Relative Distribution of Ca²⁺Channels at the Crayfish Inhibitory Neuromuscular Junction

Abstract

We investigated the Ca(2+) channel-synaptic vesicle topography at the inhibitor of the crayfish (Procambarus Clarkii) neuromuscular junction (NMJ) by analyzing the effect of different modes of Ca(2+) channel block on transmitter release. Initial identification of Ca(2+) channels revealed the presence of two classes, P and non-P-type with P-type channels governing approximately 70% of the total Ca(2+) influx. The remaining Ca(2+) influx was completely blocked by Cd(2+) but not by saturating concentrations of omega-conotoxins MVIIC and GVIA, or nifedipine and SNX-482. To examine the relative spatial distribution of Ca(2+) channels with respect to synaptic vesicles, we compared changes in inhibitory postsynaptic current amplitude and synaptic delay resulting from different spatial profiles of [Ca(2+)](i) around release sites. Specifically, addition of either [Mg(2+)](o), which decreases single-channel current, or omega-Aga IVA, which completely blocks P-type channels, prolonged synaptic delay by a similar amount when Ca(2+) 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 Ca(2+) influx was blocked by approximately 50%, omega-Aga IVA increased delay significantly more than Mg(2+), suggesting that P-type channels are located closer than non-P-type channels to synaptic vesicles. This distribution of Ca(2+) 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.

Modeling of quantal neurotransmitter release kinetics in the presence of fixed and mobile calcium buffers

“…Consistently, recent studies have demonstrated that the time course of quantal release is highly regulated and reflects the plasticity of the vesicular secretion (Sabatini and Regehr 1999;Lin and Farber 2002;Allana and Lin 2004). Thus, to understand how the reliability and strength of synaptic transmission can be regulated, we need to investigate how Ca 2+ mediates the relative timings of quantal events (Naoki et al 2005).…”

Modulation of the kinetics of evoked quantal release at mouse neuromuscular junctions by calcium and strontium

“…2000; Geiger and Jonas 2000; Lin and Faber 2002). The synaptic delay at the crayfish neuromuscular junction can also be prolonged by blockade of P‐type Ca 2+ channels with ω‐Aga IVA toxin or by increasing [Mg 2+ ] o (Allana and Lin 2004). Further, it has been shown that intraterminal Ca 2+ buffers can influence the duration of the release process (Chen and Regehr 1999; Chuhma and Ohmori 2002).…”

Increased Ca2+ influx through Na+/Ca2+ exchanger during long‐term facilitation at crayfish neuromuscular junctions

“…Allana & Lin (2004) report the presence of Ca 2+ influx through non‐P, non‐Q, non‐N, non‐L, non‐R‐type Ca 2+ channels at crayfish opener inhibitor motor nerve terminals, which appear to be more distant from vesicles than the P‐type Ca 2+ channels evoking release. This component of Ca 2+ influx did not normally support or modulate transmitter release, although it could do so under conditions of blockade of P‐type Ca 2+ channels and prolonged depolarization.…”

“…We felt that the most straightforward interpretation of this result was as follows: following an LTF‐inducing tetanus, there is an increase in the rise in [Ca 2+ ] i during a brief train, and this is likely to reflect an increased influx in Ca 2+ ions through the P‐type Ca 2+ channels opened by action potentials and supporting transmitter release (Wright et al 1996; Allana & Lin, 2004). This, then, explains immediately why the EJPs in such bursts are larger.…”