Synaptic Laminin and the Calcium Channel

描述(由申请人提供)： 我们的建议旨在阐明在神经肌肉接头(NMJ)的突触发生过程中，细胞外基质如何定位神经末梢中的神经分泌装置。NMJ的神经再生研究表明，突触基板成分告诉神经递质释放机制的位置，包括突触囊泡团和活动区，神经递质释放的位置。遗传学证据表明，含有α4或β2链的突触层粘连蛋白是定位神经分泌器的关键基板配体。电子显微镜研究表明，神经递质释放机制的跨膜锚是一种电压门控钙通道。我们已经发现包含α4、β2和Gamma1链的突触层粘连蛋白与与NMJ同源的电器官突触的电压门控钙通道之间存在跨膜联系。利用洗涤剂溶解的电子器官突触体，我们免疫分离了一个含有这种层粘连蛋白的蛋白质复合体，以及钙通道、细胞骨架蛋白光谱蛋白和几个未知的蛋白质。交联性研究表明，α4Beta2Gamma1层粘连蛋白直接与蛋白质复合体中的钙通道结合。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。我们假设突触基底膜中的α-4-β-2-层粘连蛋白与钙通道相连，而钙通道又与胞浆神经分泌器相连。α4β2层粘连蛋白可能以集聚蛋白组织突触后膜的方式来组织突触前分泌器。为了验证我们的假设，我们将提出以下问题：钙通道是否充当α4β2层粘连蛋白的受体？它是唯一的受体吗？在这个复合体中是否还有其他的层粘连蛋白结合蛋白？钙通道/α4β2γ1层粘连蛋白复合体的未知成分是什么？它们可能是信号转导蛋白，还是新的活性区域成分？运动神经元轴突表面钙通道的聚集是否会导致神经分泌器的其他成分，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，并可能对神经再生具有临床意义。

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.