The epithelial Na ؉ channel (ENaC) mediates Na ؉ transport across high resistance epithelia. This channel is assembled from three homologous subunits with the majority of the protein's mass found in the extracellular domains. Acid-sensing ion channel 1 (ASIC1) is homologous to ENaC, but a key functional domain is highly divergent. Here we present molecular models of the extracellular region of ␣ ENaC based on a large data set of mutations that attenuate inhibitory peptide binding in combination with comparative modeling based on the resolved structure of ASIC1. The models successfully rationalized the data from the peptide binding screen. We engineered new mutants that had not been tested based on the models and successfully predict sites where mutations affected peptide binding. Thus, we were able to confirm the overall general fold of our structural models. Further analysis suggested that the ␣ subunit-derived inhibitory peptide affects channel gating by constraining motions within two major domains in the extracellular region, the thumb and finger domains.Epithelial Na ϩ channels (ENaCs) 3 are members of the ENaC/degenerin family of ion channels, of which the high resolution structure of acid-sensing ion channel 1 (ASIC1) has been reported. These channels are probably trimers (1, 2) with each subunit having two transmembrane helices, large extracellular regions, and short cytosolic amino and carboxyl termini (3). The resolved structure of the extracellular region of ASIC1 is composed of core ␤-sheet domains (termed palm and ␤-ball) surrounded by peripheral ␣-helical domains (termed finger, thumb, and knuckle) (1). Channels in the ENaC/degenerin family are Na ϩ -permeable and are gated by a diverse set of stimuli, including external ligands and mechanical forces (4). As such, ENaC/degenerin family members play diverse roles in biology. For ENaC, these include the regulation of extracellular volume and blood pressure by mediating Na ϩ transport in the distal nephron of the kidney, regulation of airway surface liquid volume and mucociliary clearance by facilitating Na ϩ transport in airways, and facilitation of salt taste by transporting Na ϩ in lingual epithelium (4). ENaC is assembled from homologous ␣, ␤, and ␥ subunits and is allosterically inhibited by extracellular Na ϩ by a phenomenon referred to as Na ϩ self-inhibition (5-7). Within the ENaC/degenerin family, sequence conservation is conspicuously lacking within the finger domains of the extracellular regions of these channels (1). This fact may partly account for the diversity in the regulation of channel gating observed among gene family members and is an obstacle in building comparative models of ENaC subunits based on the resolved ASIC1 structure.Among the panoply of ENaC properties is its activation by proteolytic cleavage, which is unusual for ion channels (8). Proteolytic activation of ENaC occurs through the cleavage of both the ␣ and ␥ subunits at multiple sites within their finger domains, leading to the release of inhibitory tracts (9 -12). Pe...

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 ␣ 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 fingerthumb domain interface.The epithelial Na ϩ channel (ENaC) 2 is expressed at the apical surface of Na ϩ -transporting epithelia such as the distal nephron of the kidney, distal colon, and lung alveoli and airway. In conjunction with the Na ϩ /K ϩ -ATPase, ENaC transfers Na ϩ from the luminal to the interstitial space. This transfer is crucial in regulating blood pressure through its role in renal Na ϩ absorption and in regulating airway surface liquid volume and mucociliary clearance through its role in airway Na ϩ absorption. In accord with its role in these processes, improper ENaC function is implicated in several disorders. There is a growing body of evidence that enhanced ENaC activity in the airways of individuals with cystic fibrosis contributes to depletion of airway surface liquids resulting in poor mucociliary clearance (1-3). In the kidney, increased levels of aldosterone activate ENaC and increase the reabsorption of filtered Na ϩ (4). In both instances, increases in channel activity reflect, in part, enhanced channel proteolysis. Proteinuric states, characterized by excessive protein in the urine, are often accompanied by renal Na ϩ retention, volume expansion, and hypertension. Recent work indicates that volume expansion in proteinuric states reflects proteolytic activation of ENaC (5-7).ENaC is a trimer composed of three homologous subunits, ␣, ␤, and ␥ (8, 9). ENaC subunits are members of the much larger ENaC/Degenerin family of ion channel proteins. These channels share a few salient features as follows: 1) most are gated by ligands and/or mechanical forces; 2) they are Na ϩ -permeable and blocked by amiloride, a potassium-sparing diuretic; and 3) each subunit has two transmembrane helices (six transmembrane helices for the full channel), short intracellular N and C termini, and a large extracellular region comprised of sever...

