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.

Base of the thumb domain modulates epithelial sodium channel gating.

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 acid-sensing 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.

Constraint-based, homology model of the extracellular domain of the epithelial Na+ channel α subunit reveals a mechanism of channel activation by proteases.

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 α 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 α subunit-derived inhibitory peptide affects channel gating by constraining motions within two major domains in the extracellular region, the thumb and finger domains.

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.

Cys palmitoylation of the beta subunit modulates gating of the epithelial sodium channel.

The epithelial Na(+) channel (ENaC) is comprised of three homologous subunits (α, ß, and γ) 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 acid-exchange chemistry was used to determine whether channel subunits were Cys-palmitoylated. We observed that only the ß and γ subunits were modified by Cys palmitoylation. Analyses of ENaCs with mutant ß subunits revealed that Cys-43 and Cys-557 were palmitoylated. Xenopus oocytes expressing ENaC with a ß C43A,C557A mutant had significantly reduced amiloride-sensitive 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 ß Cys-43 is in proximity to the first transmembrane α helix, whereas ß Cys-557 is within an amphipathic α-helix contiguous with the second transmembrane domain. We propose that ß subunit palmitoylation modulates channel gating by facilitating interactions between cytoplasmic domains and the plasma membrane.

Conformational changes associated with proton-dependent gating of ASIC1a.

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.

Novel determinants of epithelial sodium channel gating within extracellular thumb domains.

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 gammaMet(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 alpha subunit (G481A, G481C, and G481M) dramatically increased the magnitude and speed of Na(+) self-inhibition. Mutations at the homologous betaAla(422) resulted in minimal or no change in Na(+) self-inhibition. Low, high, and intermediate open probabilities were observed in oocytes expressing alphaG481Mbetagamma, alphabetagammaM438V, and alphaG481M/betagammaM438V, respectively. This pair of residues map to thealpha5 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 alphaG481Cbetagamma and alphabetagammaM438C channels were inhibited by an externally applied and membrane-impermeant sulfhydryl reagent. Our results demonstrate that alphaGly(481) and gammaMet(438) are functional determinants of Na(+) self-inhibition and of ENaC gating and suggest that the thumb domain contributes to the channel gating machinery.