Browsing by Author "Lorca, RA"

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    Extracellular histidine residues identify common structural determinants in the copper/zinc P2X2 receptor modulation
    (2005) Lorca, RA; Coddou, C; Gazitúa, MC; Bull, P; Arredondo, C; Huidobro-Toro, JP
    To assess the mechanism of P2X(2) receptor modulation by transition metals, the cDNA for the wild-type receptor was injected to Xenopus laevis oocytes and examined 48-72 h later by the two-electrode voltage-clamp technique. Copper was the most potent of the trace metals examined; at 10 mu M it evoked a 25-fold potentiation of the 10 mu M ATP-gated currents. Zinc, nickel or mercury required 10-fold larger concentrations to cause comparable potentiations, while palladium, cobalt or cadmium averaged only 12- and 3-fold potentiations, respectively. Platinum was inactive. The non-additive effect of copper and zinc at 10-100 mu M suggests a common site of action; these metals also shifted to the left the ATP concentration-response curves. To define residues necessary for trace metal modulation, alanines were singly substituted for each of the nine histidines in the extracellular domain of the rat P2X(2) receptor. The H120A and H213A mutants were resistant to the modulator action of copper, zinc and other metals with the exception of mercury. Mutant H192A showed a reduction but not an abrogation of the copper or zinc potentiation. H245A showed less affinity for copper while this mutant flattened the zinc-induced potentiation. Mutant H319A reduced the copper but not the zinc-induced potentiation. In contrast, mutants H125A, H146A, H152A and H174A conserved the wild-type receptor sensitivity to trace metal modulation. We propose that His120, His192, His213 and His245 form part of a common allosteric metal-binding site of the P2X(2) receptor, which for the specific coordination of copper, but not zinc, additionally involves His319.
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    Heavy metals modulate the activity of the purinergic P2X4 receptor
    (2005) Coddou, C; Lorca, RA; Acuña-Castillo, C; Grauso, M; Rassendren, F; Huidobro-Toro, JP
    To further characterize the nature of the regulatory metal-binding sites of the rat P2X(4) receptor, several transition heavy metals were tested to examine their ability to mimic the facilitator action of zinc or the inhibitory action of copper. cDNA coding for the rat P2X(4) receptor was injected into Xenopus laevis oocytes; the two-electrode voltage-clamp technique was used to measure and quantify the ATP-evoked currents in the absence or presence of the metals. Cadmium facilitated the ATP-gated currents in a reversible and voltage-independent manner; maximal potentiation occurred within less than 1 min.
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    The human prion octarepeat fragment prevents and reverses the inhibitory action of copper in the P2X4 receptor without modifying the zinc action
    (2003) Lorca, RA; Chacón, M; Barría, MI; Inestrosa, NC; Huidobro-Toro, JP
    Human prion protein fragments (PrP60-67 or PrP59-91) prevented and reversed the inhibition elicited by 5 mum copper on the P2X(4) receptor expressed in Xenopus laevis oocytes. A 60-s pre-application of 5 muM copper caused a 69.2 +/- 2.6% inhibition of the 10 muM adenosine triphosphate (ATP)-evoked currents, an effect that was prevented by mixing 5 muM copper with 0.01-10 muM of the PrP fragments 1-min prior to application. This interaction was selective, as PrP59-91 did not alter the facilitatory action of zinc. The EC50 of PrP60-67 and PrP59-91 for the reduction of the copper inhibition were 4.6 +/- 1 and 1.3 +/- 0.4 muM, respectively. A synthetic PrP59-91 variant in which all four His were replaced by Ala was inactive. However, the replacement of Trp in each of the four putative copper-binding domains by Ala slightly decreased its potency. Furthermore, the application of 10 muM PrP59-91 reversed the copper-evoked inhibition, restoring the ATP concentration curve to the same level as the non-inhibited state. Fragment 139-157 of betaA4 amyloid precursor protein also prevented the action of copper; its EC50 was 1.6 +/- 0.1 muM; the metal chelator penicillamine was equipotent with PrP60-67, but carnosine was significantly less potent. Our findings highlight the role of PrP in copper homeostasis and hint at its possible role as a modulator of synapses regulated by this trace metal.