Molecular and Cellular Mechanisms of Salt Taste.

Salt taste, the taste of sodium chloride (NaCl), is mechanistically one of the most complex and puzzling among basic tastes. Sodium has essential functions in the body but causes harm in excess. Thus, animals use salt taste to ingest the right amount of salt, which fluctuates by physiological needs: typically, attraction to low salt concentrations and rejection of high salt. This concentration-valence relationship is universally observed in terrestrial animals, and research has revealed complex peripheral codes for NaCl involving multiple taste pathways of opposing valence. Sodium-dependent and -independent pathways mediate attraction and aversion to NaCl, respectively. Gustatory sensors and cells that transduce NaCl have been uncovered, along with downstream signal transduction and neurotransmission mechanisms. However, much remains unknown. This article reviews classical and recent advances in our understanding of the molecular and cellular mechanisms underlying salt taste in mammals and insects and discusses perspectives on human salt taste.

Posttranslational regulation of CALHM1/3 channel: N-linked glycosylation and S-palmitoylation.

Among calcium homeostasis modulator (CALHM) family members, CALHM1 and 3 together form a voltage-gated large-pore ion channel called CALHM1/3. CALHM1/3 plays an essential role in taste perception by mediating neurotransmitter release at channel synapses of taste bud cells. However, it is poorly understood how CALHM1/3 is regulated. Biochemical analyses of the two subunits following site-directed mutagenesis and pharmacological treatments established that both CALHM1 and 3 were N-glycosylated at single Asn residues in their second extracellular loops. Biochemical and electrophysiological studies revealed that N-glycan acquisition on CALHM1 and 3, respectively, controls the biosynthesis and gating kinetics of the CALHM1/3 channel. Furthermore, failure in subsequent remodeling of N-glycans decelerated the gating kinetics. Thus, the acquisition of N-glycans on both subunits and their remodeling differentially contribute to the functional expression of CALHM1/3. Meanwhile, metabolic labeling and acyl-biotin exchange assays combined with genetic modification demonstrated that CALHM3 was reversibly palmitoylated at three intracellular Cys residues. Screening of the DHHC protein acyltransferases identified DHHC3 and 15 as CALHM3 palmitoylating enzymes. The palmitoylation-deficient mutant CALHM3 showed a normal degradation rate and interaction with CALHM1. However, the same mutation markedly attenuated the channel activity but not surface localization of CALHM1/3, suggesting that CALHM3 palmitoylation is a critical determinant of CALHM1/3 activity but not its formation or forward trafficking. Overall, this study characterized N-glycosylation and S-palmitoylation of CALHM1/3 subunits and clarified their differential contributions to its functional expression, providing insights into the fine control of the CALHM1/3 channel and associated physiological processes.

Taste transduction and channel synapses in taste buds.

The variety of taste sensations, including sweet, umami, bitter, sour, and salty, arises from diverse taste cells, each of which expresses specific taste sensor molecules and associated components for downstream signal transduction cascades. Recent years have witnessed major advances in our understanding of the molecular mechanisms underlying transduction of basic tastes in taste buds, including the identification of the bona fide sour sensor H+ channel OTOP1, and elucidation of transduction of the amiloride-sensitive component of salty taste (the taste of sodium) and the TAS1R-independent component of sweet taste (the taste of sugar). Studies have also discovered an unconventional chemical synapse termed "channel synapse" which employs an action potential-activated CALHM1/3 ion channel instead of exocytosis of synaptic vesicles as the conduit for neurotransmitter release that links taste cells to afferent neurons. New images of the channel synapse and determinations of the structures of CALHM channels have provided structural and functional insights into this unique synapse. In this review, we discuss the current view of taste transduction and neurotransmission with emphasis on recent advances in the field.

CALHM1 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes.

