Localization of ATP-gated ion channels in cerebellum using P2x(2)R subunit-specific antisera

THE distribution of the P2x(2) purinoceptor subunit protein, which forms ATP-gated ion channels by homo- and hetero-multimeric assembly, was examined in the adult rat and guinea-pig cerebellum using two novel antisera generated against separate 18 amino acid sequences located in the predicted extracellular domain of this subunit. These antisera, the first available for labelling the P2x(2)R subunit protein, were validated by selective labelling of a fusion protein containing the target amino acid sequences, and in cerebellum, by peptide specific block of immunoreactivity and by comparison with the distribution of P2x(2)R mRNA. P2x(2)R-like immunoreactivity was seen in Purkinje cells, specifically the soma and dendrites, neurons in the granular and molecular layers and deep cerebellar nuclei. The identification of P2x(2)R-like immunoreactivity within the cerebellar neural circuitry is consistent with a role for extracellular ATP acting as a fast neurotransmitter in motor learning and coordination of movement. Additionally, labelling of neuroglia and fibre tracts supports a diverse role for extracellular ATP in CNS homeostasis.