Kidney-Specific CAP1/Prss8-Deficient Mice Maintain ENaC-Mediated Sodium Balance through an Aldosterone Independent Pathway.

The serine protease prostasin (CAP1/Prss8, channel-activating protease-1) is a confirmed in vitro and in vivo activator of the epithelial sodium channel ENaC. To test whether proteolytic activity or CAP1/Prss8 abundance itself are required for ENaC activation in the kidney, we studied animals either hetero- or homozygous mutant at serine 238 (S238A; Prss8cat/+ and Prss8cat/cat), and renal tubule-specific CAP1/Prss8 knockout (Prss8PaxLC1) mice. When exposed to varying Na+-containing diets, no changes in Na+ and K+ handling and only minor changes in the expression of Na+ and K+ transporting protein were found in both models. Similarly, the α- or γENaC subunit cleavage pattern did not differ from control mice. On standard and low Na+ diet, Prss8cat/+ and Prss8cat/cat mice exhibited standard plasma aldosterone levels and unchanged amiloride-sensitive rectal potential difference indicating adapted ENaC activity. Upon Na+ deprivation, mice lacking the renal CAP1/Prss8 expression (Prss8PaxLC1) exhibit significantly decreased plasma aldosterone and lower K+ levels but compensate by showing significantly higher plasma renin activity. Our data clearly demonstrated that the catalytic activity of CAP1/Prss8 is dispensable for proteolytic ENaC activation. CAP1/Prss8-deficiency uncoupled ENaC activation from its aldosterone dependence, but Na+ homeostasis is maintained through alternative pathways.

A molecular view of the function and pharmacology of acid-sensing ion channels.

The pH in the different tissues and organs of our body is kept within tight limits. Local pH changes occur, however, temporarily under physiological conditions, as for example in synapses during neuronal activity. In pathological situations, such as in ischemia, inflammation, and tumor growth, long-lasting acidification develops. Acid-sensing ion channels (ASICs) are low pH-activated Na+-permeable ion channels that are widely expressed in the central and peripheral nervous systems. ASICs act as pH sensors, leading to neuronal excitation when the pH drops. Animal studies have shown that ASICs are involved in several physiological and pathological processes, such as pain sensation, learning, fear sensing, and neurodegeneration after ischemic stroke. ASIC inhibitors could be used as analgesic and anxiolytic drugs, and as drugs for the treatment of ischemic stroke. For these reasons, ASICs have recently attracted increasing attention. Currently, no drugs are clinically used as ASIC modulators. ASICs are however targets of several peptide toxins from animals. Much effort is invested in research studying the function of these channels. We review here the available pharmacological agents acting on ASICs, which include small molecules and animal toxins. We then discuss the current understanding of the molecular mechanisms by which pH controls ASIC activity. Knowledge of the function of ASICs at the molecular level should allow the development of new pharmacological strategies for targeting these promising ion channels.

Conformational dynamics and role of the acidic pocket in ASIC pH-dependent gating.

Acid-sensing ion channels (ASICs) are proton-activated Na+ channels expressed in the nervous system, where they are involved in learning, fear behaviors, neurodegeneration, and pain sensation. In this work, we study the role in pH sensing of two regions of the ectodomain enriched in acidic residues: the acidic pocket, which faces the outside of the protein and is the binding site of several animal toxins, and the palm, a central channel domain. Using voltage clamp fluorometry, we find that the acidic pocket undergoes conformational changes during both activation and desensitization. Concurrently, we find that, although proton sensing in the acidic pocket is not required for channel function, it does contribute to both activation and desensitization. Furthermore, protonation-mimicking mutations of acidic residues in the palm induce a dramatic acceleration of desensitization followed by the appearance of a sustained current. In summary, this work describes the roles of potential pH sensors in two extracellular domains, and it proposes a model of acidification-induced conformational changes occurring in the acidic pocket of ASIC1a.

GLUTAMATE RECEPTOR-LIKE genes mediate leaf-to-leaf wound signalling.

