Synaptic Laminin and the Calcium Channel

突触层粘连蛋白和钙通道

• 批准号: 7014076
• 负责人: STEVEN Scott CARLSON
• 金额: $ 24.61万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7014076
• 关键词: SDS polyacrylamide gel electrophoresis; Torpedo; Xenopus; affinity chromatography; antigen antibody reaction; binding sites; calcium channel; chimeric proteins; embryo /fetus; extracellular matrix; immunoprecipitation; laboratory mouse; laminin; molecular cloning; motor neurons; neuroendocrine system; neuromuscular junction; protein binding; protein protein interaction; protein purification; protein structure function; receptor binding; synaptogenesis; synaptotagmin; syntaxin; western blottings

DESCRIPTION (provided by applicant): Our proposal is aimed at elucidating how the extracellular matrix localizes the neurosecretory apparatus in the nerve terminal during synaptogenesis at the neuromuscular junction (NMJ). Nerve regeneration studies at NMJ suggest that synaptic basal lamina components tell the retuming axon where to locate neurotransmitter release machinery, including synaptic vesicle clusters and active zones, the sites of neurotransmitter release. Genetic evidence suggests that synaptic laminins containing alpha4 or Beta2 chains are the critical basal lamina ligands which localize the neurosecretory apparatus. Electron microscopic studies suggest that the transmembrane anchor for the neurotransmitter release machinery is a voltage-gated calcium channel. We have found a transmembrane link between a synaptic laminin, containing alpha4, Beta2, and gamma1chains, and the voltage-gated calcium channel at the electric organ synapse, which is homologous to the NMJ. Using detergent-solubilized electric organ synaptosomes, we have immuno-isolated a protein complex containing this laminin, as well as the calcium channel, the cytoskeletal protein spectrin, and several unidentified proteins. Cross-linking studies suggest that the alpha4Beta2gamma1 laminin is directly bound to the calcium channel in the protein complex. Further, we find that this laminin binds preferentially to cultured cells expressing the calcium channel. We hypothesize that alpha4Beta2gamma1 laminin in the synaptic basal lamina attaches to calcium channel, which in turn joins to the cytosolic neurosecretory apparatus. The alpha4Beta2gamma1 laminin might act to organize the presynaptic secretory apparatus the way agrin organizes the postsynaptic membrane. To test our hypothesis we will ask the following questions: Does the calcium channel act as the receptor for the alpha4Beta2gamma1 laminin? Is it the only receptor? Are there other alpha4Beta2gamma1 laminin-binding proteins in the complex? What kinds of proteins are the unidentified components of the calcium channel/ alpha4Beta2gamma1 laminin protein complex ? Could they be signal transduction proteins, or new active zone components? Will clustering of calcium channels on the surface of motor neuron axons cause the clustering of other components of the neurosecretory apparatus, such as synaptic vesicles? This work investigates a fundamental question of neurobiology and may have clinical relevance to nerve regeneration.

描述（由申请人提供）： 我们的建议旨在阐明细胞外基质在神经肌肉接头（NMJ）突触发生过程中如何将神经分泌装置定位在神经末梢。 NMJ 的神经再生研究表明，突触基底层成分告诉返回的轴突在哪里定位神经递质释放机制，包括突触囊泡簇和活动区，即神经递质释放的部位。遗传证据表明，含有 α4 或 Beta2 链的突触层粘连蛋白是定位神经分泌装置的关键基底层配体。电子显微镜研究表明，神经递质释放机制的跨膜锚是电压门控钙通道。我们发现突触层粘连蛋白（包含 α4、Beta2 和 gamma1 链）与电器官突触处的电压门控钙通道（与 NMJ 同源）之间存在跨膜连接。使用去污剂溶解的电器官突触体，我们免疫分离了含有这种层粘连蛋白的蛋白质复合物，以及钙通道、细胞骨架蛋白血影蛋白和几种未鉴定的蛋白质。交联研究表明 alpha4Beta2gamma1 层粘连蛋白直接与蛋白质复合物中的钙通道结合。此外，我们发现这种层粘连蛋白优先与表达钙通道的培养细胞结合。我们假设突触基底层中的 α4Beta2gamma1 层粘连蛋白附着在钙通道上，钙通道又连接到胞浆神经分泌装置。 α4Beta2gamma1 层粘连蛋白可能以集聚蛋白组织突触后膜的方式组织突触前分泌装置。为了检验我们的假设，我们将提出以下问题：钙通道是否充当 alpha4Beta2gamma1 层粘连蛋白的受体？它是唯一的受体吗？复合物中是否还有其他 alpha4Beta2gamma1 层粘连蛋白结合蛋白？钙通道/α4Beta2γ1 层粘连蛋白复合物的未识别成分是什么类型的蛋白质？它们可能是信号转导蛋白，还是新的活性区成分？运动神经元轴突表面钙通道的聚集是否会导致神经分泌装置其他成分（例如突触小泡）的聚集？这项工作研究了神经生物学的一个基本问题，可能与神经再生具有临床相关性。