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The role of histidine residues in modulation of the rat P2X 2 purinoceptor by zinc and pH
Author(s) -
Clyne J. Dylan,
LaPointe Lisa D.,
Hume Richard I.
Publication year - 2002
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2001.013244
Subject(s) - histidine , extracellular , chemistry , zinc , mutant , receptor , long term potentiation , biophysics , dissociation constant , xenopus , biochemistry , voltage clamp , wild type , biology , membrane potential , enzyme , organic chemistry , gene
P2X 2 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 P2X 2 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 EC 50 of the ATP concentration‐response relation. Decreasing the extracellular pH from 7.5 to 6.5 potentiated the responses to 10 μ m ATP of wild‐type P2X 2 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 p K a (‐log of dissociation constant) of the potentiation to a more basic pH as compared with P2X 2 and revealed a substantial pH‐dependent decrease in the maximum response with a p K a near 6.0. Substituting a lysine for H319 reduced the EC 50 for ATP 40‐fold. Zinc (20 μ m ) potentiated the responses to 10 μ m ATP of wild‐type P2X 2 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.