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J. Biol. Chem., Vol. 282, Issue 51, 36879-36886, December 21, 2007

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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^*

Claudio Coddou¹, Claudio Acuña-Castillo², Paulina Bull^¶, and J. Pablo Huidobro-Toro³

From the Centro de Regulación Celular y Patología J. V. Luco, Instituto Milenio de Biología Fundamental y Aplicada MIFAB, Laboratorio de Nucleótidos, Departamento de Fisiología y, ^¶Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago 651-3677, Chile and the Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago 917-0019, Chile

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 P2X₄ receptor involved in trace metal interaction, specifically in the immediate linear vicinity of His¹⁴⁰, 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 Thr¹²³ and Thr¹⁴⁶ for alanines. cDNAs for the wild-type and the receptor mutants were expressed in Xenopus laevis oocytes and examined by the two-electrode technique. Cys¹³², but not Cys¹²⁶, 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, Asp¹³⁸, but not Asp¹³¹ was critical for copper and zinc inhibition; moreover, mutant D138A was 20-fold more reactive to zinc potentiation than wild-type receptors. Asp¹²⁹, Asp¹³¹, and Thr¹³³ 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.

Received for publication, August 20, 2007 , and in revised form, October 11, 2007.

^* This research was supported in part by the J. V. Luco Centre for Cell Regulation and Pathology; the Millennium Institute of Applied and Fundamental Biology (MIFAB) also contributed to the Centre funding. 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.

¹ Supported by a CONICYT doctoral scholarship.

² Postdoctoral fellow at the Nucleotide Research Laboratory, supported by a Bicentennial Scientific Research Grant.

³ To whom correspondence should be addressed: Nucleotide Research Laboratory, Dept. of Physiology, Faculty of Biological Sciences, P. Catholic University of Chile, Casilla 114-D, Santiago, Chile. Tel.: 562-6862858; Fax: 562-2225515; E-mail: jphuid{at}bio.puc.cl.

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