Dig and CASK at the mammalian neuromuscular junction

描述(由申请人提供)：神经递质受体在突触的突触后膜上的聚集对于有效地将信号从神经元传输到其目标是必不可少的。在胆碱能哺乳动物神经肌肉接头(NMJ)，乙酰胆碱受体聚集的机制和这些神经递质受体的生理学已经得到了很好的研究。然而，对其他受体和通道的作用知之甚少，包括NMDA谷氨酸和ErbB4受体，以及也定位于突触后膜的钠通道。一个含有PDZ蛋白基序的蛋白质家族定位于谷氨酸能果蝇NMJ和哺乳动物中枢神经系统突触的通道和受体，并被认为在这些突触的组织、结构和功能中发挥着重要作用。我们最近在哺乳动物NMJ的突触后膜上发现了几个这些PDZ结构域蛋白的定位。其中两个PDZ结构域蛋白，DLG和CASK，代表着与NMJ突触后膜上的NMDA和ErbB4受体以及钠通道相互作用的优秀候选者。目前应用的目的是确定DLG和CASK在骨骼肌中与哪些蛋白质相互作用，这两种蛋白质是否负责定位NMJ的通道和受体，以及这些蛋白质的突变是否会导致突触功能的改变。这些研究将利用免疫学和生化分离技术以及肌源性细胞培养来确定DLG和CASK的结合伙伴，并确定它们在骨骼肌中的定位和蛋白质相互作用的结构域。转基因小鼠将在体内研究DLG和CASK的功能及其PDZ结构域在骨骼肌通道和受体聚集中的作用，并为这些受体对肌肉生理特性的影响提供有价值的信息。这些研究将对PDZ结构域蛋白在NMJ的组织和功能中的作用有重要的新见解，并将对理解包括通道病在内的多种神经肌肉疾病和原因不明的疾病具有重要意义。

DESCRIPTION (provided by applicant): The clustering of neurotransmitter receptors at the post-synaptic membrane of synapses is essential for efficient transmission of a signal from a neuron to its target. At the cholinergic mammalian neuromuscular junction (NMJ) the mechanisms of acetylcholine receptor clustering and the physiology of these neurotransmitter receptors have been well studied. However, little is known about the roles of other receptors and channels, including NMDA glutamate and ErbB4 receptors, and sodium channels that are also localized to this post-synaptic membrane. A family of proteins containing a PDZ protein motif localizes channels and receptors at glutamatergic Drosophila NMJ and mammalian central nervous system synapses and are thought to play a fundamental role in the organization, structure, and function of these synapses. We have recently shown the localization of several of these PDZ domain proteins at the post-synaptic membrane of the mammalian NMJ. Two of these PDZ domain proteins, Dlg and CASK, represent excellent candidates for interacting with the NMDA and ErbB4 receptors and sodium channels at the NMJ post-synaptic membrane. The aims of the current application are to determine the proteins with which Dlg and CASK interact in skeletal muscle, whether these two proteins are responsible for localizing channels and receptors at the NMJ, and whether mutations in these proteins lead to alterations in the function of this synapse. These studies will utilize immunological and biochemical fractionation techniques together with myogenic cell culture to identify binding partners for Dlg and CASK and to determine the domains responsible for their localization and protein interactions in skeletal muscle. Transgenic mice will be used to define the functions of Dlg and CASK and their PDZ domains in channel and receptor clustering in skeletal muscle in vivo and provide valuable information about the effect of these receptors on the physiological characteristics of muscle. These studies will lead to important new insights into the roles of PDZ domain proteins in the organization and function of the NMJ and will have important implications for understanding a wide variety of neuromuscular diseases including channelopathies and those disorders for which a cause is unknown.