Recognition of sweet, bitter and umami tastes requires the non-vesicular release from taste bud cells of ATP, which acts as a neurotransmitter to activate afferent neural gustatory pathways. However, how ATP is released to fulfil this function is not fully understood. Here we show that calcium homeostasis modulator 1 (CALHM1), a voltage-gated ion channel, is indispensable for taste-stimuli-evoked ATP release from sweet-, bitter- and umami-sensing taste bud cells. Calhm1 knockout mice have severely impaired perceptions of sweet, bitter and umami compounds, whereas their recognition of sour and salty tastes remains mostly normal. Calhm1 deficiency affects taste perception without interfering with taste cell development or integrity. CALHM1 is expressed specifically in sweet/bitter/umami-sensing type II taste bud cells. Its heterologous expression induces a novel ATP permeability that releases ATP from cells in response to manipulations that activate the CALHM1 ion channel. Knockout of Calhm1 strongly reduces voltage-gated currents in type II cells and taste-evoked ATP release from taste buds without affecting the excitability of taste cells by taste stimuli. Thus, CALHM1 is a voltage-gated ATP-release channel required for sweet, bitter and umami taste perception.

Action of Protein Tyrosine Kinase Inhibitors on the Hypotonicity-Stimulated Trafficking Kinetics of Epithelial Na+ Channels (ENaC) in Renal Epithelial Cells: Analysis Using a Mathematical Model.

BACKGROUND/AIMS: Epithelial Na+ channels (ENaCs) play crucial roles in control of blood pressure by determining the total amount of renal Na+ reabsorption, which is regulated by various factors such as aldosterone, vasopressin, insulin and osmolality. The intracellular trafficking process of ENaCs regulates the amount of the ENaC-mediated Na+ reabsorption in the collecting duct of the kidney mainly by determining the number of ENaC expressed at the apical membrane of epithelial cells. Although we previously reported protein tyrosine kinases (PTKs) contributed to the ENaC-mediated epithelial Na+ reabsorption, we have no information on the role of PTKs in the intracellular ENaC trafficking. METHODS: Using the mathematical model recently established in our laboratory, we studied the effect of PTKs inhibitors (PTKIs), AG1296 (10 µM: an inhibitor of the PDGF receptor (PDGFR)) and AG1478 (10 µM: an inhibitor of the EGF receptor (EGFR)) on the rates of the intracellular ENaC trafficking in renal epithelial A6 cells endogenously expressing ENaCs. RESULTS: We found that application of PTKIs significantly reduced the insertion rate of ENaC to the apical membrane by 56%, the recycling rate of ENaC by 83%, the cumulative time of an individual ENaC staying in the apical membrane by 27%, the whole life-time after the first insertion of ENaC by 47%, and the cumulative Na+ absorption by 61%, while the degradation rate was increased to 3.8-fold by application of PTKIs. These observations indicate that PTKs contribute to the processes of insertion, recycling and degradation of ENaC in the intracellular trafficking process under a hypotonic condition. CONCLUSION: The present study indicates that application of EGFR and PDGFR-inhibitable PTKIs reduced the insertion rate (kI), and the recycling rate (kR) of ENaCs, but increased degradation rate (kD) in renal A6 epithelial cells under a hypotonic condition. These observations indicate that hypotonicity increases the surface expression of ENaCs by increasing the insertion rate (kI) and the recycling rate (kR) of ENaCs associated with a decrease in the degradation rate but without any significant effects on the endocytotic rate (kE) in EGFR and PDGFR-related PTKs-mediated pathways.

ATP Release Channels.

