P2X receptors are ligand-gated ionic channels, and can be differentially modulated by divalent metals that bind to specific allosteric sites. We previously demontrated that His-140 is a critical amino acid responsible for the inhibitory modulation exerted by copper on the P2X₄ receptor, and the facilitatory action of zinc is augmented and its typical modulation, a bell-shaped curve observed in the wild-type receptor is replaced by a sigmoid in this mutant, suggesting that zinc can bind to the inhibitory site too at high concentrations (Coddou et al., 2003). Since metal coordination requires more than one amino acid, we replaced other amino acids in the vicinity of His-140 that could form part of the inhibitory binding site and the mutated cDNAs were injected in X. laevis oocytes and tested with electrophysiological techniques. We found that the replacement of Asp-138 by an alanine (D138A) results in a loss of copper-induced inhibition (with no inhibition of ATP-evoked currents with 10 μM Cu²⁺ and a 47.6±15.5% inhibition with 300 μM Cu²⁺) and an increase of the zinc-induced potentiation (20.1±2.4 fold increase) much similar to that obseved in the H140A mutant. However mercury still inhibited the ATP-evoked currents in this mutant with an IC₅₀ of 3.7±1.3 μM (n=4), suggesting that this metal exert its effects by a different site of copper. Asp-129 had a modest contribution of copper-induced inhibition, with an IC₅₀ of 24.9±4.4 μM (n=4), whereas the zinc-induced potentiation was diminished. The D131A mutant showed no significant differences with wild type receptor on the inhibitory action of copper and a slightly increase in zinc-induced potentiation. It has been suggested a key role of Cys-132 for zinc facilitation (Xiong et al., 2003), but no other amino acids responsible for metal facilitation has been identified. In the C132A mutent the zinc-induced potentiation was completely lost and zinc inhibited the ATP-evoked currents. The copper-induced inhibition was also augmented in this mutant with and IC₅₀ of 1.5±0.9 μM (n=3). These results show that both Asp-138 and His-140 are critical for the inhibitory action of copper, zinc and cadmium and form part of the inhibitory binding-site of the P2X₄ receptor. On the other hand, Cys-132 is part of the facilitatory-site that coordinates zinc and/or cadmium. The role of Asp-129 seems to be minor contributing modestly to the binding of metals to both the inhibitory and facilitatory sites.