Involvement of the left-flipper-to-dorsal-fin interface of the zebrafish P2X4 receptor in ATP binding and structural rearrangement.

Abstract P2X receptors are trimeric ATP-activated non-selective cation channels. The ATP binding pocket is positioned between two neighboring subunits. Accompanying ligand binding, subunit–subunit contacts are most likely involved in receptor function and drive a conformational change to open the ion permeation pathway. In this way, we sought to determine the function of side chains of the zebrafish P2X4 receptor ectodomain left-flipper-to-dorsal-fin interface residues in ligand binding. By combining site-directed mutagenesis and electrophysiology methods, we showed that cysteine substitutions of I212, S215, Y216 and L217 resulted in decreased sensitivity to ATP. In addition, the ATP induced current at L217C was completely inhibited by sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES − ), indicating a role for this residue in ATP action. Deletion of residues 285–293 from the zebrafish P2X4 receptor abolished channel function. However, insertion of the same sequence frame into a homologous position of the rat P2X6 receptor did not rescue channel function, suggesting that these residues are necessary but not sufficient for achieving the correct ATP-induced conformation.