Calcium channel-synaptic vesicle organization at the crayfish inhibitory neuromuscular junction and its functional implication

The Ca2+ channel-synaptic vesicle organization and the mechanism of F2 facilitation were investigated at the crayfish inhibitory neuromuscular junction (NMJ) using electrophysiological, pharmacological and Ca2+ imaging techniques. Saturating concentrations of o-Aga IVA, a specific P-type Ca2+ channel blocker, completely suppressed release, but not Ca2+ influx under physiological conditions. The remaining non-P-type channels could not be blocked by any of the known specific Ca2+ channel blockers. The absence of release after P-type channel block, even though ∼30% Ca2+ influx remained, suggests that these channels were in closer proximity to synaptic vesicles than non-P-type channels. Changes of synaptic delay in response to lowered Ca2+ influx, probed with broad APs, further supported this topography. Specifically, 5 nM o-Aga IVA increased delay significantly more than 40 mM Mg2+ (non-specific Ca2+ channel blocker) even though both reduced Ca2+ influx by ∼50%, and EGTA (Ethylene glycol bis (beta-aminoethyl ether) N, N'-tetracetic acid), a Ca2+ buffer with a slow on-rate, was unable to prolong delay in control conditions, but significantly delayed release in the presence of o-Aga IVA.;F2 facilitation at this synapse is characterized by enhanced release and shortened synaptic delay (DeltaDelayfac). The ability to manipulate Ca2+ channel-synaptic vesicle distance provided a unique opportunity to determine whether the mechanism underlying facilitation was due to residual Ca2+ or buffer saturation. In both control and o-Aga IVA treated preparations, reducing Ca2+ influx by elevating the [Mg2+]o concentration increased facilitation, while suppressing residual [Ca2+]i by EGTA decreased it, suggesting that residual [Ca2+] i governed facilitation at this synapse regardless of the distance between Ca2+ channels and synaptic vesicles. The increasing trends of facilitation magnitude and DeltaDelayfac observed in the presence of increasing [Mg2+]o and o-Aga IVA concentrations could also be accounted for by invoking the residual Ca2+ hypothesis. Finally, the mechanism underlying DeltaDelayfac was examined in detail. Ca2+ influx, monitored using imaging techniques, was found not to be elevated during facilitation, suggesting that events downstream of Ca2+ influx caused the shortening of delay. A hypothesis that considers differences in local Ca2+ concentration, and the affinity of the Ca2+ sensors responsible for release, is proposed.