Activity of the epithelial Na؉ channel (ENaC) is modulated by Na ؉ self-inhibition, an allosteric down-regulation of channel open probability by extracellular Na ؉ . We searched for determinants of Na ؉ self-inhibition by analyzing changes in this inhibitory response resulting from specific mutations within the extracellular domains of mouse ENaC subunits. Mutations at ␥Met 438 altered the Na ؉ self-inhibition response in a substitution-specific manner. Fourteen substitutions (Ala, Arg, Asp, Cys, Gln, Glu, His, Ile, Phe, Pro, Ser, Thr, Tyr, and Val) significantly suppressed Na ؉ self-inhibition, whereas three mutations (Asn, Gly, and Leu) moderately enhanced the inhibition. Met to Lys mutation did not alter Na ؉ self-inhibition. Mutations at the homologous site in the ␣ subunit (G481A, G481C, and G481M) dramatically increased the magnitude and speed of Na ؉ self-inhibition. Mutations at the homologous ␤Ala 422 resulted in minimal or no change in Na ؉ self-inhibition. Low, high, and intermediate open probabilities were observed in oocytes expressing ␣G481M␤␥, ␣␤␥M438V, and ␣G481M/ ␤␥M438V, respectively. This pair of residues map to the ␣5 helix in the extracellular thumb domain in the chicken acid sensing ion channel 1 structure. Both residues likely reside near the channel surface because both ␣G481C␤␥ and ␣␤␥M438C channels were inhibited by an externally applied and membrane-impermeant sulfhydryl reagent. Our results demonstrate that ␣Gly 481 and ␥Met 438 are functional determinants of Na ؉ self-inhibition and of ENaC gating and suggest that the thumb domain contributes to the channel gating machinery.Maintenance of body fluid volume homeostasis requires a collaborative interaction of many Na ϩ transport mechanisms. Na ϩ transport in epithelia that line the late distal convoluted tubule, connecting tubule, and collecting tubule relies on apical Na ϩ entry through epithelial Na ϩ channels (ENaC self-inhibition (4, 6 -8). However, detailed elements regarding its mechanism have not been revealed.A logical place to search for structural elements associated with Na ϩ self-inhibition is the large extracellular domain (ECD) that connects the two transmembrane domains (M1 and M2) within each ENaC subunit. The ECD likely exists as well structured subdomains with 16 conserved Cys residues. We recently reported that point mutations at multiple ␣ and ␥ ECD Cys residues blunted Na ϩ self-inhibition, and certain double or triple mutations rendered ENaC insensitive to high concentration of extracellular Na ϩ . These results suggest that multiple Cys residues are required to establish the proper tertiary structure permitting this allosteric regulation (9). In addition, the N-terminal portion of ECD contains ␥His 239 , a previously identified residue critical for Na ϩ self-inhibition, as well as defined protease cleavage sites (4, 10 -12). Various proteases have been shown to regulate ENaC activity, in part, by interfering with Na ϩ self-inhibition (6, 7, 13). The resolved high resolution structure of the chicken acidsensing i...

The epithelial Na ؉ channel (ENaC) is comprised of three homologous subunits (␣, ␤, and ␥) that have a similar topology with two transmembrane domains, a large extracellular region, and cytoplasmic N and C termini. Although ENaC activity is regulated by a number of factors, palmitoylation of its cytoplasmic Cys residues has not been previously described. Fatty acidexchange chemistry was used to determine whether channel subunits were Cys-palmitoylated. We observed that only the ␤ and ␥ subunits were modified by Cys palmitoylation. Analyses of ENaCs with mutant ␤ subunits revealed that Cys-43 and Cys-557 were palmitoylated. Xenopus oocytes expressing ENaC with a ␤ C43A,C557A mutant had significantly reduced amiloridesensitive whole cell currents, enhanced Na ؉ self-inhibition, and reduced single channel P o when compared with wild-type ENaC, while membrane trafficking and levels of surface expression were unchanged. Computer modeling of cytoplasmic domains indicated that ␤ Cys-43 is in proximity to the first transmembrane ␣ helix, whereas ␤ Cys-557 is within an amphipathic ␣-helix contiguous with the second transmembrane domain. We propose that ␤ subunit palmitoylation modulates channel gating by facilitating interactions between cytoplasmic domains and the plasma membrane.Epithelial sodium channels (ENaCs) 4 mediate amiloridesensitive Na ϩ transport across the apical membrane of high resistance epithelia and have important roles in regulating extracellular fluid volume and blood pressure, as well as airway surface liquid volume and mucociliary clearance. The ␣, ␤, and ␥ subunits of ENaC are members of the ENaC/Degenerin family of ion channels, which include H ϩ -gated channels (referred to as acid-sensing ion channels (ASICs)) that are expressed in mammalian central and peripheral nervous systems and have a role in nociception and mechanosensation (1). ENaC and other family members are ion channels composed of subunits that have a similar topology with two transmembrane helices, a large extracellular region, including numerous conserved disulfide bridges, and cytoplasmic N and C termini. The recently resolved crystal structure of ASIC1 (2) revealed a homotrimer with highly organized extracellular regions, suggesting that ENaC is an ␣ 1 ␤ 1 ␥ 1 heterotrimer.ENaCs are assembled within the endoplasmic reticulum (ER), where they undergo N-linked glycosylation. Assembly is inefficient, and the majority of newly synthesized subunits undergo ER-associated degradation with a half-life of 1-2 h as determined by metabolic labeling (3-7). A small pool of newly synthesized subunits has a significantly longer half-life (Ͼ4 h) and represents assembled channels that have exited the ER and are present in later compartments. This latter pool contains subunits whose N-glycans have undergone both Golgi-dependent terminal processing and cleavage of ␣ and ␥ by furin, a protease localized primarily to the trans-Golgi network (5, 8, 9). ENaC exit from the ER is regulated by a signal within the C-terminal cytoplasmic domain of the ␣ su...

