Received September 5, 2001
Accepted after revision December 8, 2001

The role of histidine residues in modulation of the rat P2X₂ purinoceptor by zinc and pH

¹ Department of Molecular, Cellular and Developmental Biology, University of Michigan, 3095 Natural Science Bldg, 830 N. University Ave, Ann Arbor, MI 48109, USA
² Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA

^* To whom correspondence should be addressed.

P2X₂ receptor currents are potentiated by acidic pH and zinc. To identify residues necessary for proton and zinc modulation, alanines were singly substituted for each of the nine histidines in the extracellular domain of the rat P2X₂ receptor. Wild-type and mutant receptors were expressed in Xenopus oocytes and analysed with two-electrode voltage clamp. All mutations caused less than a 2-fold change in the EC₅₀ of the ATP concentration-response relation. Decreasing the extracellular pH from 7.5 to 6.5 potentiated the responses to 10 µm ATP of wild-type P2X₂ and eight mutant receptors more than 4-fold, but the response of the mutant receptor H319A was potentiated only 1.4-fold. The H319A mutation greatly attenuated the maximal potentiation that could be produced by a drop in pH, shifted the pK_a (-log of dissociation constant) of the potentiation to a more basic pH as compared with P2X₂ and revealed a substantial pH-dependent decrease in the maximum response with a pK_a near 6.0. Substituting a lysine for H319 reduced the EC₅₀ for ATP 40-fold. Zinc (20 µm) potentiated the responses to 10 µm ATP of wild-type P2X₂ and seven histidine mutants by ~8-fold but had virtually no effect on the responses of two mutants, H120A and H213A. Neither H120A nor H213A removed the voltage-independent inhibition caused by high concentrations of zinc. The observation that different mutations selectively eliminated pH or zinc potentiation implies that there are two independent sites of action, even though the mechanisms of pH and zinc potentiation appear similar.

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