Adenosine triphosphate (ATP) has been well established as an important extracellular ligand of autocrine signaling, intercellular communication, and neurotransmission with numerous physiological and pathophysiological roles. In addition to the classical exocytosis, non-vesicular mechanisms of cellular ATP release have been demonstrated in many cell types. Although large and negatively charged ATP molecules cannot diffuse across the lipid bilayer of the plasma membrane, conductive ATP release from the cytosol into the extracellular space is possible through ATP-permeable channels. Such channels must possess two minimum qualifications for ATP permeation: anion permeability and a large ion-conducting pore. Currently, five groups of channels are acknowledged as ATP-release channels: connexin hemichannels, pannexin 1, calcium homeostasis modulator 1 (CALHM1), volume-regulated anion channels (VRACs, also known as volume-sensitive outwardly rectifying (VSOR) anion channels), and maxi-anion channels (MACs). Recently, major breakthroughs have been made in the field by molecular identification of CALHM1 as the action potential-dependent ATP-release channel in taste bud cells, LRRC8s as components of VRACs, and SLCO2A1 as a core subunit of MACs. Here, the function and physiological roles of these five groups of ATP-release channels are summarized, along with a discussion on the future implications of understanding these channels.

Post-translational palmitoylation controls the voltage gating and lipid raft association of the CALHM1 channel.

KEY POINTS: Calcium homeostasis modulator 1 (CALHM1), a new voltage-gated ATP- and Ca2+ -permeable channel, plays important physiological roles in taste perception and memory formation. Regulatory mechanisms of CALHM1 remain unexplored, although the biophysical disparity between CALHM1 gating in vivo and in vitro suggests that there are undiscovered regulatory mechanisms. Here we report that CALHM1 gating and association with lipid microdomains are post-translationally regulated through the process of protein S-palmitoylation, a reversible attachment of palmitate to cysteine residues. Our data also establish cysteine residues and enzymes responsible for CALHM1 palmitoylation. CALHM1 regulation by palmitoylation provides new mechanistic insights into fine-tuning of CALHM1 gating in vivo and suggests a potential layer of regulation in taste and memory. ABSTRACT: Emerging roles of CALHM1, a recently discovered voltage-gated ion channel, include purinergic neurotransmission of tastes in taste buds and memory formation in the brain, highlighting its physiological importance. However, the regulatory mechanisms of the CALHM1 channel remain entirely unexplored, hindering full understanding of its contribution in vivo. The different gating properties of CALHM1 in vivo and in vitro suggest undiscovered regulatory mechanisms. Here, in searching for post-translational regulatory mechanisms, we discovered the regulation of CALHM1 gating and association with lipid microdomains via protein S-palmitoylation, the only reversible lipid modification of proteins on cysteine residues. CALHM1 is palmitoylated at two intracellular cysteines located in the juxtamembrane regions of the third and fourth transmembrane domains. Enzymes that catalyse CALHM1 palmitoylation were identified by screening 23 members of the DHHC protein acyltransferase family. Epitope tagging of endogenous CALHM1 proteins in mice revealed that CALHM1 is basally palmitoylated in taste buds in vivo. Functionally, palmitoylation downregulates CALHM1 without effects on its synthesis, degradation and cell surface expression. Mutation of the palmitoylation sites has a profound impact on CALHM1 gating, shifting the conductance-voltage relationship to more negative voltages and accelerating the activation kinetics. The same mutation also reduces CALHM1 association with detergent-resistant membranes. Our results comprehensively uncover a post-translational regulation of the voltage-dependent gating of CALHM1 by palmitoylation.

Analysis of Aprotinin, a Protease Inhibitor, Action on the Trafficking of Epithelial Na+ Channels (ENaC) in Renal Epithelial Cells Using a Mathematical Model.