Wounded leaves communicate their damage status to one another through a poorly understood process of long-distance signalling. This stimulates the distal production of jasmonates, potent regulators of defence responses. Using non-invasive electrodes we mapped surface potential changes in Arabidopsis thaliana after wounding leaf eight and found that membrane depolarizations correlated with jasmonate signalling domains in undamaged leaves. Furthermore, current injection elicited jasmonoyl-isoleucine accumulation, resulting in a transcriptome enriched in RNAs encoding key jasmonate signalling regulators. From among 34 screened membrane protein mutant lines, mutations in several clade 3 GLUTAMATE RECEPTOR-LIKE genes (GLRs 3.2, 3.3 and 3.6) attenuated wound-induced surface potential changes. Jasmonate-response gene expression in leaves distal to wounds was reduced in a glr3.3 glr3.6 double mutant. This work provides a genetic basis for investigating mechanisms of long-distance wound signalling in plants and indicates that plant genes related to those important for synaptic activity in animals function in organ-to-organ wound signalling.

International Union of Basic and Clinical Pharmacology. XCI. structure, function, and pharmacology of acid-sensing ion channels and the epithelial Na+ channel.

The epithelial Na(+) channel (ENaC) and the acid-sensing ion channels (ASICs) form subfamilies within the ENaC/degenerin family of Na(+) channels. ENaC mediates transepithelial Na(+) transport, thereby contributing to Na(+) homeostasis and the maintenance of blood pressure and the airway surface liquid level. ASICs are H(+)-activated channels found in central and peripheral neurons, where their activation induces neuronal depolarization. ASICs are involved in pain sensation, the expression of fear, and neurodegeneration after ischemia, making them potentially interesting drug targets. This review summarizes the biophysical properties, cellular functions, and physiologic and pathologic roles of the ASIC and ENaC subfamilies. The analysis of the homologies between ENaC and ASICs and the relation between functional and structural information shows many parallels between these channels, suggesting that some mechanisms that control channel activity are shared between ASICs and ENaC. The available crystal structures and the discovery of animal toxins acting on ASICs provide a unique opportunity to address the molecular mechanisms of ENaC and ASIC function to identify novel strategies for the modulation of these channels by pharmacologic ligands.

Heterotrimeric Go protein links Wnt-Frizzled signaling with ankyrins to regulate the neuronal microtubule cytoskeleton.

Drosophila neuromuscular junctions (NMJs) represent a powerful model system with which to study glutamatergic synapse formation and remodeling. Several proteins have been implicated in these processes, including components of canonical Wingless (Drosophila Wnt1) signaling and the giant isoforms of the membrane-cytoskeleton linker Ankyrin 2, but possible interconnections and cooperation between these proteins were unknown. Here, we demonstrate that the heterotrimeric G protein Go functions as a transducer of Wingless-Frizzled 2 signaling in the synapse. We identify Ankyrin 2 as a target of Go signaling required for NMJ formation. Moreover, the Go-ankyrin interaction is conserved in the mammalian neurite outgrowth pathway. Without ankyrins, a major switch in the Go-induced neuronal cytoskeleton program is observed, from microtubule-dependent neurite outgrowth to actin-dependent lamellopodial induction. These findings describe a novel mechanism regulating the microtubule cytoskeleton in the nervous system. Our work in Drosophila and mammalian cells suggests that this mechanism might be generally applicable in nervous system development and function.

Extracellular Subunit Interactions Control Transitions between Functional States of Acid-sensing Ion Channel 1a.

Acid-sensing ion channels (ASICs) are neuronal, voltage-independent Na(+) channels that are transiently activated by extracellular acidification. They are involved in pain sensation, the expression of fear, and in neurodegeneration after ischemic stroke. Our study investigates the role of extracellular subunit interactions in ASIC1a function. We identified two regions involved in critical intersubunit interactions. First, formation of an engineered disulfide bond between the palm and thumb domains leads to partial channel closure. Second, linking Glu-235 of a finger loop to either one of two different residues of the knuckle of a neighboring subunit opens the channel at physiological pH or disrupts its activity. This suggests that one finger-knuckle disulfide bond (E235C/K393C) sets the channel in an open state, whereas the other (E235C/Y389C) switches the channel to a non-conducting state. Voltage-clamp fluorometry experiments indicate that both the finger loop and the knuckle move away from the ß-ball residue Trp-233 during acidification and subsequent desensitization. Together, these observations reveal that ASIC1a opening is accompanied by a distance increase between adjacent thumb and palm domains as well as a movement of Glu-235 relative to the knuckle helix. Our study identifies subunit interactions in the extracellular loop and shows that dynamic changes of these interactions are critical for normal ASIC function.

