Coordinated Movements During ASIC1A Activity

Abstract

Acid-sensing ion channels (ASICs) are H+-gated and Na+-conducting channels predominantly expressed in the nervous system. They are involved in H+ sensing in many physiological and pathological states such as ischemia and pain perception. The crystal structure of the chicken ASIC1 isoform has been revealed in the desensitized and the open state. Functional studies indicate that protonation of key residues in the extracellular loop triggers conformational changes leading to channel opening. However the molecular mechanisms linking protonation to the opening and closing of the gate have not been clarified yet. In this study we used voltage-clamp fluorometry (VCF) to reveal activity-associated movements occurring on the different ASIC1a domains. 20 different fluorophore positions located in the thumb, palm, finger and knuckle domains and in the extracellular pore entry showed VCF signals related to conformational changes. The timing of fluorescence changes suggests a complex sequence of movements upon pH change. When the pH of the extracellular solution was lowered to activate ASICs, rapid conformational changes were observed in the thumb, finger, knuckle and extracellular pore entry, followed by slower movements in the palm. The kinetics of fluorescence and current signals were compared to each other in order to assess whether the timing of the fluorescence signal corresponded to an effective channel transition. Some of the residues tested were found to be closely related to channel opening, desensitization or recovery from desensitization. Moreover we found that an endogenous tryptophan of the β-ball quenched the fluorescence signal of probes positioned in the finger and knuckle domains. The observed increase of this signal during channel opening indicates a movement of these domains away from the β-ball. This is the first extensive analysis of activity-dependent conformational changes in ASICs which could be applied to other ENaC-Deg related channels.