Bulky Substitutions in the Thumb Domain of ASIC1a Restrict Proton-Gating

Abstract

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation selective ion channels expressed in the nervous system. Each monomer consists of two transmembrane helices with short cytoplasmic tails and a large extracellular region organized in distinct domains named the thumb, finger, knuckle, β-ball, and palm. In this report we investigated the contribution to mouse ASIC1a activation and desensitization of three pairs of acidic residues, Glu238-Asp345, Asp237-Asp349 and Glu219-Asp407. Residues in each pair are located in close proximity and together form an acidic cluster at the interface between the thumb and β-ball domains. We examined the reactivity toward methanethiosulfonate (MTS) reagents of channels carrying individual Cys substitutions at these positions. MTSET-modification of D345C channels shifted the pH of half-maximal activation (pH50) from 6.23±0.06 to 4.48±0.08, and increased the desensitization rate. A similar effect was observed with other positively-charged MTS reagents such as MTSMT, MTSPT and MTSEA, as well as with MTSES, a negatively-charged reagent. Moreover, Lys substitution at position 345 shifted pH50 toward more acidic values. Remarkably, Lys substitution at positions 219, 237 or 238 did not significantly change the pH50, suggesting that acid residues at these positions do not substantially contribute to proton activation. Our results indicate that the introduction of a bulky moiety at position 345 restricts conformational movements required for proton-gating, suggesting that the thumb-β-ball interface undergoes dynamic rearrangements during activation.