extraction efficiencies of 42.1% and 13.7%, respectively.