Abstract
Acid-sensing ion channels (ASICs) are proton-gated cation channels critical for neuronal functions. Studies of ASIC1, a major ASIC isoform and proton sensor, have identified acidic pocket, an extracellular region enriched in acidic residues, as a key participant in channel gating. While binding to this region by the venom peptide psalmotoxin modulates channel gating, molecular and structural mechanisms of ASIC gating modulation by small molecules are poorly understood. Here, combining functional, crystallographic, computational and mutational approaches, we show that two structurally distinct small molecules potently and allosterically inhibit channel activation and desensitization by binding at the acidic pocket and stabilizing the closed state of rat/chicken ASIC1. Our work identifies a previously unidentified binding site, elucidates a molecular mechanism of small molecule modulation of ASIC gating, and demonstrates directly the structural basis of such modulation, providing mechanistic and structural insight into ASIC gating, modulation and therapeutic targeting.
Highlights
Acid-sensing ion channels (ASICs) are proton-gated cation channels critical for neuronal functions
A multitude of functional studies highlight the critical role of regions in the extracellular domain (ECD), including the acidic pocket and the palm domain, in channel activation and desensitization
We report a previously unidentified binding site in ASIC1 for two structurally distinct small molecules, whose occupancy is shown for the first time, to our knowledge, to correlate with and result in modulation of ASIC1 gating
Summary
JNJ-799760 and JNJ-67869386 are potent allosteric antagonists of ASIC1a. JNJ-799760 and JNJ-67869386 are small synthetic molecules belonging to two distinct chemical series (Fig. 1a, b). A small fraction of channels still recovers slowly, resembling the kinetics of current recovery from compound inhibition in the closed state (Fig. 5a), suggesting that some channels are still compound bound at the start of recovery and that the desensitizing pulse is not long enough for complete compound dissociation to occur. This indicates that some desensitized channels are still compound bound after 14 s. Positions of the L414 and N415 side chains in ΔASIC1/JNJ–799760 are “un-swapped”
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