BACKGROUND/AIM: Epithelial Na+ channels (ENaC) play a crucial role in control of blood pressure by regulating renal Na+ reabsorption. Intracellular trafficking of ENaC is one of the key regulators of ENaC function, but a quantitative description of intracellular recycling of endogenously expressed ENaC is unavailable. We attempt here to provide a model for intracellular recycling after applying a protease inhibitor under hypotonic conditions. METHODS: We simulated the ENaC-mediated Na+ transport in renal epithelial A6 cells measured as short-circuit currents using a four-state mathematical ENaC trafficking model. RESULTS: We developed a four-state mathematical model of ENaC trafficking in the cytosol of renal epithelial cells that consists of: an insertion state of ENaC that can be trafficked to the apical membrane state (insertion rate); an apical membrane state of ENaC conducting Na+ across the apical membrane; a recycling state containing ENaC that are retrieved from the apical membrane state (endocytotic rate) and then to the insertion state (recycling rate) communicating with the apical membrane state or to a degradation state (degradation rate). We studied the effect of aprotinin (a protease inhibitor) blocking protease-induced cleavage of the extracellular loop of γ ENaC subunit on the rates of intracellular ENaC trafficking using the above-defined four-state mathematical model of ENaC trafficking and the recycling number relative to ENaC staying in the apical membrane. We found that aprotinin significantly reduced the insertion rate of ENaC to the apical membrane by 40%, the recycling rate of ENaC by 81%, the cumulative time of an individual ENaC staying in the apical membrane by 32%, the cumulative life-time after the first endocytosis of ENaC by 25%, and the cumulative Na+ absorption by 31%. The most interesting result of the present study is that cleavage of ENaC affects the intracellular ENaC trafficking rate and determines the residency time of ENaC, indicating that more active cleaved ENaCs stay longer at the apical membrane contributing to transcellular Na+ transport via an increase in recycling of ENaC to the apical membrane. CONCLUSION: The extracellular protease-induced cleavage of the extracellular loop of γ ENaC subunit increases transcellular epithelial Na+ transport by elevating the recycling rate of ENaC due to an increase in the recycling rate of ENaCs associated with increases in the insertion rate of ENaC.

Hypotonicity activates a voltage-dependent membrane conductance in N2a neuroblastoma cells.

To maintain cellular and bodily homeostasis, cells respond to extracellular stimuli including osmotic stress by activating various ion channels, which have been implicated in many physiological and pathophysiological conditions. However, cellular osmosensory mechanisms remain elusive. Here, we report a novel voltage-dependent current in N2a cells activated by exposure to hypotonic stress. After a hypotonic challenge, N2a cells sequentially develop two distinct currents. The volume-regulated anion channel (VRAC) current emerges first and, after a delay, activation of a previously uncharacterized strongly outwardly rectifying current follows. The latter, delayed current (Id) is insensitive to NPPB, a nonspecific blocker of Cl- channels, and intracellular Mg2+, which inhibits VRAC and swelling-activated TRPM3 and TRPM7 channels. Replacement of extracellular Na+ with NMDG+ reduces inward tail currents, suggesting that Id is mediated by cations. Finally, Id shows voltage-dependent activation with slow activation kinetics and half-maximal activation at +76 mV. These pharmacological and biophysical characteristics of Id are distinct from those of known osmotic cell swelling-activated ion channels. In conclusion, our data identify and characterize a novel osmotically-activated, voltage-dependent ion channel in N2a cells.

Adeno-Associated Virus-Mediated Gene Transfer into Taste Cells In Vivo.

The sense of taste is achieved by cooperation of many signaling molecules expressed in taste cells, which code and transmit information on quality and intensity of taste to the nervous system. Viral vector-mediated gene transfer techniques have been proven to be useful to study and control function of a gene product in vivo However, there is no transduction method for taste cells in live animals. Here, we have established a method for inducing foreign gene expression in mouse taste cells in vivo by recombinant adeno-associated virus (AAV) vector. First, using enhanced green fluorescent protein (EGFP) as a reporter, we screened 6 AAV serotypes along with a recombinant lentivirus vector for their ability to transduce taste cells. One week after viral injection into the submucosa of the tongue, EGFP expression in fungiform taste cells was observed only in animals injected with AAV-DJ, a synthetic serotype. Next, time course of AAV-DJ-mediated EGFP expression in fungiform taste cells was evaluated. Intragemmal EGFP signals appeared after a delay, rapidly increased until 7 days postinjection, and gradually decreased over the next few weeks probably because of the cell turnover. Finally, the taste cell types susceptible to AAV-DJ transduction were characterized. EGFP expression was observed in PLCß2-immunoreactive type II and aromatic l-amino acid decarboxylase (AADC)-immunoreactive type III taste cells as well as in cells immunonegative for both PLCß2 and AADC, demonstrating that AAV-DJ does not discriminate functional taste cell types. In conclusion, the method established in this study will be a promising tool to study the mechanism of taste.

