omega-Conotoxin binding sites and regulation of transmitter release in cerebellar granule neurons

The protective action of Ca2+ and a series of other divalent cations on heat inactivation (48 degrees C, 30 min) of [125I]omega-conotoxin (CTX) binding sites was investigated in membranes prepared from rat forebrain. Moreover, the influence of GABA (500 microM) on this protection was studied. Binding of [125I]CTX as well as its inhibitory action on K+ (55 mM) stimulated, Ca(2+)-dependent transmitter release were studied in rat cerebellar granule neurons cultured in the presence or absence of the GABAA receptor against THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol). In cells cultured in the presence of THIP (150 microM) it was investigated whether the ability of THIP to inhibit evoked transmitter release could be influenced by CTX. Ca2+ and other divalent cations could effectively protect against heat inactivation of [125I]CTX binding sites in rat forebrain membranes, but this protective action was not influenced by the presence of 500 microM GABA. The cultured cerebellar granule neurons exhibited specific binding sites for [125I]CTX, the number of which was independent of exposure of the cells to THIP during the culture period. Evoked transmitter release was inhibited by CTX with an IC50 value of 13 nM. In neurons cultured in the presence of 150 microM THIP, THIP could dose-dependently inhibit evoked transmitter release, but this inhibitory action was not influenced by CTX (20 nM). The results show that cerebellar granule neurons exhibit functionally meaningful CTX binding sites. An association between such sites and GABA receptors is not apparent.