Acid-sensing ion channel 1a drives AMPA receptor plasticity following ischaemia and acidosis in hippocampal CA1 neurons.

KEY POINTS: The hippocampal CA1 region is highly vulnerable to ischaemic stroke. Two forms of AMPA receptor (AMPAR) plasticity - an anoxic form of long-term potentiation and a delayed increase in Ca(2+) -permeable (CP) AMPARs - contribute to this susceptibility by increasing excitotoxicity. In CA1, the acid-sensing ion channel 1a (ASIC1a) is known to facilitate LTP and contribute to ischaemic acidotoxicity. We have examined the role of ASIC1a in AMPAR ischaemic plasticity in organotypic hippocampal slice cultures exposed to oxygen glucose deprivation (a model of ischaemic stroke), and in hippocampal pyramidal neuron cultures exposed to acidosis. We find that ASIC1a activation promotes both forms of AMPAR plasticity and that neuroprotection, by inhibiting ASIC1a, circumvents any further benefit of blocking CP-AMPARs. Our observations establish a new interaction between acidotoxicity and excitotoxicity, and provide insight into the role of ASIC1a and CP-AMPARs in neurodegeneration. Specifically, we propose that ASIC1a activation drives certain post-ischaemic forms of CP-AMPAR plasticity. ABSTRACT: The CA1 region of the hippocampus is particularly vulnerable to ischaemic damage. While NMDA receptors play a major role in excitotoxicity, it is thought to be exacerbated in this region by two forms of post-ischaemic AMPA receptor (AMPAR) plasticity - namely, anoxic long-term potentiation (a-LTP), and a delayed increase in the prevalence of Ca(2+) -permeable GluA2-lacking AMPARs (CP-AMPARs). The acid-sensing ion channel 1a (ASIC1a), which is expressed in CA1 pyramidal neurons, is also known to contribute to post-ischaemic neuronal death and to physiologically induced LTP. This raises the question does ASIC1a activation drive the post-ischaemic forms of AMPAR plasticity in CA1 pyramidal neurons? We have tested this by examining organotypic hippocampal slice cultures (OHSCs) exposed to oxygen glucose deprivation (OGD), and dissociated cultures of hippocampal pyramidal neurons (HPNs) exposed to low pH (acidosis). We find that both a-LTP and the delayed increase in the prevalence of CP-AMPARs are dependent on ASIC1a activation during ischaemia. Indeed, acidosis alone is sufficient to induce the increase in CP-AMPARs. We also find that inhibition of ASIC1a channels circumvents any potential neuroprotective benefit arising from block of CP-AMPARs. By demonstrating that ASIC1a activation contributes to post-ischaemic AMPAR plasticity, our results identify a functional interaction between acidotoxicity and excitotoxicity in hippocampal CA1 cells, and provide insight into the role of ASIC1a and CP-AMPARs as potential drug targets for neuroprotection. We thus propose that ASIC1a activation can drive certain forms of CP-AMPAR plasticity, and that inhibiting ASIC1a affords neuroprotection.

Calcium entry via TRPV1 but not ASICs induces neuropeptide release from sensory neurons.

Inflammatory mediators induce neuropeptide release from nociceptive nerve endings and cell bodies, causing increased local blood flow and vascular leakage resulting in edema. Neuropeptide release from sensory neurons depends on an increase in intracellular Ca(2+) concentration. In this study we investigated the role of two types of pH sensors in acid-induced Ca(2+) entry and neuropeptide release from dorsal root ganglion (DRG) neurons. The transient receptor potential vanilloid 1 channel (TRPV1) and acid-sensing ion channels (ASICs) are both H(+)-activated ion channels present in these neurons, and are therefore potential pH sensors for this process. We demonstrate with in situ hybridization and immunocytochemistry that TRPV1 and several ASIC subunits are co-expressed with neuropeptides in DRG neurons. The activation of ASICs and of TRPV1 led to an increase in intracellular Ca(2+) concentration. While TRPV1 has a high Ca(2+) permeability and allows direct Ca(2+) entry when activated, we show here that ASICs of DRG neurons mediate Ca(2+) entry mostly by depolarization-induced activation of voltage-gated Ca(2+) channels and only to a small extent via the pore of Ca(2+)-permeable ASICs. Extracellular acidification led to the release of the neuropeptide calcitonin gene-related peptide from DRG neurons. The pH dependence and the pharmacological profile indicated that TRPV1, but not ASICs, induced neuropeptide secretion. In conclusion, this study shows that although both TRPV1 and ASICs mediate Ca(2+) influx, TRPV1 is the principal sensor for acid-induced neuropeptide secretion from sensory neurons.

