P2X2 receptors are ATP-gated ion channels widely expressed by neurons. Thr339 lies in the second of the two transmembrane domains of the rat P2X2 receptor protein, and is likely to be close to the narrowest part of the pore. Single-channel and whole-cell recording after expression in human embryonic kidney 293 cells showed that P2X2[T339S] receptors had pronounced spontaneous channel openings that were never seen in wild-type P2X2 receptors. P2X2[T339S] receptors were 10-fold more sensitive than wild type to exogenous ATP, and ßmeATP also increased channel opening. Two conserved ectodomain lysine residues (Lys69 and Lys308) are critical for function and have been proposed to contribute to the ATP binding site of P2X receptors. The spontaneous opening of P2X2[K69A/T339S] receptors was not different than that seen in P2X2[T339S], but for P2X2[K308A/T339S] the spontaneous activity was absent. Suramin, which is a noncompetitive antagonist at wild-type P2X2 receptors, had a pronounced agonist action at both P2X2[T339S] and P2X2[K69A/T339S] receptors but not at P2X2[K308A/T339S]. 2',3'-O-O-(2,4,6-Trinitrophenyl)-ATP (TNP-ATP), which is a competitive agonist at wild-type receptors, was also an agonist at P2X2[T339S] receptors, but not at either double mutant. The results indicate that the T339S mutation substantially destabilizes the closed channel and suggest an important role in channel gating. The correction of this gating defect, in the absence of any agonist, by the second mutation K308A shows that Lys308 is also involved in channel gating. A similar interpretation can account for the results with suramin and TNP-ATP.