Binding and functions of extracellular ATP in cultured cerebellar granule neurons

Selected purinoceptor modulators were previously shown to prevent glutamate-evoked cytotoxicity in cerebellar granule neurons. In the same cellular system, we now identify and characterize the presence of P₂ receptors. The binding of [³H]ATP to membranes of cerebellar granule neurons grows monotonically as a function of neuronal differentiation, is saturable and reaches steady state within 6 min. Scatchard plot of the equilibrium saturation data is curvilinear with a K(d) value of 28 nM and a B(max) value of 87 pmol/mg of protein for the high affinity binding sites and a K(d) value of 1.5 μM and B(max) value of 1.2 nmol/mg of protein, for the more numerous low affinity binding sites. We also show that extracellular ATP increases the release, but not the uptake, of [³H]D-aspartate and that it furthermore potentiates the release of [³H]D-aspartate evoked by glutamate and KCl. ATP itself is released by cerebellar granule cultures and such release grows monotonically as a function of neuronal differentiation. These data are consistent with the role that ATP is believed to play as a cotransmitter for the central nervous system.