Ionotropic ATP receptors are widely expressed in mammalian CNS. Despite extensive functional characterization of neuronal homomeric P2X receptors in heterologous expression systems, the subunit composition of native central P2X ATP-gated channels remains to be elucidated. P2X4 and P2X6 are major central subunits with highly overlapping mRNA distribution at both regional and cellular levels. When expressed alone in Xenopus oocytes, P2X6 subunits do not assemble into surface receptors responsive to ATP applications. On the other hand, P2X4 subunits assemble into bona fide ATP-gated channels, slowly desensitizing and weakly sensitive to the partial agonist alpha,beta-methylene ATP and to noncompetitive antagonists suramin and pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid. We demonstrate here that the coexpression of P2X4 and P2X6 subunits in Xenopus oocytes leads to the generation of a novel pharmacological phenotype of ionotropic ATP receptors. Heteromeric P2X4+6 receptors are activated by low-micromolar alpha, beta-methylene ATP (EC50 = 12 microM) and are blocked by suramin and by Reactive Blue 2, which has the property, at low concentrations, to potentiate homomeric P2X4 receptors. The assembly of P2X4 with P2X6 subunits results from subunit-dependent interactions, as shown by their specific copurification from HEK-293 cells transiently transfected with various epitope-tagged P2X channel subunits. Our data strongly suggest that the numerous cases of neuronal colocalizations of P2X4 and P2X6 subunits observed in mammalian CNS reflect the native expression of heteromeric P2X4+6 channels with unique functional properties.

Small changes of extracellular pH activate depolarizing inward currents in most nociceptive neurons. It has been recently proposed that acid sensitivity of sensory as well as central neurons is mediated by a family of proton-gated cation channels structurally related to Caenorhabditis elegans degenerins and mammalian epithelial sodium channels. We describe here the molecular cloning of a novel human proton receptor, hASIC3, a 531-amino acid-long subunit homologous to rat DRASIC. Expression of homomeric hASIC3 channels in Xenopus oocytes generated biphasic inward currents elicited at pH Ͻ5, providing the first functional evidence of a human proton-gated ion channel. Contrary to the DRASIC current phenotype, the fast desensitizing early component and the slow sustained late component differed both by their cationic selectivity and by their response to the antagonist amiloride, but not by their pH sensitivity (pH 50 ϭ 3.66 vs. 3.82). Using RT-PCR and mRNA blot hybridization, we detected hASIC3 mRNA in sensory ganglia, brain, and many internal tissues including lung and testis, so hASIC3 gene expression was not restricted to peripheral sensory neurons. These functional and anatomical data strongly suggest that hASIC3 plays a major role in persistent proton-induced currents occurring in physiological and pathological conditions of pH changes, likely through a tissue-specific heteropolymerization with other members of the proton-gated channel family.

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