Na+ Inhibits the Epithelial Na+ Channel by Binding to a Site in an Extracellular Acidic Cleft

The epithelial Na⁺ channel (ENaC) has a key role in the regulation of extracellular fluid volume and blood pressure. ENaC belongs to a family of ion channels that sense the external environment. These channels have large extracellular regions that are thought to interact with environmental cues, such as Na⁺, Cl⁻, protons, proteases, and shear stress, which modulate gating behavior. We sought to determine the molecular mechanism by which ENaC senses high external Na⁺ concentrations, resulting in an inhibition of channel activity. Both our structural model of an ENaC α subunit and the resolved structure of an acid-sensing ion channel (ASIC1) have conserved acidic pockets in the periphery of the extracellular region of the channel. We hypothesized that these acidic pockets host inhibitory allosteric Na⁺ binding sites. Through site-directed mutagenesis targeting the acidic pocket, we modified the inhibitory response to external Na⁺. Mutations at selected sites altered the cation inhibitory preference to favor Li⁺ or K⁺ rather than Na⁺. Channel activity was reduced in response to restraining movement within this region by cross-linking structures across the acidic pocket. Our results suggest that residues within the acidic pocket form an allosteric effector binding site for Na⁺. Our study supports the hypothesis that an acidic cleft is a key ligand binding locus for ENaC and perhaps other members of the ENaC/degenerin family.     

Allosteric Inhibition of the Epithelial Na+ Channel through Peptide Binding at Peripheral Finger and Thumb Domains

The epithelial Na⁺ channel (ENaC) mediates the rate-limiting step in transepithelial Na⁺ transport in the distal segments of the nephron and in the lung. ENaC subunits are cleaved by proteases, resulting in channel activation due to the release of inhibitory tracts. Peptides derived from these tracts inhibit channel activity. The mechanism by which these intrinsic inhibitory tracts reduce channel activity is unknown, as are the sites where these tracts interact with other residues within the channel. We performed site-directed mutagenesis in large portions of the predicted periphery of the extracellular region of the α subunit and measured the effect of mutations on an 8-residue inhibitory tract-derived peptide. Our data show that the inhibitory peptide likely binds to specific residues within the finger and thumb domains of ENaC. Pairwise interactions between the peptide and the channel were identified by double mutant cycle experiments. Our data suggest that the inhibitory peptide has a specific peptide orientation within its binding site. Extended to the intrinsic inhibitory tract, our data suggest that proteases activate ENaC by removing residues that bind at the finger-thumb domain interface.     

Role of the Wrist Domain in the Response of the Epithelial Sodium Channel to External Stimuli

The epithelial Na⁺ channel (ENaC) is regulated by a variety of external factors that alter channel activity by inducing conformational changes within its large extracellular region that are transmitted to the gate. The wrist domain consists of small linkers connecting the extracellular region to the transmembrane domains, where the channel pore and gate reside. We employed site-directed mutagenesis combined with two-electrode voltage clamp to investigate the role of the wrist domain in channel gating in response to extracellular factors. Channel inhibition by external Na⁺ was reduced by selected mutations within the wrist domain of the α subunit, likely reflecting an increase in channel open probability. The most robust changes were observed when Cys was introduced at αPro-138 and αSer-568, sites immediately adjacent to the palm domain. In addition, one of these Cys mutants exhibited an enhanced response to shear stress. In the context of channels that have a low open probability due to retention of an inhibitory tract, the response to external Na⁺ was reduced by Cys substitutions at both αPro-138 and αSer-568. We observed a significant correlation between changes in channel inhibition by external Na⁺ and the relative response to shear stress for the α subunit mutants that were examined. Mutants that exhibited reduced inhibition by external Na⁺ also showed an enhanced response to shear stress. Together, our data suggest that the wrist domain has a role in modulating the channel''s response to external stimuli.     

