Finger-Thumb Interdomain Interactions Influence ASIC1a Proton Activation

Abstract

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation-selective channels expressed in the nervous system. Each monomer consists of two transmembrane helices, short cytoplasmic tails and a large extracellular region organized in distinct domains named the thumb, finger, knuckle, β-ball, and palm. Despite the structural information available for ASIC1, there is limited understanding of the molecular mechanism that allows these channels to sense and respond to drops in extracellular pH. Here, we employed the substituted cysteine accessibility method (SCAM) and site-directed mutagenesis to identify molecular determinants for proton activation in the extracellular region of mouse ASIC1a. We mutated individually residues in the acidic pocket of ASIC1a to Cys and proved its reactivity toward MTSET. We found that the modification of Cys residues at positions 238 and 345 by MTSET reduced apparent proton affinity for activation. Glu238 and Asp345 reside in the finger and thumb domains, respectively. In the desensitized conformation these residues are positioned in closed proximity to each other. Surprisingly, D345K and D345R mutants, as well as D345C channels treated with MTSEA, displayed biphasic dose-response activation curves. In contrast, E238K and D345N channels showed monophasic dose-response curves with apparent proton affinities for activation similar to those from the controls. Together, these results suggest that finger-thumb interactions indirectly affect the function of a proton-binding site. To examine conformational movements in the thumb domain associated with activation, we mutated the residues in the alpha-helix 5 to Cys and assessed their reactivity toward MTSET. The modification by MTSET of A344C, L348C, L351C and V352C channels reduced the response to extracellular acidification. These residues are located on the same side of the alpha-helix 5 facing the finger domain. In conclusion, our results indicate that extracellular acidification drives conformation movements in the finger and thumb domains of ASIC1a.