Molecular determinants of desensitization in an ENaC/degenerin channel.

Epithelial Na(+) channel (ENaC)/degenerin family members are involved in mechanosensation, blood pressure control, pain sensation, and the expression of fear. Several of these channel types display a form of desensitization that allows the channel to limit Na(+) influx during prolonged stimulation. We used site-directed mutagenesis and chemical modification, functional analysis, and molecular dynamics simulations to investigate the role of the lower palm domain of the acid-sensing ion channel 1, a member of the ENaC/degenerin family. The lower palm domains of this trimeric channel are arranged around a central vestibule, at ∼20 Šabove the plasma membrane and are covalently linked to the transmembrane channel parts. We show that the lower palm domains approach one another during desensitization. Residues in the palm co-determine the pH dependence of desensitization, its kinetics, and the stability of the desensitized state. Mutations of palm residues impair desensitization by preventing the closing movement of the palm. Overexpression of desensitization-impaired channel mutants in central neurons allowed--in contrast to overexpression of wild type--a sustained signaling response to rapid pH fluctuations. We identify and describe here the function of an important regulatory domain that most likely has a conserved role in ENaC/degenerin channels.

Identification of the modulatory Ca2+-binding sites of acid-sensing ion channel 1a.

Acid-sensing ion channels (ASICs) are neuronal Na+-permeable ion channels activated by extracellular acidification. ASICs are involved in learning, fear sensing, pain sensation and neurodegeneration. Increasing the extracellular Ca2+ concentration decreases the H+ sensitivity of ASIC1a, suggesting a competition for binding sites between H+ and Ca2+ ions. Here, we predicted candidate residues for Ca2+ binding on ASIC1a, based on available structural information and our molecular dynamics simulations. With functional measurements, we identified several residues in cavities previously associated with pH-dependent gating, whose mutation reduced the modulation by extracellular Ca2+ of the ASIC1a pH dependence of activation and desensitization. This occurred likely owing to a disruption of Ca2+ binding. Our results link one of the two predicted Ca2+-binding sites in each ASIC1a acidic pocket to the modulation of channel activation. Mg2+ regulates ASICs in a similar way as does Ca2+. We show that Mg2+ shares some of the binding sites with Ca2+. Finally, we provide evidence that some of the ASIC1a Ca2+-binding sites are functionally conserved in the splice variant ASIC1b. Our identification of divalent cation-binding sites in ASIC1a shows how Ca2+ affects ASIC1a gating, elucidating a regulatory mechanism present in many ion channels.

Subtype-specific modulation of acid-sensing ion channel (ASIC) function by 2-guanidine-4-methylquinazoline.

Acid-sensing ion channels (ASICs) are neuronal Na(+)-selective channels that are transiently activated by extracellular acidification. ASICs are involved in fear and anxiety, learning, neurodegeneration after ischemic stroke, and pain sensation. The small molecule 2-guanidine-4-methylquinazoline (GMQ) was recently shown to open ASIC3 at physiological pH. We have investigated the mechanisms underlying this effect and the possibility that GMQ may alter the function of other ASICs besides ASIC3. GMQ shifts the pH dependence of activation to more acidic pH in ASIC1a and ASIC1b, whereas in ASIC3 this shift goes in the opposite direction and is accompanied by a decrease in its steepness. GMQ also induces an acidic shift of the pH dependence of inactivation of ASIC1a, -1b, -2a, and -3. As a consequence, the activation and inactivation curves of ASIC3 but not other ASICs overlap in the presence of GMQ at pH 7.4, thereby creating a window current. At concentrations >1 mM, GMQ decreases maximal peak currents by reducing the unitary current amplitude. Mutation of residue Glu-79 in the palm domain of ASIC3, previously shown to be critical for channel opening by GMQ, disrupted the GMQ effects on inactivation but not activation. This suggests that this residue is involved in the consequences of GMQ binding rather than in the binding interaction itself. This study describes the mechanisms underlying the effects of a novel class of ligands that modulate the function of all ASICs as well as activate ASIC3 at physiological pH.

