Abstract
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.
Highlights
Extracellular acidification promotes structural rearrangements in the ectodomain of ASIC1a that result in activation and desensitization
Glu-79 is conserved within Acid-sensing ion channels (ASICs) subunits that generate proton-gated currents as homomers, whereas Glu-416 is present in virtually all ASIC isoforms
These results suggest that negatively charged residues at positions 79 and 416 facilitate pore opening in response to extracellular acidification
Summary
Extracellular acidification promotes structural rearrangements in the ectodomain of ASIC1a that result in activation and desensitization. These studies suggest that the transmembrane-extracellular region junction experiences significant structural changes during channel gating In agreement with these studies, psalmotoxin-ASIC complexes in an apparently permeable conformation illustrate an expansion of the conductive pathway concomitantly with a contraction of the extracellular vestibule in the lower palm domain [18]. To define conformational changes in the lower palm domain that occur in response to extracellular acidification, we conducted studies of accessibility with thiol-reactive reagents on channels bearing Cys at positions 79 and 416. The results of these studies indicate that the palm domain of ASIC1a resides in an expanded conformation at resting pH and transitions to a contracted conformation following extracellular acidification
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
