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

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... shout-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.