Interaction of SNARE complexes with P/Q-type calcium channels in rat cerebellar synaptosomes

P- and Q-type calcium channels, which trigger rapid neurotransmitter release at many mammalian synapses, are blocked by ω-conotoxin MVIIC. ¹²⁵I-ω-Conotoxin MVIIC binding to rat cerebellar synaptosomes was not displaced by ω-conotoxins GVIA or MVIIA (K(i) > 1 μM), which are selective for N-type calcium channels. Solubilized ¹²⁵I-ω-conotoxin MVIIC receptors were specifically recognized by antibodies directed against α₁A calcium channel subunits, proteins known to constitute a pore with P/Q-like channel properties. Antibodies against syntaxin 1, SNAP 25, and VAMP 2 (synaptobrevin) each immunoprecipitated a similar fraction (20-40%) of ω- conotoxin MVIIC receptors. Immunoprecipitation was not additive, suggesting that heterotrimeric (SNARE) complexes containing these three proteins interact with P/Q-type calcium channels. Immobilized monoclonal anti-syntaxin antibodies retained α₁A calcium channel subunits of 220, 180 and 160 kDa monitored by immunoblotting with site directed antibodies. Synaptotagmin was detected in channel-associated complexes, but not synaptophysin, Rab 3A nor rat cysteine string protein. Trimeric SNARE complexes are implicated in calcium-dependent exocytosis, a process thought to be regulated by synaptotagmin. Our results indicate that these proteins interact with P/Q- type calcium channels, which may optimize their location within domains of calcium influx.