Identification of the SPLUNC1 ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airway epithelial cultures.

The epithelial sodium channel (ENaC) is responsible for Na(+) and fluid absorption across colon, kidney, and airway epithelia. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is a secreted, innate defense protein and an autocrine inhibitor of ENaC that is highly expressed in airway epithelia. While SPLUNC1 has a bactericidal permeability-increasing protein (BPI)-type structure, its NH2-terminal region lacks structure. Here we found that an 18 amino acid peptide, S18, which corresponded to residues G22-A39 of the SPLUNC1 NH2 terminus inhibited ENaC activity to a similar degree as full-length SPLUNC1 (∼2.5 fold), while SPLUNC1 protein lacking this region was without effect. S18 did not inhibit the structurally related acid-sensing ion channels, indicating specificity for ENaC. However, S18 preferentially bound to the ßENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Unlike control, CF human bronchial epithelial cultures (HBECs) where airway surface liquid (ASL) height was abnormally low (4.2 ± 0.6 µm), addition of S18 prevented ENaC-led ASL hyperabsorption and maintained CF ASL height at 7.9 ± 0.6 µm, even in the presence of neutrophil elastase, which is comparable to heights seen in normal HBECs. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, suggesting that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the S18 peptide suggests that this peptide may be suitable for treating CF lung disease.

Identification of SPLUNC1's ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airways.

The epithelial sodium channel (ENaC) is responsible for Na+ and fluid absorption across colon, kidney, and airway epithelia. We have previously identified SPLUNC1 as an autocrine inhibitor of ENaC. We have now located the ENaC inhibitory domain of SPLUNC1 to SPLUNC1's N terminus, and a peptide corresponding to this domain, G22-A39, inhibited ENaC activity to a similar degree as full-length SPLUNC1 (∼2.5 fold). However, G22-A39 had no effect on the structurally related acid-sensing ion channels, indicating specificity for ENaC. G22-A39 preferentially bound to the ß-ENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Addition of G22-A39 to CF human bronchial epithelial cultures (HBECs) resulted in an increase in airway surface liquid height from 4.2±0.6 to 7.9±0.6 µm, comparable to heights seen in normal HBECs, even in the presence of neutrophil elastase. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, which suggests that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the G22-A39 peptide suggests that this peptide may be suitable for treating CF lung disease.

ASIC1a affects hypothalamic signaling and regulates the daily rhythm of body temperature in mice.

The body temperature of mice is higher at night than during the day. We show here that global deletion of acid-sensing ion channel 1a (ASIC1a) results in lower body temperature during a part of the night. ASICs are pH sensors that modulate neuronal activity. The deletion of ASIC1a decreased the voluntary activity at night of mice that had access to a running wheel but did not affect their spontaneous activity. Daily rhythms of thyrotropin-releasing hormone mRNA in the hypothalamus and of thyroid-stimulating hormone ß mRNA in the pituitary, and of prolactin mRNA in the hypothalamus and pituitary were suppressed in ASIC1a-/- mice. The serum thyroid hormone levels were however not significantly changed by ASIC1a deletion. Our findings indicate that ASIC1a regulates activity and signaling in the hypothalamus and pituitary. This likely leads to the observed changes in body temperature by affecting the metabolism or energy expenditure.

The Concise Guide to PHARMACOLOGY 2023/24: Ion channels.

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.

Probing conformational changes during activation of ASIC1a by an optical tweezer and by methanethiosulfonate-based cross-linkers.

Acid-sensing ion channels (ASICs) are neuronal, proton-gated, Na+-selective ion channels. They are involved in various physiological and pathological processes such as neurodegeneration after stroke, pain sensation, fear behavior and learning. To obtain information on the activation mechanism of ASIC1a, we attempted in this study to impose distance constraints between paired residues in different channel domains by using cross-linkers reacting with engineered Cys residues, and we measured how this affected channel function. First, the optical tweezer 4'-Bis(maleimido)azobenzene (BMA) was used, whose conformation changes depending on the wavelength of applied light. After exposure of channel mutants to BMA, an activation of the channel by light was only observed with a mutant containing a Cys mutation in the extracellular pore entry, I428C. Western blot analysis indicated that BMA did not cross-link Cys428 residues. Extracellular application of methanethiosulfonate (MTS) cross-linkers of different lengths changed the properties of several Cys mutants, in many cases likely without cross-linking two Cys residues. Our observations suggest that intersubunit cross-linking occurred in the wrist mutant A425C and intrasubunit cross-linking in the acidic pocket mutant D237C/I312C. In these mutants, exposure to cross-linkers favored a non-conducting channel conformation and induced an acidic shift of the pH dependence and a decrease of the maximal current amplitude. Overall, the cross-linking approaches appeared to be inefficient, possibly due to the geometrical requirements for successful reactions of the two ends of the cross-linking compound.

