Intersubunit Salt-Bridge Formation during Gating of Rasic1a

Acid-sensing ion channels (ASICs) are proton-gated cation channels, which are expressed in the nervous system. They contribute to synaptic transmission, learning and memory, nociception, and neurodegeneration associated with brain ischemia.Previous work has shown that the highly conserved amino acids His72, His73, and Asp78 are important for proper gating of ASIC1a. The relatively close position of these amino acids in the crystal structure of cASIC1 let to the hypothesis that one histidine forms a salt bridge with an aspartate of an adjacent subunit.In this study, we used double-mutant cycle analysis to explore whether the formation of this salt bridge is energetically favorable in a particular state during gating (open, closed, desensitized) of rat ASIC1a. We substituted H72, H73, and D78 individually and in combination by alanine and determined the time-constants for activation and deactivation of these mutants, using outside-out patches and a piezo-driven application system, which assures a solution exchange under 2 ms. Moreover, we determined the pH values for half-maximal activation and the time-constants for desensitization and recovery of the channel, using two-electrode voltage clamp (TEVC). So far our results indicate that D78 can form salt bridges with each histidine in particular states during gating.To corroborate these findings, we currently investigate the accessibility of substituted Cys residues at positions 72, 73 and 78 to methanethiosulfonate (MTS) reagents. Accessibility is compared in the closed and the desensitized state to find additional evidence for movement of these residues during gating.This study will provide information on the role of these three highly conserved amino acids in gating of ASIC1a.