The overall objective of this project is to understand the molecular interactions that appear to exist between specific types of Ca2+ channels and components of the protein complex supporting transmitter release. The general background is growing knowledge about the diversity of voltage- gated Ca2+ channels, including a novel channel labelled Q-type, and the structural determinants of key processes such as Ca2+ channel inactivation, the shutting off of the channel during a maintained depolarization. In the proposed experiments, we will focus on interactions between synataxin, a key component of the fusion machinery, and N- and Q-type Ca2+ channels which act in concert at CA3-CA1 hippocampal synapses to trigger synaptic transmission. The immediate impetus is our recent observation that coexpression of syntaxin 1A with N-type channels in Xenopus oocytes promotes inactivation of the channels by causing a about 20 m V hyperpolarizing shift in the voltage-dependence of their availability. Like N-type channels, Q-type channels were also affected by syntaxin, whereas inactivation of l-type channels was unchanged. Previous biochemical studies had already demonstrated t hat syntaxin is capable of binding to N-type Ca2+ channels, but this is the first evidence for any effect on the channels' functional properties and any interaction with Q- type channels. Thus, our data raise the possibility that syntaxin may modulate Ca2+ entry via key presynaptic Ca2+ channels, over and above its putative role as a docking site for synaptic vesicles. During the grant support period, we intend to (1) analyze syntaxin action according to the modulated receptor hypothesis and determine which types of Ca2+ channels can be influenced by syntaxin, (2) delineate regions of Ca2+ channels that allow the interaction, with specific attention to those domains that are dominant in governing the speed or extent of inactivation, (3) characterize the molecular determinants that allow syntaxin to influence Ca2+ channels, (4) find out whether syntaxin's actions on Ca2+ channels in oocytes can be prevented by the botulinum toxin BoTx/C1, which selectively cleaves syntaxin, or by other protein components (e.g. cysteine string proteins, synaptotagmin, VAMP, n-secl, alpha-SNAP, NSF etc. (5) determine the importance of syntaxin -Ca2+ channel signalling in nerve terminals by testing effects of BoTx/C1 on Ca2+ influx on synaptosomes. It is interesting to ask whether syntaxin effects on the gating of N- and Q- type channels might represent a novel signaling pathway from exocytotic machinery to Ca2+ channels that would allow Ca2+ entry at sites where vesicles have been readied for exocytosis. This would be an efficient system for controlling transmitter release while minimizing possible Ca2+ overload and associated neurotoxicity.
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