Acid-sensing ion channels are proton-gated Na ؉ channels expressed predominantly in neurons. How channel structure translates an environmental stimulus into changes in pore permeability remains largely undefined. The pore of ASIC1 is defined by residues in the second transmembrane domain (TM2), although a segment of the outer vestibule is formed by residues of TM1. We used the voltage clamp fluorometry technique to define the role of the region preceding TM2 (pre-TM2) in activation and desensitization of mouse ASIC1a. Oocytes expressing E425C channels labeled with Alexa Fluor 488 C5-maleimide showed a change in the emission of the fluorescent probe in response to extracellular acidification. The time course of the change in fluorescence correlated with activation but not desensitization of E425C channels. The fluorescence emission did not change following extracellular acidification in oocytes carrying an inactivating mutation (W287G/E425C), although these channels were labeled and expressed at the plasma membrane. Our data indicate that pore opening occurs in conjunction with a conformational rearrangement of the pre-TM2. We observed a change in the emission of the fluorescent probe when labeled E425C channels transition from the desensitized to the resting state. The substituted-cysteine-accessibility method was used to determine whether the pre-TM2 has different conformations in the resting and desensitized states. State-dependent changes in accessibility to 2-[(trimethylammonium)ethyl]methanethiosulfonate bromide modification were observed in oocytes expressing K421C, K422C, Y424C, and E425C channels. Our results suggest that the pre-TM2 of ASIC1a undergoes dynamic conformational rearrangements during proton-dependent gating.Acid-sensing ion channels (ASICs) 3 are members of the epithelial sodium channel (ENaC)/degenerin family expressed in neurons of the peripheral and central nervous system. To date, seven ASIC subunits have been cloned, including ASIC1a (1), ASIC1b (2, 3), ASIC1b2 (4), ASIC2a (5-7), ASIC2b (8), ASIC3 (9 -12), and ASIC4 (13,14). In the peripheral nervous system, ASICs are expressed in sensory nerve endings where they participate in normal touch sensation and pain perception (15)(16)(17)(18)(19)(20)(21)(22)(23)(24). In the central nervous system, ASIC1a contributes to synaptic plasticity in the hippocampus, hippocampus-dependent spatial memory, spatial learning, and neural mechanisms of fear conditioning (25)(26)(27). Recently, ASICs have been implicated in the pathogenesis and migration of malignant glioma cells (28 -30).The first high resolution structure of the extracellular and membrane-spanning domains of Gallus gallus (chicken) acidsensing ion channel 1 (cASIC1) was recently reported (31). cASIC1 is organized as a homotrimer. Each subunit has a short intracellular N terminus and C terminus and two transmembrane domains (TMs) connected by a large extracellular region protruding from the plane of the membrane. The extracellular region is organized in discrete domains and, using the anal...

