Presynaptic calcium channels and field-evoked transmitter exocytosis from cultured cerebellar granule cells

Regulated exocytosis from cultured rat cerebellar granule cells can be localized by the vesicle specific marker FM2-10 to specific sites, the highest density of which are at visible varicosities coinciding with neurite–neurite contacts. Exocytosis can be evoked by uniform electrical field pulses, which initiate tetrodotoxin-sensitive action potentials, or by elevated KCl. [ 3H] d-Aspartate is an authentic false transmitter in this preparation, judged by sensitivity of release to bafilomycin A1 and tetanus toxin. The coupling of presynaptic voltage-activated Ca 2+ channels to [ 3H] d-aspartate exocytosis was determined during field stimulation. The peak cytoplasmic free Ca 2+ concentration achieved in the varicosities was proportional to Ca 2+ entry during a 10 s train of pulses. L-type Ca 2+ channels did not contribute to either Ca 2+ entry or [ 3H] d-aspartate exocytosis. The P-type Ca 2+ channel antagonist ω-agatoxin-IVA (30 nM) only inhibited at 75% of the varicosities, although a mean 15% inhibition of Ca 2+ entry caused a 39% inhibition of exocytosis. In contrast the N-type Ca 2+ channel inhibitor ω-conotoxin-GVIA (1 μM), which inhibited at virtually all varicosities, caused mean inhibitions of Ca 2+ entry and exocytosis of 26% and 24% respectively. The toxin ω-conotoxin-MVIIC (5 μM), which inhibits N-, P- and Q-type Ca 2+ channels, was effective at all varicosities. The Q-type component of Ca 2+ entry was calculated to be only 5–10%; however, the additional inhibition of exocytosis was 30%. Thus P-type and particularly Q-type channels appear to be more closely coupled to exocytosis than N-type Ca 2+ channels. The residual Ca 2+ entry following 5 μM ω-conotoxin-MVIIC is scarcely coupled to release. The ω-agatoxin-IVA and ω-conotoxin-GVIA inhibitions of both Ca 2+ entry and exocytosis were additive and varied stochastically between individual varicosities. These results demonstrate that both Q- and P-type Ca 2+ channels are highly efficient in their coupling to amino acid exocytosis, with N-type less efficient, and L-type channels not at all. The Ca 2+ channel types coupled to exocytosis are also able to support exocytosis when evoked by either brief field-evoked action potentials or prolonged depolarization with KCl, indicating that these presynaptic channels, in contrast to those on the somata of the cells, can respond to widely different patterns of activation.