Dissecting the facilitator and inhibitor allosteric metal sites of the P2X4 receptor channel -: Critical roles of Cys132 for zinc potentiation and Asp138 for copper inhibition

Zinc and copper are atypical modulators of ligand-gated ionic channels in the central nervous system. We sought to identify the amino acids of the rat P2X(4) receptor involved in trace metal interaction, specifically in the immediate linear vicinity of His(140), a residue previously identified as being critical for copper-induced inhibition of the ATP-evoked currents. Site-directed mutagenesis replaced conspicuous amino acids located within the extracellular domain region between Thr(123) and Thr(146) for alanines. cDNAs for the wild-type and the receptor mutants were expressed in Xenopus laevis oocytes and examined by the two-electrode technique. Cys(132), but not Cys(126), proved crucial for zinc-induced potentiation of the receptor activity, but not for copper-induced inhibition. Zinc inhibited in a concentration-dependent manner the ATP-gated currents of the C132A mutant. Likewise, Asp(138), but not Asp(131) was critical for copper and zinc inhibition; moreover, mutant D138A was 20-fold more reactive to zinc potentiation than wild-type receptors. Asp(129), Asp131, and Thr(133) had minor roles in metal modulation. We conclude that this region of the P2X4 receptor has a pocket for trace metal coordination with two distinct and separate facilitator and inhibitor metal allosteric sites. In addition, Cys132 does not seem to participate exclusively as a structural receptor channel folding motif but plays a role as a ligand for zinc modulation highlighting the role of trace metals in neuronal excitability.