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 Ca(2+) 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 [Mg(2+)](o), but decreased by pressure-injected EGTA. Blocking P-type channels with 5 nM omega-Aga IVA, which reduced total Ca(2+) influx and release to levels comparable to that recorded in 40 mM [Mg(2+)](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 Ca(2+) channel-vesicle separation could potentially enhance the role of endogenous buffers. The characteristics of facilitation in synapses treated with omega-Aga IVA were probed with broad action potentials in the presence of K(+) channel blockers. After Ca(2+) channel-vesicle separation was increased by omega-Aga IVA, facilitation probed with broad action potential was still decreased by EGTA injection and increased by 40 mM [Mg(2+)](o). EGTA-AM perfusion was used to test the impact of EGTA over a range of concentration in omega-Aga IVA-poisoned preparations. The results showed a concentration dependent decrease in facilitation as EGTA concentration rose. Thus, probing facilitation with EGTA and reduced Ca(2+) influx showed that characteristics of facilitation are not changed after the role of endogenous buffer is enhanced by increasing Ca(2+) channel-vesicle separation. There is no clear indication that buffer saturation has become the dominant mechanism for facilitation after omega-Aga IVA poisoning. Finally, we sought correlation between residual Ca(2+) and the magnitude of facilitation. Using fluorescence transients of a low affinity Ca(2+) indicator, we calculated the ratio of fluorescence amplitude measured immediately before test pulse (residual Ca(2+)) to that evoked during action potential (local Ca(2+)). This ratio provides an estimate of relative changes between residual Ca(2+) and local Ca(2+) important for release. There is a significant increase in the ratio when Ca(2+) influx is reduced by 40 mM [Mg(2+)](o). The magnitude of facilitation exhibited a clear and positive correlation with the ratio, regardless of separation between Ca(2+) channels and releasable vesicles. This correlation suggests the importance of relative changes between residual and local Ca(2+) and lends support to the residual Ca(2+) hypothesis of facilitation.