Interactions of Presynaptic Ca2+ Channels and Snare Proteins in Neurotransmitter Release

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Abstract
N‐ and P/Q‐type Ca2+ channels are localized in high density in presynaptic nerve terminals and are crucial elements in neuronal excitation‐secretion coupling. In addition to mediating Ca2+ entry to initiate transmitter release, they are thought to interact directly with proteins of the synaptic vesicle docking/fusion machinery. These Ca2+ channels can be purified from brain as a complex with SNARE proteins, which are involved in exocytosis. In addition, N‐type and P/Q‐type Ca2+ channels are colocalized with syntaxin in high‐density clusters in nerve terminals. The synaptic protein interaction (synprint) sites in the intracellular loop II‐III (LII‐III) of both α1B and α1A subunits of N‐type and P/Q‐type Ca2+ channels bind to syntaxin, SNAP‐25, and synaptotagmin.Ca2+ has a biphasic effect on the interactions of N‐type Ca2+ channels with SNARE complexes, stimulating optimal binding in the range of 10–30 μM. PKC or CaM KII phosphorylation of the N‐type synprint peptide inhibits interactions with SNARE complexes containing syntaxin and SNAP‐25. Introduction of the synprint peptides into presynaptic superior cervical ganglion neurons reversibly inhibits EPSPs from synchronous transmitter release by 42%. At physiological Ca2+ concentrations, synprint peptides significantly reduce transmitter release in injected frog neuromuscular junctions in cell culture, consistent with detachment of 70% of the docked vesicles from Ca2+ channels as analyzed by a theoretical model. Together, these studies suggest that presynaptic Ca2+ channels not only provide the Ca2+ signal required by the exocytotic mechinery, but also contain structural elements that are integral to vesicle docking, priming, and fusion processes.