We investigated the Ca2+ channel-synaptic vesicle topography at the inhibitor of the crayfish ( Procambarus Clarkii) neuromuscular junction (NMJ) by analyzing the effect of different modes of Ca2+ channel block on transmitter release. Initial identification of Ca2+ channels revealed the presence of two classes, P and non-P-type with P-type channels governing ∼70% of the total Ca2+ influx. The remaining Ca2+ influx was completely blocked by Cd2+ but not by saturating concentrations of ω-conotoxins MVIIC and GVIA, or nifedipine and SNX-482. To examine the relative spatial distribution of Ca2+ channels with respect to synaptic vesicles, we compared changes in inhibitory postsynaptic current amplitude and synaptic delay resulting from different spatial profiles of [Ca2+]i around release sites. Specifically, addition of either [Mg2+]o, which decreases single-channel current, or ω-Aga IVA, which completely blocks P-type channels, prolonged synaptic delay by a similar amount when Ca2+ influx block was <40%. Because non-P-type channels are able to compensate for blocked P-type channels, it suggests that these channels overlap considerably in their distribution. However, when Ca2+ influx was blocked by ∼50%, ω-Aga IVA increased delay significantly more than Mg2+, suggesting that P-type channels are located closer than non-P-type channels to synaptic vesicles. This distribution of Ca2+ channels was further supported by the observations that non-P-type channels are unable to trigger release in physiological saline and EGTA preferentially prolongs synaptic delay dominated by non-P-type channels when transmitter release is evoked with broad action potentials. We therefore conclude that although non-P-type channels do not directly trigger release under physiological conditions, their distribution partially overlaps with P-type channels.
Transmitter release is evoked with low probability predominately by calcium flux through single channel openings at the frog neuromuscular junction
