Zinc and Copper Modulate Differentially the P2X 4 Receptor

The rat ATP P2X 4 receptor was expressed in Xenopus laevis oocytes to assess the effect of zinc and copper aspossible regulators of purinergic mechanisms. ATP applied for 20 s evoked aninward cationic current with a median effective concentration(EC 50 ) of 21.4 ± 2.8 μ M and a Hill coefficient( n H ) of 1.5 ± 0.1. Coapplication of ATP plus 10μ M zinc displaced leftward, in a parallel fashion, the ATPconcentration‐response curve, reducing the EC 50 to 8.4 ± 1.8μ M ( p < 0.01) without altering the receptor n H . The zinc potentiation was fast in onset, easilyreversible, and voltage‐independent and did not require metal preexposure. Thezinc EC 50 was 2‐5 μ M , with a bell‐shaped curve. Atconcentrations of 100‐300 μ M , zinc produced less potentiation, andat 1 m M , it inhibited 50% the ATP current. The effect of zinc wasmimicked by cadmium. In contrast, copper inhibited the ATP‐evoked currents ina time‐ and concentration‐dependent fashion, reducing the maximal current( I max ) without altering the EC 50 . Thecopper‐induced inhibition was slow in onset, slowly reversible, andvoltage‐independent. Whereas coapplication of 300 μ M copper plusATP reduced I max to 36.2 ± 5%, the coapplicationof, or 60‐s preexposure by, 10 μ M copper reduced I max to 79 ± 9.2% ( p < 0.05) and 39.6± 8.7% ( p < 0.01), respectively. The inhibition wasnoncompetitive in nature and mimicked by mercury. Cobalt, barium, andmanganese did not modify significantly the ATP‐evoked current, demonstratingmetal specificity. The simultaneous 1‐min preapplication of both metalsrevealed that the 10 μ M zinc‐induced potentiation was obliteratedby 10 μ M copper, whereas 30 μ M copper not only reducedthe potentiation, but inhibited the ATP response. Following coapplication ofboth metals for 20 s with ATP, at least 100 μ M copper was requiredto counteract the 10 μ M zinc‐induced potentiation. Thesimultaneous preincubation with both metals provided evidence for anoncompetitive interaction. We hypothesize the existence of metal bindingsite(s), which are most likely localized in the extracellular domain of theP2X 4 receptor structure. These sites are selective and accessibleto extracellular metal applications and bind micromolar concentrations ofmetals. The present results are compatible with the working hypothesis thattrace metals, such as copper and zinc, are physiological modulators of theP2X 4 receptor. The modulation of brain purinergic transmission by physiologically and toxicologically relevant trace metal cations is highlighted.