Central P2X₄and P2X₆Channel Subunits Coassemble into a Novel Heteromeric ATP Receptor

<jats:p>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<jats:sub>4</jats:sub>and P2X<jats:sub>6</jats:sub>are major central subunits with highly overlapping mRNA distribution at both regional and cellular levels. When expressed alone in<jats:italic>Xenopus</jats:italic>oocytes, P2X<jats:sub>6</jats:sub>subunits do not assemble into surface receptors responsive to ATP applications. On the other hand, P2X<jats:sub>4</jats:sub>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<jats:sub>4</jats:sub>and P2X<jats:sub>6</jats:sub>subunits in<jats:italic>Xenopus</jats:italic>oocytes leads to the generation of a novel pharmacological phenotype of ionotropic ATP receptors. Heteromeric P2X<jats:sub>4+6</jats:sub>receptors are activated by low-micromolar α,β-methylene ATP (EC<jats:sub>50</jats:sub>= 12 μ<jats:sc>m</jats:sc>) and are blocked by suramin and by Reactive Blue 2, which has the property, at low concentrations, to potentiate homomeric P2X<jats:sub>4</jats:sub>receptors. The assembly of P2X<jats:sub>4</jats:sub>with P2X<jats:sub>6</jats:sub>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<jats:sub>4</jats:sub>and P2X<jats:sub>6</jats:sub>subunits observed in mammalian CNS reflect the native expression of heteromeric P2X<jats:sub>4+6</jats:sub>channels with unique functional properties.</jats:p>