Synaptic Laminin and the Calcium Channel

描述（由申请人提供）： 我们的建议是旨在阐明如何在神经肌肉接头（NMJ）突触发生过程中的细胞外基质定位的神经末梢的神经分泌装置。NMJ的神经再生研究表明，突触基底层成分告诉恢复轴突在哪里定位神经递质释放机制，包括突触囊泡簇和活性区，神经递质释放的位点。遗传证据表明，含有α 4或β 2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，跨膜锚的神经递质释放机制是一个电压门控钙通道。我们已经发现了一个跨膜连接之间的突触层粘连蛋白，含有α 4，β 2和γ 1链，电压门控钙通道在电器官突触，这是同源的NMJ。使用洗涤剂溶解的电器官突触体，我们已经免疫分离的蛋白质复合物含有这种层粘连蛋白，以及钙通道，细胞骨架蛋白血影蛋白，和几个身份不明的蛋白质。交联研究表明，α 4 β 2 γ 1层粘连蛋白直接结合到蛋白复合物中的钙通道。此外，我们发现这种层粘连蛋白优先结合表达钙通道的培养细胞。我们推测，α 4 β 2 γ 1层粘连蛋白在突触基底层附着钙通道，这反过来又加入到胞质神经分泌装置。α 4 β 2 γ 1层粘连蛋白可能以聚集蛋白组织突触后膜的方式组织突触前分泌装置。为了验证我们的假设，我们将提出以下问题：钙通道是否作为α 4 β 2 γ 1层粘连蛋白的受体？它是唯一的受体吗？复合物中是否存在其他的α 4 β 2 γ 1层粘连蛋白结合蛋白？钙通道/α 4 β 2 γ 1层粘连蛋白蛋白复合物的未鉴定组分是什么样的蛋白质？它们可能是信号传导蛋白，或者是新的活性区成分吗？运动神经元轴突表面钙通道的聚集是否会引起神经分泌器的其他成分（如突触囊泡）的聚集？这项工作研究了神经生物学的一个基本问题，可能与神经再生有临床相关性。

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.