Identification of epithelial Na+ channel (ENaC) intersubunit Cl- inhibitory residues suggests a trimeric alpha gamma beta channel architecture

The extracellular domain of the epithelial Na(+) channel (ENaC) is exposed to a wide range of anion concentrations in the kidney. We have previously demonstrated that extracellular Cl(-) inhibits ENaC activity. To identify sites involved in Cl(-) inhibition, we mutated residues in the extracellular domain of α-, β-, and γENaC that are homologous to the Cl(-) binding site in acid-sensing ion channel 1a and tested the effect of Cl(-) on the activity of ENaC expressed in Xenopus oocytes. We identified two Cl(-) inhibitory sites in ENaC. One is formed by residues in the thumb domain of αENaC and the palm domain of βENaC. Mutation of residues at this interface decreased Cl(-) inhibition and decreased Na(+) self-inhibition. The second site is formed by residues at the interface of the thumb domain of βENaC and the palm domain of γENaC. Mutation of these residues also decreased Cl(-) inhibition yet had no effect on Na(+) self-inhibition. In contrast, mutations in the thumb domain of γENaC and palm of αENaC had little or no effect on Cl(-) inhibition or Na(+) self-inhibition. The data demonstrate that Cl(-) inhibits ENaC activity by two distinct Na(+)-dependent and Na(+)-independent mechanisms that correspond to the two functional Cl(-) inhibitory sites. Furthermore, based on the effects of mutagenesis on Cl(-) inhibition, the additive nature of mutations, and on differences in the mechanisms of Cl(-) inhibition, the data support a model in which ENaC subunits assemble in an αγβ orientation (listed clockwise when viewed from the top).     

Role of Epithelial Sodium Channels and Their Regulators in Hypertension

The kidney has a central role in the regulation of blood pressure, in large part through its role in the regulated reabsorption of filtered Na⁺. Epithelial Na⁺ channels (ENaCs) are expressed in the most distal segments of the nephron and are a target of volume regulatory hormones. A variety of factors regulate ENaC activity, including several aldosterone-induced proteins that are present within an ENaC regulatory complex. Proteases also regulate ENaC by cleaving the channel and releasing intrinsic inhibitory tracts. Polymorphisms or mutations within channel subunits or regulatory pathways that enhance channel activity may contribute to an increase in blood pressure in individuals with essential hypertension.     

Cysteine Palmitoylation of the γ Subunit Has a Dominant Role in Modulating Activity of the Epithelial Sodium Channel

The epithelial sodium channel (ENaC) is composed of three homologous subunits (α, β, and γ) with cytoplasmic N and C termini. Our previous work revealed that two cytoplasmic Cys residues in the β subunit, βCys-43 and βCys-557, are Cys-palmitoylated. ENaCs with mutant βC43A/C557A exhibit normal surface expression but enhanced Na⁺ self-inhibition and reduced channel open probability. Although the α subunit is not palmitoylated, we now show that the two cytoplasmic Cys residues in the γ subunit are palmitoylated. ENaCs with mutant γC33A, γC41A, or γC33A/C41A exhibit reduced activity compared with wild type channels but normal surface expression and normal levels of α and γ subunit-activating cleavage. These mutant channels have significantly enhanced Na⁺ self-inhibition and reduced open probability compared with wild type ENaCs. Channel activity was enhanced by co-expression with the palmitoyltransferase DHHC2 that also co-immunoprecipitates with ENaCs. Secondary structure prediction of the N terminus of the γ subunit places γCys-33 within an α-helix and γCys-44 on a coil before the first transmembrane domain within a short tract that includes a well conserved His-Gly motif, where mutations have been associated with altered channel gating. Our current and previous results suggest that palmitoylation of the β and γ subunits of ENaCs enhances interactions of their respective cytoplasmic domains with the plasma membrane and stabilizes the open state of the channel. Comparison of activities of channels lacking palmitoylation sites in individual or multiple subunits revealed that γ subunit palmitoylation has a dominant role over β subunit palmitoylation in modulating ENaC gating.     

Intracellular Na+ Regulates Epithelial Na+ Channel Maturation

Epithelial Na⁺ channel (ENaC) function is regulated by the intracellular Na⁺ concentration ([Na⁺]_i) through a process known as Na⁺ feedback inhibition. Although this process is known to decrease the expression of proteolytically processed active channels on the cell surface, it is unknown how [Na⁺]_i alters ENaC cleavage. We show here that [Na⁺]_i regulates the posttranslational processing of ENaC subunits during channel biogenesis. At times when [Na⁺]_i is low, ENaC subunits develop mature N-glycans and are processed by proteases. Conversely, glycan maturation and sensitivity to proteolysis are reduced when [Na⁺]_i is relatively high. Surface channels with immature N-glycans were not processed by endogenous channel activating proteases, nor were they sensitive to cleavage by exogenous trypsin. Biotin chase experiments revealed that the immature surface channels were not converted into mature cleaved channels following a reduction in [Na⁺]_i. The hypothesis that [Na⁺]_i regulates ENaC maturation within the biosynthetic pathways is further supported by the finding that Brefeldin A prevented the accumulation of processed surface channels following a reduction in [Na⁺]_i. Therefore, increased [Na⁺]_i interferes with ENaC N-glycan maturation and prevents the channel from entering a state that allows proteolytic processing.     