Galvani Offset Potential and Constant-pH Simulations of Membrane Proteins.

A central problem in computational biophysics is the treatment of titratable residues in molecular dynamics simulations of large biological macromolecular systems. Conventional simulation methods ascribe a fixed ionization state to titratable residues in accordance with their pKa and the pH of the system, assuming that an effective average model will be able to capture the predominant behavior of the system. While this assumption may be justifiable in many cases, it is certainly limited, and it is important to design alternative methodologies allowing a more realistic treatment. Constant-pH simulation methods provide powerful approaches to handle titratable residues more realistically by allowing the ionization state to vary statistically during the simulation. Extending the molecular mechanical (MM) potential energy function to a family of potential functions accounting for different ionization states, constant-pH simulations are designed to sample all accessible configurations and ionization states, properly weighted according to their Boltzmann factor. Because protonation and deprotonation events correspond to a change in the total charge, difficulties arise when the long-range Coulomb interaction is treated on the basis of an idealized infinite simulation model and periodic boundary conditions with particle-mesh Ewald lattice sums. Charging free-energy calculations performed under these conditions in aqueous solution depend on the Galvani potential of the bulk water phase. This has important implications for the equilibrium and nonequilibrium constant-pH simulation methods grounded in the relative free-energy difference corresponding to the protonated and unprotonated residues. Here, the effect of the Galvani potential is clarified, and a simple practical solution is introduced to address this issue in constant-pH simulations of the acid-sensing ion channel (ASIC).

Calcium regulates acid-sensing ion channel 3 activation by competing with protons in the channel pore and at an allosteric binding site.

The extracellular Ca2+ concentration changes locally under certain physiological and pathological conditions. Such variations affect the function of ion channels of the nervous system and consequently also neuronal signalling. We investigated here the mechanisms by which Ca2+ controls the activity of acid-sensing ion channel (ASIC) 3. ASICs are neuronal, H+-gated Na+ channels involved in several physiological and pathological processes, including the expression of fear, learning, pain sensation and neurodegeneration after ischaemic stroke. It was previously shown that Ca2+ negatively modulates the ASIC pH dependence. While protons are default activators of ASIC3, this channel can also be activated at pH7.4 by the removal of the extracellular Ca2+. Two previous studies concluded that low pH opens ASIC3 by displacing Ca2+ ions that block the channel pore at physiological pH. We show here that an acidic residue, distant from the pore, together with pore residues, controls the modulation of ASIC3 by Ca2+. Our study identifies a new regulatory site in ASIC3 and demonstrates that ASIC3 activation involves an allosteric mechanism together with Ca2+ unbinding from the channel pore. We provide a molecular analysis of a regulatory mechanism found in many ion channels.

The contact region between three domains of the extracellular loop of ASIC1a is critical for channel function.

Acid-sensing ion channels are members of the epithelial Na(+) channel/degenerin family. They are neuronal nonvoltage-gated Na(+) channels that are activated by extracellular acidification. In this study, we investigated the role of a highly conserved region of the extracellular part of ASIC1a that forms the contact between the finger domain, the adjacent beta-ball, and the upper palm domain in ASIC1a. The finger domain contributes to the pH-dependent gating and is linked via this contact zone to the rest of the protein. We found that mutation to Cys of residues in this region led to decreased channel expression and current amplitudes. Exposure of the engineered Cys residues to Cd(2+) or to charged methane thiosulfonate sulfhydryl reagents further reduced current amplitudes. This current inhibition was not due to changes in acid-sensing ion channel pH dependence or unitary conductance and was likely due to a decrease of the probability of channel opening. For some mutants, the effect of sulfhydryl reagents depended on the pH of exposure in the range 7.4 to 6.8, suggesting that this zone undergoes conformational changes during inactivation. Our study identifies a region in ASIC1a whose integrity is required for normal channel function.