Insights into the Mechanism of Pore Opening of Acid-sensing Ion Channel 1A

Abstract

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, ...

Independent Contribution of Extracellular Proton Binding Sites to ASIC1a Activation

“…1 (21,22). We previously showed that mASIC1a resides in a responsive closed state at pH values above 7.4, but it desensitizes when the extracellular pH decreases below 7.2 (19). Thus, we probed the reactivity toward MTSET of channels bearing individual Cys mutations at selected sites in the acidic cluster at pH 7.0, i.e.…”

Contribution of Residues in Second Transmembrane Domain of ASIC1a Protein to Ion Selectivity

“…These channels desensitize over a narrow range of pH. At pH values above 7.4, ASIC1a exists in the closed state, whereas at pH values below 7.2, it resides in the desensitized state (21,24). To define structural changes associated with pore opening, we explored the reactivity of MTSET with channels bearing single Cys mutations in the tract 426 -438 at pH 6.5, i.e.…”

“…To contribute to ion selectivity, residues in the (G/S)XS tract must undergo a considerable movement toward the center of pore during opening. We have proposed that a rotation of the TM2 segments could widen the closed gate and move toward the center of the pore residues that contribute to ion selectivity in the distal region of the TM2 segments (24).…”

The Thumb Domain Mediates Acid-sensing Ion Channel Desensitization

“…The half-time of hydrolysis of MTSET in the recording solution of pH 7.0 is 130 min (36). Therefore, solutions buffered at pH 7.0 containing MTS reagents were used within 30 min of preparation.…”