Probing the Structural Basis of Zn2+ Regulation of the Epithelial Na+ Channel

Extracellular Zn²⁺ activates the epithelial Na⁺ channel (ENaC) by relieving Na⁺ self-inhibition. However, a biphasic Zn²⁺ dose response was observed, suggesting that Zn²⁺ has dual effects on the channel (i.e. activating and inhibitory). To investigate the structural basis for this biphasic effect of Zn²⁺, we examined the effects of mutating the 10 extracellular His residues of mouse γENaC. Four mutations within the finger subdomain (γH193A, γH200A, γH202A, and γH239A) significantly reduced the maximal Zn²⁺ activation of the channel. Whereas γH193A, γH200A, and γH202A reduced the apparent affinity of the Zn²⁺ activating site, γH239A diminished Na⁺ self-inhibition and thus concealed the activating effects of Zn²⁺. Mutation of a His residue within the palm subdomain (γH88A) abolished the low-affinity Zn²⁺ inhibitory effect. Based on structural homology with acid-sensing ion channel 1, γAsp⁵¹⁶ was predicted to be in close proximity to γHis⁸⁸. Ala substitution of the residue (γD516A) blunted the inhibitory effect of Zn²⁺. Our results suggest that external Zn²⁺ regulates ENaC activity by binding to multiple extracellular sites within the γ-subunit, including (i) a high-affinity stimulatory site within the finger subdomain involving His¹⁹³, His²⁰⁰, and His²⁰² and (ii) a low-affinity Zn²⁺ inhibitory site within the palm subdomain that includes His⁸⁸ and Asp⁵¹⁶.     

A Single Amino Acid Tunes Ca2+ Inhibition of Brain Liver Intestine Na+ Channel (BLINaC)

Ion channels of the degenerin/epithelial Na⁺ channel gene family are Na⁺ channels that are blocked by the diuretic amiloride and are implicated in several human diseases. The brain liver intestine Na⁺ channel (BLINaC) is an ion channel of the degenerin/epithelial Na⁺ channel gene family with unknown function. In rodents, it is expressed mainly in brain, liver, and intestine, and to a lesser extent in kidney and lung. Expression of rat BLINaC (rBLINaC) in Xenopus oocytes leads to small unselective currents that are only weakly sensitive to amiloride. Here, we show that rBLINaC is inhibited by micromolar concentrations of extracellular Ca²⁺. Removal of Ca²⁺ leads to robust currents and increases Na⁺ selectivity of the ion pore. Strikingly, the species ortholog from mouse (mBLINaC) has an almost 250-fold lower Ca²⁺ affinity than rBLINaC, rendering mBLINaC constitutively active at physiological concentrations of extracellular Ca²⁺. In addition, mBLINaC is more selective for Na⁺ and has a 700-fold higher amiloride affinity than rBLINaC. We show that a single amino acid in the extracellular domain determines these profound species differences. Collectively, our results suggest that rBLINaC is opened by an unknown ligand whereas mBLINaC is a constitutively open epithelial Na⁺ channel.     

Extracellular finger domain modulates the response of the epithelial sodium channel to shear stress

The epithelial sodium channel (ENaC) is regulated by multiple extracellular stimuli, including shear stress. Previous studies suggest that the extracellular finger domains of ENaC α and γ subunits contain allosteric regulatory modules. However, the role of the finger domain in the shear stress response is unknown. We examined whether mutations of specific residues in the finger domain of the α subunit altered the response of channels to shear stress. We observed that Trp substitutions at multiple sites within the tract αLys-250-αLeu-290 altered the magnitude or kinetics of channel activation by shear stress. Consistent with these findings, deletion of two predicted peripheral β strands (αIle-251-αTyr-268) led to slower channel activation by shear stress, suggesting that these structures participate in the shear stress response. The effects of mutations on the shear stress response did not correlate with their effects on allosteric Na(+) inhibition (i.e. Na(+) self-inhibition), indicating a divergence within the finger domain regarding mechanisms by which the channel responds to these two external stimuli. This result contrasts with well correlated effects we previously observed at sites near the extracellular mouth of the pore, suggesting mechanistic convergence in proximity to the pore. Our results suggest that the finger domain has an important role in the modulation of channel activity in response to shear stress.     

