CA CHANNEL INACTICATION AND MOLECULAR CONTROL OF TRANSMITTER RELEASE

本项目的总体目标是了解分子 似乎存在于特定类型的Ca 2+通道之间的相互作用 以及支持递质释放的蛋白质复合物的组分。 的 总的背景是关于电压的多样性的知识不断增长， 门控Ca 2+通道，包括标记为Q型的新通道，以及 关键过程的结构决定因素，如Ca 2+通道失活， 在维持去极化期间关闭通道。 在 建议的实验，我们将集中在突触蛋白， 融合机制的关键组成部分，以及N型和Q型Ca 2+通道 它们共同作用于CA 3-CA 1海马突触， 传输 直接的推动力是我们最近的观察， 突触融合蛋白1A与N型通道在爪蟾卵母细胞中的共表达 促进通道的失活，引起约20 m V 它们的可用性的电压依赖性的超极化转变。 与N型通道一样，Q型通道也受到突触融合蛋白的影响， 而L-型通道的失活没有改变。 先前 生物化学研究已经证明，突触融合蛋白能够 结合到N型Ca 2+通道，但这是第一个证据，任何 对通道功能特性的影响以及与Q的任何相互作用， 类型频道。 因此，我们的数据提出了突触融合蛋白可能 通过关键的突触前Ca 2+通道调节Ca 2+进入，超过其 作为突触囊泡的对接点的假定作用。 在资助期间，我们打算（1）分析syntaxin的作用 根据调节受体假说，并确定哪些类型的 Ca 2+通道可受突触融合蛋白影响，（2）描绘Ca 2+区域 允许互动的渠道，特别注意那些 在控制失活的速度或程度方面占主导地位的结构域， (3)描述了使突触融合蛋白 对Ca ~（2+）通道的影响;（4）研究syntaxin对Ca ~（2+）通道的影响 肉毒杆菌毒素BoTx/C1可以阻止卵母细胞中的通道， 选择性切割突触融合蛋白，或通过其它蛋白组分（例如半胱氨酸 串蛋白、突触结合蛋白、VAMP、n-secl、α-SNAP、NSF等（5） 确定突触融合蛋白-Ca 2+通道信号传导在神经中的重要性 检测BoTx/C1对突触体Ca ~（2+）内流的影响。 它 有趣的是，问syntaxin是否影响N-和Q-的门控， 型通道可能代表了一种新的细胞外吞信号通路 机械到Ca 2+通道，允许Ca 2+进入的网站， 囊泡已准备好进行胞吐作用。 这将是一个有效的 用于控制发射器释放同时使可能的Ca 2+最小化的系统 超负荷和相关的神经毒性。

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.