Central P2X₄and P2X₆Channel Subunits Coassemble into a Novel Heteromeric ATP Receptor

Abstract

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. P2X 4 and P2X 6 are major central subunits with highly overlapping mRNA distribution at both regional and cellular levels. When expressed alone in Xenopus oocytes, P2X 6 subunits do not assemble into surface receptors responsive to ATP applications. On the other hand, P2X 4 subunits assemble into bona fide ATP-gated channels, slowly desensitizing and weakly sensitive to the partial agonist α,β-methylene ATP and to noncompetitive antagonists suramin and pyridoxal-5-phosphate-6-azophenyl-2′,4′-disulfonic acid. We demonstrate here that the coexpression of P2X 4 and P2X 6 subunits in Xenopus oocytes leads to the generation of a novel pharmacological phenotype of ionotropic ATP receptors. Heteromeric P2X 4+6 receptors are activated by low-micromolar α,β-methylene ATP (EC 50 = 12 μ m ) and are blocked by suramin and by Reactive Blue 2, which has the property, at low concentrations, to potentiate homomeric P2X 4 receptors. The assembly of P2X 4 with P2X 6 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 P2X 4 and P2X 6 subunits observed in mammalian CNS reflect the native expression of heteromeric P2X 4+6 channels with unique functional properties.