Extracellular histidine residues identify common structural determinants in the copper/zinc P2X₂ receptor modulation

Abstract

To assess the mechanism of P2X 2 receptor modulation by transition metals, the cDNA for the wild-type receptor was injected to Xenopus laevis oocytes and examined 48-72 h later by the two-electrode voltage-clamp technique. Copper was the most potent of the trace metals examined; at 10 lM it evoked a 25-fold potentiation of the 10 lM ATP-gated currents. Zinc, nickel or mercury required 10-fold larger concentrations to cause comparable potentiations, while palladium, cobalt or cadmium averaged only 12-and 3-fold potentiations, respectively. Platinum was inactive. The non-additive effect of copper and zinc at 10-100 lM suggests a common site of action; these metals also shifted to the left the ATP concentration-response curves. To define residues necessary for trace metal modulation, alanines were singly substituted for each of the nine histidines in the extracellular domain of the rat P2X 2 receptor. The H120A and H213A mutants were resistant to the modulator action of copper, zinc and other metals with the exception of mercury. Mutant H192A showed a reduction but not an abrogation of the copper or zinc potentiation. H245A showed less affinity for copper while this mutant flattened the zinc-induced potentiation. Mutant H319A reduced the copper but not the zincinduced potentiation. In contrast, mutants H125A, H146A, H152A and H174A conserved the wild-type receptor sensitivity to trace metal modulation. We propose that His120, His192, His213 and His245 form part of a common allosteric metalbinding site of the P2X 2 receptor, which for the specific coordination of copper, but not zinc, additionally involves His319.

Activation and Regulation of Purinergic P2X Receptor Channels

“…P2X2 receptor. Zinc and copper both exhibit a biphasic effect on rP2X2R function: the ATP-induced responses are significantly potentiated at 10 to 100 M and inhibition at millimolar concentrations (Xiong et al, 1999;Clyne et al, 2002a;He et al, 2003b;Lorca et al, 2005). Zinc also potentiates the ATP responses of native rP2X2Rs and rP2X2/3Rs expressed in neurons from parasympathetic ganglia , as well as in neurons from the rat hypothalamus (Vorobjev et al, 2003) and dorsal motor nucleus (Ueno et al, 2001).…”

“…In both cases, the actions of mercury resemble the actions of copper, suggesting that these metals may bind to the same allosteric site. However, this hypothesis was discarded when in was reported that the copper-resistant mutants were still modulated by mercury Lorca et al, 2005). Using chimeric P2X2/X4Rs, we recently found that the primary site of action for mercury is an intracellular cysteine at position 430 (Coddou et al, 2009 P2X7R Virginio et al, 1997).…”

“…Cobalt and nickel also potentiate the activity of rP2X2R, but the potential inhibitory effect at high concentrations was not tested . Site-directed mutagenesis revealed the critical role of extracellular His 120 and His 213 in the positive modulation by zinc and copper (Clyne et al, 2002a;Lorca et al, 2005 contributing to the stabilization of the allosteric site . Nagaya et al (2005), using concatamers, showed that the allosteric sites for zinc and probably copper are formed by His 120 and His 213 of adjacent subunits.…”

“…This was inferred from competition studies using zinc and cadmium and from experiments with zinc-resistant mutants Lorca et al, 2005). Other metals, including lead, barium, palladium, silver, platinum, and gallium, had no effect on the ATP-evoked currents in P2X2R-and P2X4R-expressing cells, indicating that the allosteric sites have strict structural requirements Lorca et al, 2005).…”

Insights into the channel gating of P2X receptors from structures, dynamics and small molecules

“…The superimposition of closed and open structures revealed that the RF domain does not undergo conformational changes during the channel gating of P2X receptors ( Figure 2F); however, it does not imply that the RF domain is irrelevant to channel gating. One piece of evidence is that H245, a residue in the RF domain of rP2X2, is involved in zinc/copper potentiation [60] . E249K (rP2X4 numbering) shows sensitivity to PPADS, a chemical that has no effect on the wild-type rP2X4 receptor [61] , indicating that RF may act as an anchor when PPADS binds to the channel.…”