Browsing by Author "Huidobro-Toro, J. Pablo"

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    Differential role of extracellular histidines in copper, zinc, magnesium and proton modulation of the P2X7 purinergic receptor
    (2007) Acuna-Castillo, Claudio; Coddou, Claudio; Bull, Paulina; Brito, Jocelyn; Huidobro-Toro, J. Pablo
    The P2X(7) receptor is a non-selective cationic channel activated by extracellular ATP, belonging to the P2X receptor family. To assess the role of extracellular histidines on the allosteric modulation of the rat P2X(7) receptor by divalent metals (copper, zinc and magnesium) and protons, these amino acid residues were singly substituted for corresponding alanines. Wild-type and mutated receptors were injected to Xenopus laevis oocytes; metal-related effects were evaluated by the two-electrode voltage-clamp technique. Copper inhibited the ATP-gated currents with a median inhibitory concentration of 4.4 +/- 1.0 mu mol/L. The inhibition was non-competitive and time-dependent; copper was 60-fold more potent than zinc. The mutant H267A, resulted in a copper resistant receptor; mutants H201A and H130A were less sensitive to copper inhibition (p < 0.05). The rest of the mutants examined, H62A, H85A, and H219A, conserved the copper-induced inhibition. Only mutants H267A and H219A were less sensitive to the modulator action of zinc. Moreover, the magnesium-induced inhibition was abolished exclusively on the H130A and H201A mutants, suggesting that this metal may act at a novel cationic modulator site. Media acidification inhibited the ATP-gated current 87 +/- 3%; out of the six mutants examined, only H130A was significantly less sensitive to the change in pH, suggesting that His-130 could be involved as a pH sensor. In conclusion, while His-267 is critically involved in the copper or zinc allosteric modulation, the magnesium inhibitory effects is related to His-130 and His-201, His-130 is involved in proton sensing, highlighting the role of defined extracellular histidines in rat P2X(7) receptor allosteric modulation.
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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
    (2007) Coddou, Claudio; Acuna-Castillo, Claudio; Bull, Paulina; Huidobro-Toro, J. Pablo
    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 P2X(4) receptor involved in trace metal interaction, specifically in the immediate linear vicinity of His(140), 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 Thr(123) and Thr(146) for alanines. cDNAs for the wild-type and the receptor mutants were expressed in Xenopus laevis oocytes and examined by the two-electrode technique. Cys(132), but not Cys(126), 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, Asp(138), but not Asp(131) was critical for copper and zinc inhibition; moreover, mutant D138A was 20-fold more reactive to zinc potentiation than wild-type receptors. Asp(129), Asp131, and Thr(133) 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.