The activity of the epithelial sodium channel (ENaC) is modulated by multiple external factors, including proteases, cations, anions and shear stress. The resolved crystal structure of acidsensing ion channel 1 (ASIC1), a structurally related ion channel, and mutagenesis studies suggest that the large extracellular region is involved in recognizing external signals that regulate channel gating. The thumb domain in the extracellular region of ASIC1 has a cylinder-like structure with a loop at its base that is in proximity to the tract connecting the extracellular region to the transmembrane domains. This loop has been proposed to have a role in transmitting proton-induced conformational changes within the extracellular region to the gate. We examined whether loops at the base of the thumb domains within ENaC subunits have a similar role in transmitting conformational changes induced by external Na ؉ and shear stress. Mutations at selected sites within this loop in each of the subunits altered channel responses to both external Na ؉ and shear stress.The most robust changes were observed at the site adjacent to a conserved Tyr residue. In the context of channels that have a low open probability due to retention of an inhibitory tract, mutations in the loop activated channels in a subunit-specific manner. Our data suggest that this loop has a role in modulating channel gating in response to external stimuli, and are consistent with the hypothesis that external signals trigger movements within the extracellular regions of ENaC subunits that are transmitted to the channel gate.Epithelial Na ϩ channels (ENaCs) 5 are expressed in the distal segments of the nephron, airway and alveolae, and distal colon, where they mediate Na ϩ flux across apical membranes of epithelial cells. These channels have important roles in the regulation of airway surface liquid volume and mucociliary clearance, and in the regulation of extracellular fluid volume and blood pressure. ENaCs are composed of three homologous subunits, termed ␣, ␤, and ␥, which have two hydrophobic transmembrane domains linked by large extracellular regions that include conserved Cys-rich domains (1).Members of the ENaC/degenerin family are regulated by extracellular factors. For ENaC, these factors include proteases, protons, metals (including Na ϩ ), Cl Ϫ , and mechanical forces such as shear stress (1-6). ENaCs share about 20% identity at the amino acid level with acid-sensing ion channels (ASICs), members of the ENaC/degenerin family that are activated by extracellular acidification. The resolved structure of the extracellular and transmembrane portions of ASIC1 has provided clues into mechanisms by which external factors could modulate channel gating (7,8). The extracellular domain of ASIC1 resembles an outstretched hand containing a ball, and has defined subdomains termed wrist, finger, thumb, palm, ␤-ball, and knuckle. Proton binding was proposed to lead conformational changes within specific extracellular domains, including the thumb domain (7).A loop that li...

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. Acid-sensing ion channels (ASICs)2 are voltage-independent ligand-gated ion channels expressed throughout neurons of the mammalian central and peripheral nervous systems (1, 2). ASICs are members of the Epithelial Sodium Channel/Degenerin (ENaC/Deg) family, a large group of proteins that are implicated in mechanosensation, pain sensation, regulation of extracellular fluid volume, and airway surface liquid volume (2-5). These channels are organized as homo-or hetero-trimers. Each subunit has two transmembrane segments (TMs) connected by a large extracellular region with the N and C termini on the intracellular side (6, 7). ASICs are constitutively closed and undergo activation and desensitization in response to extracellular acidification. Seven ASIC subunits are expressed in mammals: ASIC1a, ASIC1b, ASIC1b2, ASIC2a, ASIC2b, ASIC3, and ASIC4 (2). The sensitivity of activation and desensitization by protons depend on the specific subunits forming the channel complex.The pore of ASIC1 in the presumed desensitized-like state has an hourglass-like shape defined mainly by residues from TM2, although residues from TM1 form a portion of the outer vestibule (6, 7). The TM2 helices are tilted by a ϳ50°angle from the membrane normal, and their intersection likely constitutes the desensitization gate. The TM1 segments are in contact with the lipid bilayer and with the TM2 segments in their own and neighboring subunits. Compelling evidence suggests that ASIC activation involves protonation of multiple residues within the extracellular region of the protein (6, 8 -10). A fundamental question regarding the mechanism of gating of ENaC/Deg channels is how extracellular cues trigger pore opening and closing events. The extracellular region of ASIC1 is organized in discrete subdomains referred to as the palm, ...

The epithelial Na+ channel (ENaC) serves as the final site of renal Na+ absorption in the distal nephron, where the fine tuning of its activity regulates extracellular fluid volume and blood pressure. The three homologous subunits of ENaC (alpha, beta and gamma) have a similar topology with two transmembrane domains, a large extracellular loop, and cytoplasmic N‐ and C‐termini. While various lipids are known regulators of ENaC activity, cytoplasmic Cys‐palmitoylation of ENaC has not been previously described. Palmitoylation of individual epitope‐tagged subunits was assessed with fatty acid‐exchange chemistry, whereby palmitate is replaced by biotin and biotinylated subunits were analyzed by immunoblotting. We observed that only the beta and gamma subunits were modified. The function of palmitoylation adjacent to the membrane was studied by expressing ENaC containing a beta C43,557A mutant in Xenopus oocytes. These mutant channels exhibited significantly enhanced Na+ self inhibition and reduced open probability, when compared with wild type ENaC. In contrast, normal membrane trafficking was observed. Our results indicate that palmitoylation of the beta subunit enhances ENaC gating. (Funding from DK065161, DK065521 and AHA).