To study the roles of the Lys(313)-Ile(333) ectodomain sequence of the rat P2X(4) receptor in ATP binding and transduction of signals to the channel gate, the conserved Lys(313), Tyr(315), Gly(316), Ike(317), Arg(318), Asp(320), Val(323), Lys(329), Phe(330), and Ile(333) residues were mutated. Current recordings were done on lifted cells and ATP was applied using an ultrafast solution-switching system. The rates of wild type channel opening and closing in the presence of ATP, but not the rate of washout-induced closing, were dependent on agonist concentration. All mutants other than I317A were expressed in the plasma membrane at comparable levels. The majority of mutants showed significant changes in the peak amplitude of responses and the EC(50) values for ATP. When stimulated with the supramaximal (1.4 mm) ATP concentration, mutants also differed in the kinetics of their activation, deactivation, and/or desensitization. The results suggest a critical role of the Lys(313) residue in receptor function other than coordination of the phosphate group of ATP and possible contribution of the Tyr(315) residue to the agonist binding module. The pattern of changes of receptor function by mutation of other residues was consistent with the operation of the Gly(316)-Ile(333) sequence as a signal transduction module between the ligand binding domain and the channel gate in the second transmembrane domain.