The manner in which presynaptic Ca2+ influx controls the release of neurotransmitter was investigated at the granule cell to Purkinje cell synapse in rat cerebellar slices. Excitatory postsynaptic currents were measured using whole-cell voltage clamp, and changes in presynaptic Ca2+ influx were determined with the Ca(2+)-sensitive dye furaptra. We manipulated presynaptic Ca2+ entry by altering external Ca2+ levels and by blocking Ca2+ channels with Cd2+ or with the toxins omega-conotoxin GVIA and omega-Aga-IVA. For all of the manipulations, other than the application of omega-Aga-IVA, the relationship between Ca2+ influx and release was well approximated by a power law, n approximately 2.5. When omega-Aga-IVA was applied, release appeared to be more steeply dependent on Ca2+ (n approximately 4), suggesting that omega-Aga-IVA-sensitive channels are more effective at triggering release. Based on interactive effects of toxins on synaptic currents, we conclude that multiple types of Ca2+ channels synergistically control individual release sites.

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Voltage-dependent calcium channels mediate calcium entry into neurons, which is crucial for many processes in the brain including synaptic transmission, dendritic spiking, gene expression and cell death. Many types of calcium channels exist in mammalian brains, but high-affinity blockers are available for only two types, L-type channels (targeted by nimodipine and other dihydropyridine channel blockers) and N-type channels (targeted by omega-conotoxin). In a search for new channel blockers, we have identified a peptide toxin from funnel web spider venom, omega-Aga-IVA, which is a potent inhibitor of both calcium entry into rat brain synaptosomes and of 'P-type' calcium channels in rat Purkinje neurons. omega-Aga-IVA will facilitate characterization of brain calcium channels resistant to existing channel blockers and may assist in the design of neuroprotective drugs.

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