BRAIN SPECTRIN AND SYNAPTIC TRANSMISSION

这是研究脑内血影蛋白作用的新应用。 突触传递。我们将确定幽灵蛋白是否扮演着 直接作用于去极化引起的神经递质释放 突触前终末。需要检验的具体假设是 该血影蛋白是一种必需的对接蛋白和钙感应蛋白。 在钙调节的囊泡融合和神经递质释放中 50 nm直径的球形突触小泡。目标是(1) 测定血影蛋白的合成、周转和组装速度 静息状态和去极化状态的海马神经元(/-钙)。 (2)确定建议的突触素I结合位点是否在 脑β-血影蛋白(Res.207-445的β(Sp)IIsigma1)可以结合 突触小泡，并定义最小功能亚域和 附着物必需的氨基酸。同时确定突触是否 囊泡结合取代了幽灵蛋白和f-肌动蛋白的附着 在“抛线”假说中提出。最终确定 突触小泡与脑β-光谱的相互作用 由游离钙水平直接调节。(3)确定是否 显微注射β-血影蛋白重组多肽及其人工合成 含有突触囊泡结合域的多肽(和Fab 针对这些合成肽的片段)改变神经递质 培养的成对海马神经元释放。多肽和 抗体将被注入突触前神经元，我们将 应用膜片钳技术和量子分析研究心肌细胞的变化 小球形突触囊泡释放递质。Dr。 古德曼和他的合作者处于一个独特的位置，能够成功地 进行这些研究是因为他们已经开发出必要的 血影蛋白DNA构建物、重组多肽、合成多肽和 进行拟议的研究的抗体。他们也有 在神经细胞培养，分子生物学， 蛋白质化学、电生理学和长期的工作历史 与幽灵蛋白分子的关系。

This is a new application to study the role of brain spectrin in synaptic transmission. We will determine whether spectrin plays a direct role in depolarization-induced release of neurotransmitter from the presynaptic terminal. The specific hypothesis to be tested is that spectrin is an essential docking protein and Ca2+ sensor protein in the Ca2+-regulated vesicle fusion and neurotransmitter release from 50 nm diameter spherical synaptic vesicles. The aims are to (1) Determine the rate of spectrin synthesis, turnover, and assembly in hippocampal neurons in the resting and depolarized state (+/-Ca2+). (2) Determine whether the proposed binding site for synapsin I on brain beta spectrin (res. 207-445 of beta(Sp)IIsigma1) can bind synaptic vesicles, and define the smallest functional subdomain and essential amino acids for attachment. Also determine whether synaptic vesicle binding displaces the attachment of spectrin and f-actin as proposed in the "casting the line" hypothesis. Finally determine whether the interaction of synaptic vesicles with brain beta spectrin is directly regulated by free Ca2+ levels. (3) Determine whether microinjection of beta spectrin recombinant peptides and synthetic peptides which contain the synaptic vesicle binding domain (and FAB fragments against these synthetic peptides) alters neurotransmitter release from paired hippocampal neurons in culture. Peptides and antibodies will be injected into the presynaptic neurons and we will use patch clamp techniques and quantal analysis to study changes in transmitter release from small spherical synaptic vesicles. Dr. Goodman and his collaborators are in a unique position to successfully conduct these studies because they have developed the necessary spectrin DNA constructs, recombinant peptides, synthetic peptides and antibodies to perform the proposed studies. They also have the requisite expertise in neuronal cell culture, molecular biology, protein chemistry, electrophysiology and a long history of working with the spectrin molecule.