Actions of Quercetin, a Polyphenol, on Blood Pressure.

Disorder of blood pressure control causes serious diseases in the cardiovascular system. This review focuses on the anti-hypertensive action of quercetin, a flavonoid, which is one of the polyphenols characterized as the compounds containing large multiples of phenol structural units, by varying the values of various blood pressure regulatory factors, such as vascular compliance, peripheral vascular resistance, and total blood volume via anti-inflammatory and anti-oxidant actions. In addition to the anti-inflammatory and anti-oxidant actions of quercetin, we especially describe a novel mechanism of quercetin's action on the cytosolic Cl- concentration ([Cl-]c) and novel roles of the cytosolic Cl- i.e.: (1) quercetin elevates [Cl-]c by activating Na⁺-K⁺-2Cl- cotransporter 1 (NKCC1) in renal epithelial cells contributing to Na⁺ reabsorption via the epithelial Na⁺ channel (ENaC); (2) the quercetin-induced elevation of [Cl-]c in renal epithelial cells diminishes expression of ENaC leading to a decrease in renal Na⁺ reabsorption; and (3) this reduction of ENaC-mediated Na⁺ reabsorption in renal epithelial cells drops volume-dependent elevated blood pressure. In this review, we introduce novel, unique mechanisms of quercetin's anti-hypertensive action via activation of NKCC1 in detail.

Calcium homeostasis modulator (CALHM) ion channels.

Calcium homeostasis modulator 1 (CALHM1), formerly known as FAM26C, was recently identified as a physiologically important plasma membrane ion channel. CALHM1 and its Caenorhabditis elegans homolog, CLHM-1, are regulated by membrane voltage and extracellular Ca(2+) concentration ([Ca(2+)]o). In the presence of physiological [Ca(2+)]o (∼1.5 mM), CALHM1 and CLHM-1 are closed at resting membrane potentials but can be opened by strong depolarizations. Reducing [Ca(2+)]o increases channel open probability, enabling channel activation at negative membrane potentials. Together, voltage and Ca(2+) o allosterically regulate CALHM channel gating. Through convergent evolution, CALHM has structural features that are reminiscent of connexins and pannexins/innexins/LRRC8 (volume-regulated anion channel (VRAC)) gene families, including four transmembrane helices with cytoplasmic amino and carboxyl termini. A CALHM1 channel is a hexamer of CALHM1 monomers with a functional pore diameter of ∼14 Å. CALHM channels discriminate poorly among cations and anions, with signaling molecules including Ca(2+) and ATP able to permeate through its pore. CALHM1 is expressed in the brain where it plays an important role in cortical neuron excitability induced by low [Ca(2+)]o and in type II taste bud cells in the tongue that sense sweet, bitter, and umami tastes where it functions as an essential ATP release channel to mediate nonsynaptic neurotransmitter release. CLHM-1 is expressed in C. elegans sensory neurons and body wall muscles, and its genetic deletion causes locomotion defects. Thus, CALHM is a voltage- and Ca(2+) o-gated ion channel, permeable to large cations and anions, that plays important roles in physiology.

How do taste cells lacking synapses mediate neurotransmission? CALHM1, a voltage-gated ATP channel.

