Mg²⁺ Inhibites ATP-Activated Current Mediated By P2X₄Receptors Expressed In Xenopus Oocytes: New Target For Pain?

Objective: To investigate the modulation of Mg²⁺on rat P2X₄receptors and relatedmodulatory site.Methods: The Asp280of the wild type P2X₄receptors were mutated into glutamine （D280Q）,glutamate （D280E） or removed Asp280from P2X₄receptors by site-directed mutagenesistechnique. Then the cDNA coding for the wild type and the mutant P2X₄receptors weretranscribed in vitro to cRNA, which were injected into the digested oocytes of an Xenopuslaevis by microinjection technique. The effects of extracellular Mg²⁺on ATP-activatedcurrents (I_ATP) mediated by P2X₄receptors were recorded by two-electrode voltage clamptechnique.Results:（1） Applying ATP could evoke a fast inward I_ATPin oocytes injected with the cRNAof P2X₄receptors.（2） The purinoceptor antagonists PPADS or suramin had no significanteffect on I_ATP.（3） In oocytes expressing P2X₄receptors, Mg²⁺（0.5-10mmol/L） inhibited theamplitude of I_ATPinduced by100μmol/L ATP in a concentration-dependent manner. Thecalculated Mg²⁺concentration that produced50%inhibitory （IC50） was （1.24±0.07） mmol/L.The inhibition of Mg²⁺was reversible，the I_ATPcould recover after washing10min.（4） Mg²⁺（1mmol/L） shifted the dose-response curve for I_ATPright-downward, reducing the maximalcurrent （Emax） by （42±2.1）%without changing the EC50.（5） The inhibitory effect of Mg²⁺reached the maximum after the cells was preincubated by Mg²⁺for80s.（6） The Mg²⁺inhibition of I_ATPwas independent of membrane potential from-120mV to+60mV.（7）Compared with the100μmol/L ATP-activated current in the wild type receptors, currentactivated by100μmol/L ATP mediated by D280Q mutant was much smaller. The peakcurrent was only （4.12±0.15）%of that seen in the wild type receptors. However, the D280Emutant responded to ATP stimulation with a current similar to that observed in cellsexpressing wild type receptors.（8） When lacking of Asp280, the current amplitude increasedobviously, and Mg²⁺（0.5-10mmol/L） did not affect the I_ATPsignificantly.Conclusion:（1） The inhibitory effect of Mg²⁺might be realized by acting on the site Asp280of the P2X₄receptors;（2） The inhibition of Mg²⁺on I_ATPmediated P2X₄receptors maycontribute to the function of Mg²⁺on pain. Objective: To study the effect of ethanol on ATP-activated current (I_ATP) mediated by ratP2X₄receptors.Methods: The rat P2X₄receptors were expressed in Xenopus oocytes by microinjectiontechnique. The effects of ethanol on P2X₄receptors were recorded by two-electrode voltageclamp.Results:（1） Ethanol （1-500mmol/L） inhibited I_ATPmediated by rat P2X₄receptors in aconcentration-dependent manner. After the extracellular ethanol was washed away, thecurrent could recover.（2） Ethanol （60mmol/L） shifted the dose-response curve to right in aparallel manner, and increased the EC50from （15.93±2.15） mmol/L to （24.56±3.12）mmol/L. The Emaxdid not change. When the concentration of ethanol was greater than500mmol/L, the EC50did not increase any more.（3） Changing membrane potential from-80mV to+20mV had not significant effect on ethanol inhibition of I_ATP, and the reversalpotential was not changed by applying ethanol.（4） Mg²⁺（1mmol/L） and ethanol （10mmol/L）could inhibit I_ATPby （39±3.8）%and （19±1.6）%respectively, when co-applying themcould produce a synergistic effect.Conclusion:（1） Ethanol may inhibit I_ATPmediated by rat P2X₄receptors through allostericregulation.（2） Mg²⁺and ethanol might have different modulatory sites on the ectodomain ofP2X₄receptors.