The neurotransmitter release machinery: a new target general anesthetics

描述（由申请人提供）：已知全身麻醉剂（GAs）部分通过激活/促进突触后GABA(A)受体抑制中枢神经系统活动。虽然GABA(A)受体确实有助于麻醉作用，但GAs也影响突触前机制。然而，大多数研究几乎完全集中在突触前离子通道上。虽然是间接的，但在我的实验室进行的先前实验表明，GAs依托咪酯和异氟醚直接抑制神经递质释放机制。我从渗透性PC12细胞中收集的电流测量数据表明，依托咪酯实际上能够通过与哺乳动物释放机制的直接相互作用来抑制儿茶酚胺的释放。我的建议旨在结合分子，电生理和电子显微镜（EM）为基础的实验来进一步验证这一假设，并确定释放机制的组成部分，参与对气体的反应。为了确定对释放机制的抑制是否代表GAs的普遍特性，我还将研究异氟醚是否影响渗透PC12细胞的神经递质释放。然后，我将使用笼状钙的光解来更准确地表征异氟烷对PC12细胞释放机制的影响。这种技术可以更好地控制[Ca2+]i增加的持续时间和幅度，并允许细胞作为它们自己的控制。我还将确定syntaxin 1A或其激活剂UNC-13是否参与介导GAs在哺乳动物细胞中的作用。这将通过用先前发现的影响秀丽隐杆线虫GA敏感性的突变体取代这些蛋白质的内源性形式来实现。异氟醚对囊泡运输的影响也将使用EM进行研究。总之，这些实验有可能确定作为GAs重要新靶点的释放机制。如果麻醉药是通过这种机制抑制谷氨酸（或其他神经递质）在中枢突触的释放，这将为设计新的麻醉药提供重要的信息。公共卫生相关性：为了生产更有效、副作用更少的全麻，我们必须首先了解这些麻醉剂是如何抑制哺乳动物神经系统内的活动的。根据我的初步数据，似乎全身麻醉剂能够通过与一组蛋白质的直接相互作用来抑制哺乳动物细胞中的神经递质释放，这些蛋白质被称为SNAREs，负责突触囊泡融合和神经递质释放。如果这种相互作用被证明是麻醉药一般作用的重要组成部分，这将是设计和测试新麻醉药的重要信息。

DESCRIPTION (provided by applicant): General anesthetics (GAs) are known to suppress central nervous system activity in part through the activation/facilitation of postsynaptic GABA(A) receptors. While GABA(A) receptors do contribute to anesthetic action, GAs also influence presynaptic mechanisms as well. Most studies, however, have focused almost exclusively on presynaptic ion channels. Although indirect, previous experiments performed in my lab have indicated that the GAs etomidate and isoflurane inhibit the neurotransmitter release machinery directly. Amperometric data I have since collected from permeabilized PC12 cells suggests that etomidate is, in fact, capable of inhibiting the release of catecholamines via a direct interaction with the mammalian release machinery. My proposal aims to combine molecular, electrophysiological and electron microscope (EM)-based experiments to further test this hypothesis as well as identify the components of the release machinery that are involved in the response to GAs. To determine if the inhibition of the release machinery represents a universal property of GAs I will also investigate whether isoflurane affects neurotransmitter release from permeabilized PC12 cells. I will then employ photolysis of caged calcium to more accurately characterize the effects of isoflurane on the release machinery in PC12 cells. This technique allows for better control of the duration and magnitude of increases in [Ca2+]i and will allow cells to serve as their own controls. I will also determine whether syntaxin 1A or its activator, UNC-13, are involved in mediating the effects of GAs in mammalian cells. This will be done by replacing the endogenous forms of these proteins with mutants previously found to influence GA sensitivity in C. elegans. The effects of isoflurane on vesicular trafficking will also be investigated using EM. Together, these experiments have the potential to identify the release machinery as an important new target for GAs. If anesthetics were to inhibit glutamate (or other neurotransmitters) release at central synapses via this mechanism, this would provide vital information for designing new anesthetics. PUBLIC HEALTH RELEVANCE: To produce more effective general anesthetics with fewer side effects we must first understand how these anesthetics suppress activity within the mammalian nervous system. Based on my preliminary data it appears that general anesthetics are capable of suppressing neurotransmitter release in mammalian cells through a direct interaction with a group of proteins, known as SNAREs, that are responsible for synaptic vesicle fusion and neurotransmitter release. If this interaction proves to be an important part of the general action of anesthetics, this would be vital information for designing and testing new anesthetics.