The P2X receptors are widely expressed in CNS and other tissues. ATP-evoked currents in P2X₂ receptor are potentiated by 1-300μM copper or zinc; this receptor posses nine extracellular histidines, two of which, H120 and H213, were reported to be involved in the zinc-induced potentiation (Clyne et al. 2002). However it remains unknown which of the extracellular histidines are necessary for the copper-induced potentiation. This study was aimed to determining whether the same extracellular histidines are also critical for copper action, suggesting a common metal coordination site and similar mechanisms of potentiation of the P2X₂ receptor. Wild-type (wt) P2X₂ receptors and nine single amino acid site-directed mutants in which histidines were replaced by alanines, were expressed in Xenopus laevis oocytes. We used the two electrode voltage-clamp technique to study the copper and zinc-induced potentiation of the ATP-evoked currents. ATP was applied for 10 sec; ATP concentration-response curve protocols were performed in wt as well as the mutant receptors. In addition, trace metal concentration-response curves were performed using 10μM ATP in wt or the mutant receptors. The ATP EC₅₀ in wt receptors, derived from concentration-response curves, was 33.3±2.5 (n=30), 10μM copper or zinc preincubated for 1 min potentiated the ATP-evoked currents and reduced the ATP EC₅₀ (4.3±0.9 and 16.8±2.7 respectively, p<0.01, Kruskal-Wallis test). The 10μM ATP-evoked current was potentiated by 10μM copper or zinc (28±5.9 and 6.2±1-fold). In the H120A, H213A or H245A mutants we consistently observed that 10μM copper or zinc did not potentiate the ATP-evoked currents, neither changed the ATP EC₅₀. However, in the H192A and H319A mutants, the magnitude of the 10μM copper or zinc potentiation was diminished in comparison with the wt receptor, but these metals still potentiated the ATP-gated currents (3-10 fold for copper, and 3-4 fold for zinc). The other mutants examined (H125A, H146A, H152A and H174A) demonstrated that these metals potentiated the ATP responses similarly to the wt receptors. These results suggest that copper and zinc interact at a common binding-site composed of three critical histidines (H120, H213 and H245) plus two accesory histidines (H192 and H319). We conclude that the identified histidine residues conform the metal-binding site which coordinate trace metals causing a conformational change responsible for the observed increase in the ATP-gated currents.