Functional Roles of Clusters of Hydrophobic and Polar Residues in the Epithelial Na+ Channel Knuckle Domain

The extracellular regions of epithelial Na⁺ channel subunits are highly ordered structures composed of domains formed by α helices and β strands. Deletion of the peripheral knuckle domain of the α subunit in the αβγ trimer results in channel activation, reflecting an increase in channel open probability due to a loss of the inhibitory effect of external Na⁺ (Na⁺ self-inhibition). In contrast, deletion of either the β or γ subunit knuckle domain within the αβγ trimer dramatically reduces epithelial Na⁺ channel function and surface expression, and impairs subunit maturation. We systematically mutated individual α subunit knuckle domain residues and assessed functional properties of these mutants. Cysteine substitutions at 14 of 28 residues significantly suppressed Na⁺ self-inhibition. The side chains of a cluster of these residues are non-polar and are predicted to be directed toward the palm domain, whereas a group of polar residues are predicted to orient their side chains toward the space between the knuckle and finger domains. Among the mutants causing the greatest suppression of Na⁺ self-inhibition were αP521C, αI529C, and αS534C. The introduction of Cys residues at homologous sites within either the β or γ subunit knuckle domain resulted in little or no change in Na⁺ self-inhibition. Our results suggest that multiple residues in the α subunit knuckle domain contribute to the mechanism of Na⁺ self-inhibition by interacting with palm and finger domain residues via two separate and chemically distinct motifs.     

Proteolytic Activation of the Human Epithelial Sodium Channel by Trypsin IV and Trypsin I Involves Distinct Cleavage Sites

Proteolytic activation is a unique feature of the epithelial sodium channel (ENaC). However, the underlying molecular mechanisms and the physiologically relevant proteases remain to be identified. The serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant activating protease in ENaC-expressing tissues in vivo. Herein, we investigated whether human trypsin IV, a form of trypsin that is co-expressed in several extrapancreatic epithelial cells with ENaC, can activate human ENaC. In Xenopus laevis oocytes, we monitored proteolytic activation of ENaC currents and the appearance of γENaC cleavage products at the cell surface. We demonstrated that trypsin IV and trypsin I can stimulate ENaC heterologously expressed in oocytes. ENaC cleavage and activation by trypsin IV but not by trypsin I required a critical cleavage site (Lys-189) in the extracellular domain of the γ-subunit. In contrast, channel activation by trypsin I was prevented by mutating three putative cleavage sites (Lys-168, Lys-170, and Arg-172) in addition to mutating previously described prostasin (RKRK¹⁷⁸), plasmin (Lys-189), and neutrophil elastase (Val-182 and Val-193) sites. Moreover, we found that trypsin IV is expressed in human renal epithelial cells and can increase ENaC-mediated sodium transport in cultured human airway epithelial cells. Thus, trypsin IV may regulate ENaC function in epithelial tissues. Our results show, for the first time, that trypsin IV can stimulate ENaC and that trypsin IV and trypsin I activate ENaC by cleavage at distinct sites. The presence of distinct cleavage sites may be important for ENaC regulation by tissue-specific proteases.     

Gating Transitions in the Palm Domain of ASIC1a

Acid-sensing ion channels (ASICs) are trimeric cation-selective proton-gated ion channels expressed in the central and peripheral nervous systems. The pore-forming transmembrane helices in these channels are linked by short loops to the palm domain in the extracellular region. Here, we explore the contribution to proton gating and desensitization of Glu-79 and Glu-416 in the palm domain of ASIC1a. Engineered Cys, Lys, and Gln substitutions at these positions shifted apparent proton affinity toward more acidic values. Double mutant cycle analysis indicated that Glu-79 and Glu-416 cooperatively facilitated pore opening in response to extracellular acidification. Channels bearing Cys at position 79 or 416 were irreversibly modified by thiol-reactive reagents in a state-dependent manner. Glu-79 and Glu-416 are located in β-strands 1 and 12, respectively. The covalent modification by (2-(trimethylammonium)ethyl) methanethiosulfonate bromide of Cys at position 79 impacted conformational changes associated with pore closing during desensitization, whereas the modification of Cys at position 416 affected conformational changes associated with proton gating. These results suggest that β-strands 1 and 12 contribute antagonistically to activation and desensitization of ASIC1a. Site-directed mutagenesis experiments indicated that the lower palm domain contracts in response to extracellular acidification. Taken together, our studies suggest that the lower palm domain mediates conformational movements that drive pore opening and closing events.     

