Role of Lys313‐Ile333 ectodomain sequence in P2X4 receptor activity

We characterized rat P2X4 receptor activation, desensitization, and deactivation properties using an ultrafast solution‐switching system and site‐directed mutagenesis. The focus in study is on K313‐I333 sequence of P2X4 receptor, which lies between the proposed ligand binding domain and gate of channel. The conserved residues K313, Y315, G316, I317, R318, D320, V323, K329, F330, and I333 were mutated to alanine and compared with wild type P2X4 receptor. Experiments with wild type receptor revealed dose‐dependent effects of ATP on activation and desensitization kinetics, a strong correlation between activation and desensitization values, and the independence of kinetic of receptor deactivation of ATP concentration and duration of agonist application. The pattern of receptor activity was significantly altered in the majority of mutants. The parallelism and dissociation in perturbing effects of mutation on ATP efficacy and channel activity were consistent with the three‐dimensional model of receptor where Y315 is the last C‐terminal ectodomain residue contributing to the agonist binding pocket and the sequence I317–I333 operates as a linker that connects the binding site to the intrapore gate, with G316 as a possible hinge between the binding and linking regions. Individual residues in the linker sequence appeared to play specific roles in transduction of signaling from the binding site to the channel gate. Supported by the Intramural Research Program of the NICHD, NIH.