CALHM1 was recently demonstrated to be a voltage-gated ATP-permeable ion channel and to serve as a bona fide conduit for ATP release from sweet-, umami-, and bitter-sensing type II taste cells. Calhm1 is expressed in taste buds exclusively in type II cells and its product has structural and functional similarities with connexins and pannexins, two families of channel protein candidates for ATP release by type II cells. Calhm1 knockout in mice leads to loss of perception of sweet, umami, and bitter compounds and to impaired gustatory nerve responses to these tastants. These new studies validate the concept of ATP as the primary neurotransmitter from type II cells to gustatory neurons. Furthermore, they identify voltage-gated ATP release through CALHM1 as an essential molecular mechanism of ATP release in taste buds. We discuss these new findings, as well as unresolved issues in peripheral taste signaling that we hope will stimulate future research.

Shared gustatory sensor for acids and ammonium.

In a recent study, Liang, Wilson, and colleagues demonstrated that the H+-selective ion channel OTOP1, responsible for sour taste transduction, also functions as a gustatory sensor for ammonium in mice. Additionally, this research revealed a novel mode of channel activation by intracellular alkalinization, which is conserved across vertebrate species.

Liver lipophagy ameliorates nonalcoholic steatohepatitis through extracellular lipid secretion.

Nonalcoholic steatohepatitis (NASH) is a progressive disorder with aberrant lipid accumulation and subsequent inflammatory and profibrotic response. Therapeutic efforts at lipid reduction via increasing cytoplasmic lipolysis unfortunately worsens hepatitis due to toxicity of liberated fatty acid. An alternative approach could be lipid reduction through autophagic disposal, i.e., lipophagy. We engineered a synthetic adaptor protein to induce lipophagy, combining a lipid droplet-targeting signal with optimized LC3-interacting domain. Activating hepatocyte lipophagy in vivo strongly mitigated both steatosis and hepatitis in a diet-induced mouse NASH model. Mechanistically, activated lipophagy promoted the excretion of lipid from hepatocytes, thereby suppressing harmful intracellular accumulation of nonesterified fatty acid. A high-content compound screen identified alpelisib and digoxin, clinically-approved compounds, as effective activators of lipophagy. Administration of alpelisib or digoxin in vivo strongly inhibited the transition to steatohepatitis. These data thus identify lipophagy as a promising therapeutic approach to prevent NASH progression.

A light-induced small G-protein gem limits the circadian clock phase-shift magnitude by inhibiting voltage-dependent calcium channels.

Calcium signaling is pivotal to the circadian clockwork in the suprachiasmatic nucleus (SCN), particularly in rhythm entrainment to environmental light-dark cycles. Here, we show that a small G-protein Gem, an endogenous inhibitor of high-voltage-activated voltage-dependent calcium channels (VDCCs), is rapidly induced by light in SCN neurons via the calcium (Ca2+)-mediated CREB/CRE transcriptional pathway. Gem attenuates light-induced calcium signaling through its interaction with VDCCs. The phase-shift magnitude of locomotor activity rhythms by light, at night, increases in Gem-deficient (Gem-/-) mice. Similarly, in SCN slices from Gem-/- mice, depolarizing stimuli induce larger phase shifts of clock gene transcription rhythms that are normalized by the application of an L-type VDCC blocker, nifedipine. Voltage-clamp recordings from SCN neurons reveal that Ca2+ currents through L-type channels increase in Gem-/- mice. Our findings suggest that transcriptionally activated Gem feeds back to suppress excessive light-evoked L-type VDCC activation, adjusting the light-induced phase-shift magnitude to an appropriate level in mammals.

Regulation of epithelial sodium transport via epithelial Na+ channel.

Renal epithelial Na+ transport plays an important role in homeostasis of our body fluid content and blood pressure. Further, the Na+ transport in alveolar epithelial cells essentially controls the amount of alveolar fluid that should be kept at an appropriate level for normal gas exchange. The epithelial Na+ transport is generally mediated through two steps: (1) the entry step of Na+ via epithelial Na+ channel (ENaC) at the apical membrane and (2) the extrusion step of Na+ via the Na+, K+-ATPase at the basolateral membrane. In general, the Na+ entry via ENaC is the rate-limiting step. Therefore, the regulation of ENaC plays an essential role in control of blood pressure and normal gas exchange. In this paper, we discuss two major factors in ENaC regulation: (1) activity of individual ENaC and (2) number of ENaC located at the apical membrane.