Contribution of residues in second transmembrane domain of ASIC1a protein to ion selectivity

Acid-sensing ion channels (ASICs) are proton-gated cation-selective channels expressed in the peripheral and central nervous systems. The ion permeation pathway of ASIC1a is defined by residues 426–450 in the second transmembrane (TM2) segment. The gate, formed by the intersection of the TM2 segments, localizes near the extracellular boundary of the plasma membrane. We explored the contribution to ion permeation and selectivity of residues in the TM2 segment of ASIC1a. Studies of accessibility with positively charged methanethiosulfonate reagents suggest that the permeation pathway in the open state constricts below the gate, restricting the passage to large ions. Substitution of residues in the intracellular vestibule at positions 437, 438, 443, or 446 significantly increased the permeability to K⁺ versus Na⁺. ASIC1a shows a selectivity sequence for alkali metals of Na⁺>Li⁺>K⁺≫Rb⁺>Cs⁺. Alanine and cysteine substitutions at position 438 increased, to different extents, the relative permeability to Li⁺, K⁺, Rb⁺, and Cs⁺. For these mutants, ion permeation was not a function of the diameter of the nonhydrated ion, suggesting that Gly-438 encompasses an ion coordination site that is essential for ion selectivity. M437C and A443C mutants showed slightly increased permeability to K⁺, Rb⁺, and Cs⁺, suggesting that substitutions at these positions influence ion discrimination by altering molecular sieving. Our results indicate that ion selectivity is accomplished by the contribution of multiple sites in the pore of ASIC1a.     

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.     

Proteolytic Regulation of Epithelial Sodium Channels by Urokinase Plasminogen Activator: CUTTING EDGE AND CLEAVAGE SITES*

Plasminogen activator inhibitor 1 (PAI-1) level is extremely elevated in the edematous fluid of acutely injured lungs and pleurae. Elevated PAI-1 specifically inactivates pulmonary urokinase-type (uPA) and tissue-type plasminogen activators (tPA). We hypothesized that plasminogen activation and fibrinolysis may alter epithelial sodium channel (ENaC) activity, a key player in clearing edematous fluid. Two-chain urokinase (tcuPA) has been found to strongly stimulate heterologous human αβγ ENaC activity in a dose- and time-dependent manner. This activity of tcuPA was completely ablated by PAI-1. Furthermore, a mutation (S195A) of the active site of the enzyme also prevented ENaC activation. By comparison, three truncation mutants of the amino-terminal fragment of tcuPA still activated ENaC. uPA enzymatic activity was positively correlated with ENaC current amplitude prior to reaching the maximal level. In sharp contrast to uPA, neither single-chain tPA nor derivatives, including two-chain tPA and tenecteplase, affected ENaC activity. Furthermore, γ but not α subunit of ENaC was proteolytically cleaved at (¹⁷⁷GR↓KR¹⁸⁰) by tcuPA. In summary, the underlying mechanisms of urokinase-mediated activation of ENaC include release of self-inhibition, proteolysis of γ ENaC, incremental increase in opening rate, and activation of closed (electrically “silent”) channels. This study for the first time demonstrates multifaceted mechanisms for uPA-mediated up-regulation of ENaC, which form the cellular and molecular rationale for the beneficial effects of urokinase in mitigating mortal pulmonary edema and pleural effusions.     

Identification of Extracellular Domain Residues Required for Epithelial Na+ Channel Activation by Acidic pH

A growing body of evidence suggests that the extracellular domain of the epithelial Na⁺ channel (ENaC) functions as a sensor that fine tunes channel activity in response to changes in the extracellular environment. We previously found that acidic pH increases the activity of human ENaC, which results from a decrease in Na⁺ self-inhibition. In the current work, we identified extracellular domain residues responsible for this regulation. We found that rat ENaC is less sensitive to pH than human ENaC, an effect mediated in part by the γ subunit. We identified a group of seven residues in the extracellular domain of γENaC (Asp-164, Gln-165, Asp-166, Glu-292, Asp-335, His-439, and Glu-455) that, when individually mutated to Ala, decreased proton activation of ENaC. γ_E455 is conserved in βENaC (Glu-446); mutation of this residue to neutral amino acids (Ala, Cys) reduced ENaC stimulation by acidic pH, whereas reintroduction of a negative charge (by MTSES modification of Cys) restored pH regulation. Combination of the seven γENaC mutations with β_E446A generated a channel that was not activated by acidic pH, but inhibition by alkaline pH was intact. Moreover, these mutations reduced the effect of pH on Na⁺ self-inhibition. Together, the data identify eight extracellular domain residues in human β- and γENaC that are required for regulation by acidic pH.     

