BRAIN SPECTRIN AND SYNAPTIC TRANSMISSION

脑血影蛋白和突触传递

• 批准号: 2873212
• 负责人: STEVEN Richard GOODMAN
• 金额: $ 22.82万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-02-01 至 2001-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2873212
• 关键词: actins; brain metabolism; calcium; hippocampus; intermolecular interaction; laboratory rat; neural transmission; protein biosynthesis; protein structure function; proteolysis; recombinant proteins; site directed mutagenesis; spectrin; synapses; synaptic vesicles; synthetic peptide; tissue /cell culture; voltage /patch clamp

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.

这是一个新的应用研究脑血影蛋白的作用， 突触传递 我们将确定spectrin是否发挥了 在去极化诱导的神经递质释放中的直接作用 突触前末梢 待检验的具体假设是 血影蛋白是一种重要的对接蛋白和钙离子传感蛋白 在Ca 2+调节的囊泡融合和神经递质释放中， 直径50 nm的球形突触囊泡。 目标是（1） 确定血影蛋白合成、周转和组装的速率， 静息和去极化状态下的海马神经元（+/-Ca 2+）。 (2)确定突触蛋白I的拟议结合位点是否位于 脑β血影蛋白（β（Sp）II σ 1的res.207 -445）可以结合 突触囊泡，并定义最小的功能子域， 必需的氨基酸。同时确定突触 囊泡结合取代血影蛋白和F-肌动蛋白的附着， 在“铸造线”假说中提出。最终确定 突触囊泡与脑β血影蛋白的相互作用是否 直接受游离Ca 2+水平调节。 (3)确定是否 显微注射β血影蛋白重组肽和合成的 含有突触囊泡结合结构域（和FAB）的肽 针对这些合成肽的片段）改变神经递质 从培养的成对海马神经元中释放。 肽和 将抗体注射到突触前神经元， 用膜片钳技术和定量分析研究 从小的球形突触囊泡中释放的递质。 博士 古德曼和他的合作者处于一个独特的位置， 进行这些研究是因为他们已经开发了必要的 血影蛋白DNA构建体、重组肽、合成肽和 抗体进行拟议的研究。他们也有 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 血影蛋白分子