title: 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.
abstract: 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.
authors.affiliation: Centro de Regulación Celular y Patología J. V. Luco, Instituto Milenio de Biología Fundamental y Aplicada MIFAB, Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile.
authors.forename: Claudio
authors.initials: C
authors.lastname: Coddou
authors.forename: Claudio
authors.initials: C
authors.lastname: Acuña-Castillo
authors.forename: Paulina
authors.initials: P
authors.lastname: Bull
authors.forename: J Pablo
authors.initials: JP
authors.lastname: Huidobro-Toro
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000495:Allosteric Site / Q000235:genetics; D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D000818:Animals; D003300:Copper / Q000378:metabolism / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005260:Female; D015640:Ion Channel Gating / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D009865:Oocytes / Q000166:cytology; D017434:Protein Structure, Tertiary / Q000235:genetics; D058919:Purinergic P2 Receptor Antagonists; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058848:Receptors, Purinergic P2X4; D014982:Xenopus laevis; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D000596:Amino Acids; C548228:P2rx4 protein, rat; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058848:Receptors, Purinergic P2X4; D003300:Copper; D000255:Adenosine Triphosphate; D015032:Zinc