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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The mammalian P2X receptor gene family encodes two-transmembrane domain nonselective cation channels gated by extracellular ATP. Anatomical localization data obtained by in situ hybridization and immunocytochemistry have shown that neuronal P2X subunits are expressed in specific but overlapping distribution patterns. Therefore, the native ionotropic ATP receptors diversity most likely arises from interactions between different P2X subunits that generate hetero-multimers phenotypically distinct from homomeric channels. Rat P2X 1 and P2X 5 mRNAs are localized within common subsets of peripheral and central sensory neurons as well as spinal motoneurons. The present study demonstrates a functional association between P2X 1 and P2X 5 subunits giving rise to hybrid ATP-gated channels endowed with the pharmacology of P2X 1 and the kinetics of P2X 5 . When expressed in Xenopus oocytes, hetero-oligomeric P2X 1؉5 ATP receptors were characterized by slowly desensitizing currents highly sensitive to the agonist ␣,␤-methylene ATP (EC 50 ‫؍‬ 1.1 M) and to the antagonist trinitrophenyl ATP (IC 50 ‫؍‬ 64 nM), observed with neither P2X 1 nor P2X 5 alone. Direct physical evidence for P2X 1؉5 co-assembly was provided by reciprocal subunitspecific co-purifications between epitope-tagged P2X 1 and P2X 5 subunits transfected in HEK-293A cells.Ionotropic ATP receptors constitute a unique class of neurotransmitter-gated ion channels generated from the assembly of P2X subunits having two transmembrane-spanning domains and a protein architecture similar to the one of the amiloridesensitive sodium channels (1, 2). Functional characterization studies of the seven mammalian cloned P2X subunits heterologously expressed as homomeric channels allowed to classify them in three groups according to their properties of desensitization and to their sensitivity to the agonist ␣,␤-methylene ATP (␣␤m-ATP) 1 : (i) rapidly desensitizing and ␣␤m-ATP-sensitive receptors including P2X 1 and P2X 3 (3-5), (ii) moderately desensitizing and ␣␤m-ATP-insensitive receptors including P2X 4 and P2X 6 (6 -12), and (iii) nondesensitizing as well as ␣␤m-ATP-insensitive receptors including P2X 2 , P2X 5 , and P2X 7 (11)(12)(13)(14). Results from Northern blots and in situ hybridization data (11) have indicated that the six neuronal P2X subunits genes are transcribed in specific but overlapping populations in the central and peripheral nervous system (1, 11). This strongly suggests that neuronal P2X subunits belonging to different functional groups might co-assemble into heteromultimeric channels.All P2X subunits have been detected in peripheral sensory ganglia, reinforcing the view that synaptically or lytically released ATP could play an important signaling role in sensory pathways (1,11,15). Rat P2X 3 subunits have been reported to be exclusively expressed in small to medium-sized isolectin B4-positive nociceptive neurons in nodose, trigeminal, and dorsal root ganglia (4, 5, 15). A significant proportion of sensory neurons are thought to express hetero-oligomeric P2X...

A novel member of the ionotropic ATP receptor gene family has been identified in human brain. This 422 amino acid long P2X receptor subunit has 62% sequence identity with rat P2X 5 . Several characteristic motifs of ATP-gated channels are present in its primary structure, but this P2Xs-related subunit displays a single transmembrane domain. Heterologous expression of chimeric subunits containing the ( -terminal domain of rat P2Xs leads to the formation of desensitizing functional ATPgated channels in Xenopus oocytes. The developmental^ regulated mRNA, found in two splicing variant forms, is expressed at high levels in brain and immune system.

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