Effects of increasing Ca2+ channel-vesicle separation on facilitation at the crayfish inhibitory neuromuscular junction

We investigated the mechanism of facilitation at the crayfish inhibitory neuromuscular junction before and after blocking P-type Ca2+ channels. P-type channels have been shown to be closer to releasable synaptic vesicles than non-P-type channels at this synapse. Prior to the block of P-type channels, facilitation evoked by a train of 10 action potentials at 100 Hz was increased by application of 40 mM [Mg2+]o, but decreased by pressure-injected EGTA. Blocking P-type channels with 5 nM ω-Aga IVA, which reduced total Ca2+ influx and release to levels comparable to that recorded in 40 mM [Mg2+]o, did not change the magnitude of facilitation. We explored whether this observation could be attributed to the buffer saturation model of facilitation, since increasing the Ca2+ channel-vesicle separation could potentially enhance the role of endogenous buffers.The characteristics of facilitation in synapses treated with ω-Aga IVA were probed with broad action potentials in the presence of K+ channel blockers. After Ca2+ channel-vesicle separation was increased by ω-Aga IVA, facilitation probed with broad action potential was still decreased by EGTA injection and increased by 40 mM [Mg2+]o. EGTA-AM perfusion was used to test the impact of EGTA over a range of concentration in ω-Aga IVA-poisoned preparations. The results showed a concentration dependent decrease in facilitation as EGTA concentration rose. Thus, probing facilitation with EGTA and reduced Ca2+ influx showed that characteristics of facilitation are not changed after the role of endogenous buffer is enhanced by increasing Ca2+ channel-vesicle separation. There is no clear indication that buffer saturation has become the dominant mechanism for facilitation after ω-Aga IVA poisoning.Finally, we sought correlation between residual Ca2+ and the magnitude of facilitation. Using fluorescence transients of a low affinity Ca2+ indicator, we calculated the ratio of fluorescence amplitude measured immediately before test pulse (residual Ca2+) to that evoked during action potential (local Ca2+). This ratio provides an estimate of relative changes between residual Ca2+ and local Ca2+ important for release. There is a significant increase in the ratio when Ca2+ influx is reduced by 40 mM [Mg2+]o. The magnitude of facilitation exhibited a clear and positive correlation with the ratio, regardless of separation between Ca2+ channels and releasable vesicles. This correlation suggests the importance of relative changes between residual and local Ca2+ and lends support to the residual Ca2+ hypothesis of facilitation.