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J. Biol. Chem., Vol. 278, Issue 38, 36777-36785, September 19, 2003
Histidine 140 Plays a Key Role in the Inhibitory Modulation of the P2X4 Nucleotide Receptor by Copper but Not Zinc*![]() ![]() ![]() ![]() ![]() ||
From the
To elucidate the role of extracellular histidines in the modulation of the rat P2X4 receptor by trace metals, we generated single, double, and triple histidine mutants for residues 140, 241, and 286, replacing them with alanines. cDNAs for the wild-type and receptor mutants were expressed in Xenopus laevis oocytes and in human embryonic kidney 293 cells and examined by the two electrode and patch clamp techniques, respectively. Whereas copper inhibited concentration-dependently the ATP-gated currents in the wild-type and in the single or double H241A and H286A receptor mutants, all receptors containing H140A were insensitive to copper in both cell systems. The characteristic bell-shaped concentration-response curve of zinc observed in the wild-type receptor became sigmoid in both oocytes and human embryonic kidney cells expressing the H140A mutant; in these mutants, the zinc potentiation was 2.54-fold larger than in the wild-type. Results with the H140T and H140R mutants further support the importance of a histidine residue at this position. We conclude that His-140 is critical for the action of copper, indicating that this histidine residue, but not His-241 or His-286, forms part of the inhibitory allosteric metal-binding site of the P2X4 receptor, which is distinct from the putative zinc facilitator binding site.
Received for publication, May 16, 2003 * This work was supported in part by FONDAP-Bio-Medicine Grant 13980001 and International Copper Association Grant 20012003. The Millennium Institute for Fundamental and Applied Biology also contributed with funds. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. || To whom correspondence should be addressed: Dept. of Physiology, Neurohumoral Regulation Unit, Faculty of Biological Sciences, P. Catholic University of Chile, Alameda 340, Casilla 114-D, Santiago 1, Chile. Tel.: 56-2-686-2867; Fax: 56-2-222-5515; E-mail: jphuid{at}genes.bio.puc.cl.
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