Bidirectional modulation of transmitter release by calcium channel/syntaxin interactions in vivo.

Protein interactions within the active zone of the nerve terminal are critical for regulation of transmitter release. The SNARE protein syntaxin 1A, primarily known for important interactions that control vesicle fusion, also interacts with presynaptic voltage-gated calcium channels. Based on recordings of calcium channel function in vitro, it has been hypothesized that syntaxin 1A-calcium channel interactions could alter calcium channel function at synapses. However, results at synapses in vitro suggest two potentially opposing roles: enhancement of neurotransmitter release by positioning docked vesicles near calcium channels and inhibition of calcium channel function by interaction with SNARE proteins. We have examined the possibility that these two effects of syntaxin can occur at synapses by studying the effects on transmitter release of manipulating syntaxin 1A-calcium channel interactions at Xenopus tadpole tail neuromuscular synapses in vivo. Introduction of synprint peptides, which competitively perturb syntaxin 1A-calcium channel interactions, decreased quantal content at these synapses and increased paired-pulse and tetanic facilitation. In contrast, injecting mRNA for mutant (A240V, V244A) syntaxin 1A, which reduces calcium channel modulation but not binding in vitro, increased quantal content and decreased paired-pulse and tetanic facilitation. Injection of wild-type syntaxin 1A mRNA had no effect. The opposing effects of synprint peptides and mutant syntaxin 1A provide in vivo support for the hypothesis that these interactions serve both to colocalize calcium channels with the release machinery and to modulate the functional state of the calcium channel. As such, these two effects of syntaxin on calcium channels modulate transmitter release in a bidirectional manner.

Spatial Distribution of Calcium Entry Evoked by Single Action Potentials within the Presynaptic Active Zone.

The nature of presynaptic calcium (Ca(2+)) signals that initiate neurotransmitter release makes these signals difficult to study, in part because of the small size of specialized active zones within most nerve terminals. Using the frog motor nerve terminal, which contains especially large active zones, we show that increases in intracellular Ca(2+) concentration within 1 msec of action potential invasion are attributable to Ca(2+) entry through N-type Ca(2+) channels and are not uniformly distributed throughout active zone regions. Furthermore, changes in the location and magnitude of Ca(2+) signals recorded before and after experimental manipulations (omega-conotoxin GVIA, diaminopyridine, and lowered extracellular Ca(2+)) support the hypothesis that there is a remarkably low probability of a single Ca(2+) channel opening within an active zone after an action potential. The trial-to-trial variability observed in the spatial distribution of presynaptic Ca(2+) entry also supports this conclusion, which differs from the conclusions of previous work in other synapses.

Repolarization of the presynaptic action potential and short-term synaptic plasticity in the chick ciliary ganglion.

Stimulation-induced increases in synaptic efficacy have been described as being composed of multiple independent processes that arise from the activation of distinct mechanisms at the presynaptic terminal. In the chick ciliary ganglion, four components of short-term synaptic plasticity have been described: F1 and F2 components of facilitation, augmentation, and potentiation. In the present study, intracellular recording from the presynaptic calyciform nerve terminal of the chick ciliary ganglion revealed that the late repolarization and afterhypolarization (AHP) phases of the presynaptic action potential are affected by repetitive stimulation and that the time course of these effects parallel that of facilitation. The effects of these changes in the presynaptic action potential time course on calcium influx were tested by using the recorded action potential waveforms as voltage command stimuli during whole-cell patch-clamp recordings from acutely isolated chick ciliary ganglion neurons. The "facilitated" action potential waveform (slowed repolarization, decreased AHP amplitude) evoked calcium current with slightly but significantly greater total calcium influx. Taken together, these results are consistent with the hypothesis that activity-dependent changes in the presynaptic action potential are one of several mechanisms contributing to the facilitation phase of stimulation-induced increases in transmitter release in this preparation.

Presynaptic calcium influx, neurotransmitter release, and neuromuscular disease.

The synapse between a spinal motor neuron and a muscle cell is normally very effective at eliciting muscle contraction. A reliable connection between these two cells occurs because a single action potential reaching the motor nerve terminal normally releases hundreds of packets of transmitter containing thousands of chemical transmitter molecules, which cross the synapse and encounter a specialized region of postsynaptic muscle. Within the muscle membrane are thousands of receptor proteins specific for this transmitter. Activation of these postsynaptic receptors allows positively charged ions to cross the muscle membrane, generating a muscle cell action potential that leads to muscle contraction. Because of its size, contraction of a muscle cell requires the activation of an exceptionally large number of neurotransmitter receptors. To understand the regulation of this reliable communication and to elucidate details of pathological conditions that lead to muscle weakness, we have studied the subcellular mechanisms that govern synaptic transmission at the neuromuscular junction (NMJ). This article will review recent electron microscopic, electrophysiological, and imaging data in a discussion of the function of the motor nerve terminal in both normal and diseased states. Taken together, the existing data lead us to hypothesize that a small fraction of available calcium channels open within the transmitter releasing regions of the NMJ and that each vesicle fusion event is triggered by calcium flux through a single channel opening.