Insights into the mechanism of pore opening of acid-sensing ion channel 1a

Acid-sensing ion channels (ASICs) are trimeric cation channels that undergo activation and desensitization in response to extracellular acidification. The underlying mechanism coupling proton binding in the extracellular region to pore gating is unknown. Here we probed the reactivity toward methanethiosulfonate (MTS) reagents of channels with cysteine-substituted residues in the outer vestibule of the pore of ASIC1a. We found that positively-charged MTS reagents trigger pore opening of G428C. Scanning mutagenesis of residues in the region preceding the second transmembrane spanning domain indicated that the MTSET-modified side chain of Cys at position 428 interacts with Tyr-424. This interaction was confirmed by double-mutant cycle analysis. Strikingly, Y424C-G428C monomers were associated by intersubunit disulfide bonds and were insensitive to MTSET. Despite the spatial constraints introduced by these intersubunit disulfide bonds in the outer vestibule of the pore, Y424C-G428C transitions between the resting, open, and desensitized states in response to extracellular acidification. This finding suggests that the opening of the ion conductive pathway involves coordinated rotation of the second transmembrane-spanning domains.     

Binding Site and Inhibitory Mechanism of the Mambalgin-2 Pain-relieving Peptide on Acid-sensing Ion Channel 1a

Acid-sensing ion channels (ASICs) are neuronal proton-gated cation channels associated with nociception, fear, depression, seizure, and neuronal degeneration, suggesting roles in pain and neurological and psychiatric disorders. We have recently discovered black mamba venom peptides called mambalgin-1 and mambalgin-2, which are new three-finger toxins that specifically inhibit with the same pharmacological profile ASIC channels to exert strong analgesic effects in vivo. We now combined bioinformatics and functional approaches to uncover the molecular mechanism of channel inhibition by the mambalgin-2 pain-relieving peptide. Mambalgin-2 binds mainly in a region of ASIC1a involving the upper part of the thumb domain (residues Asp-349 and Phe-350), the palm domain of an adjacent subunit, and the β-ball domain (residues Arg-190, Asp-258, and Gln-259). This region overlaps with the acidic pocket (pH sensor) of the channel. The peptide exerts both stimulatory and inhibitory effects on ASIC1a, and we propose a model where mambalgin-2 traps the channel in a closed conformation by precluding the conformational change of the palm and β-ball domains that follows proton activation. These data help to understand inhibition by mambalgins and provide clues for the development of new optimized blockers of ASIC channels.     

Nonproton ligand sensing domain is required for paradoxical stimulation of acid-sensing ion channel 3 (ASIC3) channels by amiloride

Acid-sensing ion channels (ASICs), which belong to the epithelial sodium channel/degenerin family, are activated by extracellular protons and are inhibited by amiloride (AMI), an important pharmacological tool for studying all known members of epithelial sodium channel/degenerin. In this study, we reported that AMI paradoxically opened homomeric ASIC3 and heteromeric ASIC3 plus ASIC1b channels at neutral pH and synergistically enhanced channel activation induced by mild acidosis (pH 7.2 to 6.8). The characteristic profile of AMI stimulation of ASIC3 channels was reminiscent of the channel activation by the newly identified nonproton ligand, 2-guanidine-4-methylquinazoline. Using site-directed mutagenesis, we showed that ASIC3 activation by AMI, but not its inhibitory effect, was dependent on the integrity of the nonproton ligand sensing domain in ASIC3 channels. Moreover, the structure-activity relationship study demonstrated the differential requirement of the 5-amino group in AMI for the stimulation or inhibition effect, strengthening the different interactions within ASIC3 channels that confer the paradoxical actions of AMI. Furthermore, using covalent modification analyses, we provided strong evidence supporting the nonproton ligand sensing domain is required for the stimulation of ASIC3 channels by AMI. Finally, we showed that AMI causes pain-related behaviors in an ASIC3-dependent manner. These data reinforce the idea that ASICs can sense nonproton ligands in addition to protons. The results also indicate caution in the use of AMI for studying ASIC physiology and in the development of AMI-derived ASIC inhibitors for treating pain syndromes.