Analysis of blocker-labeled channels reveals the dependence of recycling rates of ENaC on the total amount of recycled channels.

Trafficking is one of the primary mechanisms of epithelial Na(+) channel (ENaC) regulation. Although it is known that ENaCs are recycled between the apical membrane and the intracellular channel pool, it has been difficult to investigate the recycling of ENaCs; especially endogenously expressed ENaCs. The aim of the present study is to investigate if the recycling rates of ENaCs depend on the total amount of recycled ENaCs. To accomplish this point, we established a novel method to estimate the total amount of recycled ENaCs and the ENaC recycling rates by using a specific blocker (benzamil) of ENaC with a high-affinity for functional label of the channels in recycling. Applying this method, we studied if a decrease in the total amount of ENaCs caused by brefeldin A (5 µg/mL, 1 h) affects respectively the rates of insertion and endocytosis of ENaCs to and from the apical membrane in monolayers of renal epithelial A6 cells. Our observations indicate that: 1) both insertion and endocytosis rates of ENaC increase when the total amount of ENaCs decreases, 2) the increase in the insertion rate is larger than that in the endocytosis rate, and 3) this larger increase in the insertion rate than the endocytosis rate caused by the decrease in the total amount of ENaCs plays an important role in preventing Na(+) transport from drastically diminishing due to a decrease in the total amount of ENaCs. The newly established analysis of blocker-labeled ENaCs in the present study provides a useful tool to investigate the recycling of endogenously expressed ENaCs.

All-Electrical Ca2+-Independent Signal Transduction Mediates Attractive Sodium Taste in Taste Buds.

Sodium taste regulates salt intake. The amiloride-sensitive epithelial sodium channel (ENaC) is the Na+ sensor in taste cells mediating attraction to sodium salts. However, cells and intracellular signaling underlying sodium taste in taste buds remain long-standing enigmas. Here, we show that a subset of taste cells with ENaC activity fire action potentials in response to ENaC-mediated Na+ influx without changing the intracellular Ca2+ concentration and form a channel synapse with afferent neurons involving the voltage-gated neurotransmitter-release channel composed of calcium homeostasis modulator 1 (CALHM1) and CALHM3 (CALHM1/3). Genetic elimination of ENaC in CALHM1-expressing cells as well as global CALHM3 deletion abolished amiloride-sensitive neural responses and attenuated behavioral attraction to NaCl. Together, sodium taste is mediated by cells expressing ENaC and CALHM1/3, where oral Na+ entry elicits suprathreshold depolarization for action potentials driving voltage-dependent neurotransmission via the channel synapse. Thus, all steps in sodium taste signaling are voltage driven and independent of Ca2+ signals. This work also reveals ENaC-independent salt attraction.

Cryo-EM structures of calcium homeostasis modulator channels in diverse oligomeric assemblies.

Calcium homeostasis modulator (CALHM) family proteins are Ca2+-regulated adenosine triphosphate (ATP)-release channels involved in neural functions including neurotransmission in gustation. Here, we present the cryo-electron microscopy (EM) structures of killifish CALHM1, human CALHM2, and Caenorhabditis elegans CLHM-1 at resolutions of 2.66, 3.4, and 3.6 Å, respectively. The CALHM1 octamer structure reveals that the N-terminal helix forms the constriction site at the channel pore in the open state and modulates the ATP conductance. The CALHM2 undecamer and CLHM-1 nonamer structures show the different oligomeric stoichiometries among CALHM homologs. We further report the cryo-EM structures of the chimeric construct, revealing that the intersubunit interactions at the transmembrane domain (TMD) and the TMD-intracellular domain linker define the oligomeric stoichiometry. These findings advance our understanding of the ATP conduction and oligomerization mechanisms of CALHM channels.