Conformational Changes Occurring in the Acidic Pocket during ASIC Activity

Abstract

Acid-Sensing Ion Channels (ASICs) are H+-gated and Na+-conducting channels. They are widely expressed throughout the central and peripheral nervous systems where they play important roles in several physiological and pathological processes. The crystal structure of chicken ASIC1 has been determined in the desensitized and open states; the conformation of the closed state is however not known. The acidic pocket is the binding site of several animal toxins, and, since it contains diverse negatively charged amino acids, it is likely that it contributes to proton sensing.In this project we investigated the conformational changes occurring in the acidic pocket during ASIC activity. In order to detect changes in fluorescence, we generated Cysteine-Tryptophan pairs; the engineered Cys was used as docking site for the specific labeling with the fluorophore and the Trp as a quencher of the fluorescence. The mutants were expressed in Xenopus laevis oocytes and the voltage-clamp fluorometry technique was used to simultaneously measure current and fluorescence signals. Changes in fluorescence intensity reflect changes in distance between Cys and Trp residues, providing clues about the structural rearrangements occurring in this region. A negative fluorescence signal would indicate an approaching of the two residues, and a positive suggests that Cys and Trp amino acids move away from each other. Interestingly, some of the tested mutants showed a composite fluorescence signal containing a rapid and a slow component. Comparison of the kinetics of current and fluorescence signals indicates that some observed movements are specifically related to the channel opening and others to the desensitization. Together these experiments allow us to predict the conformational changes in the acidic pocket occurring during ASIC activity and provide the basis for the design of new drugs acting on these channels.