The mechanism by which ethanol inhibits rat P2X4 receptors is altered by mutation of histidine 241

1 We investigated ethanol inhibition of the rat P2X4 receptor and the contribution of the three histidine residues in the extracellular loop of this receptor to ethanol inhibition of receptor function, using site-directed mutagenesis and electrophysiological characterization of recombinant receptors. 2 In the wild-type receptor, 50, 200 and 500 mM ethanol increasingly shifted the ATP concentration–response curve to the right in a parallel manner, increasing the EC50 value without affecting Emax. However, 750 or 900 mM ethanol did not produce a further increase in the EC50 value of the ATP concentration–response curve, suggesting that this inhibition is not competitive. 3 The P2X4 receptor mutations H140A and H286A did not significantly alter ethanol inhibition of ATP-activated current. By contrast, the mutation H241A changed the mechanism by which ethanol inhibits receptor function; viz., ethanol inhibition was not associated with an increased EC50 value of the ATP concentration–response curve, instead, ethanol decreased the maximal response to ATP without affecting the EC50 value of the ATP concentration–response curve. 4 Ethanol inhibition of the H241A mutant was voltage independent between −60 and +20 mV and ethanol did not alter the reversal potential of ATP-activated current. In addition, ethanol decreased the desensitization rate of the H241A-mediated current. 5 The purinoceptor antagonists, suramin and pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), did not alter the magnitude of ethanol inhibition of ATP-activated current in the H241A mutant. 6 The results suggest that ethanol inhibits the wild-type rat P2X4 receptor by an allosteric action to increase the EC50 value of the ATP concentration–response curve, the P2X4 receptor mutation H241A alters the mechanism by which ethanol inhibits P2X4 receptor function, and ethanol and PPADS or suramin appear to inhibit H241A-mutated receptors at independent sites. British Journal of Pharmacology (2005) 145, 576–586. doi:10.1038/sj.bjp.0706192