Proteomic analysis of the electrical synapse

神经系统使用两种形式的快速突触传递，化学的和电的， 到创造思想、感觉和行动的动态计算。化学突触研究得很好， 并且神经递质释放和接收的生化机制已被很好地理解。通过 相比之下，我们对电突触的大分子复合体知之甚少。电 突触由成千上万的间隙连接通道组成，这些通道产生直接的，低阻力的通路， 神经元之间的细胞质通讯。它们对神经回路的复杂功能有贡献 计算，它们通过一些短期和长期机制显示可塑性， 在发育过程中受到调控。总之，这些都表明了一种复杂的大分子结构， 然而，在这一领域取得进展的一个关键障碍仍然是确定 电突触的蛋白质。该提案的总体目标是建立斑马鱼电气 突触作为一个模型来了解它们的蛋白质组多样性。aim 1将证明电突触 蛋白质可以使用基因组工程改造的斑马鱼来鉴定， 关于TurboID TurboID是一种进化的大肠杆菌蛋白质，其允许在体内，依赖于邻近标记 蛋白质与生物素这样的生物素化的蛋白质然后可以从动物中有效地分离并分析 使用质谱法。然后，Aim 2将评估生物化学相互作用和细胞定位， 使用蛋白质-蛋白质相互作用的表达系统和体内免疫组织化学鉴定蛋白质 在斑马鱼中。如果成功，这项资助将从根本上改变对电突触的理解，揭示 参与运输途径、突触结构和功能调节的蛋白质。拟议的研究 将为脊椎动物模型中电突触的分子复合物提供新的见解， 为鉴定治疗神经发育障碍的靶点提供了基础。

The nervous system uses two forms of fast synaptic transmission, chemical and electrical, that both contribute to the dynamic computations that create thought, feelings, and actions. Chemical synapses are well studied, and the biochemical mechanisms by which neurotransmitter is released and received are well understood. By contrast, we know relatively little about the macromolecular complex of the electrical synapse. Electrical synapses are made from tens to thousands of gap junction channels that create direct, low-resistance routes of cytoplasmic communication between neurons. They contributed to sophisticated function in neural circuit computation, they display plasticity through a number of short- and long-term mechanisms, and their assembly is regulated during development. Together, this all suggest a complex macromolecular structure that controls their formation and function, yet a critical barrier to progress in the field remains in the identification of the proteins of the electrical synapse. The overarching goal of the proposal is to establish zebrafish electrical synapses as a model to understand their proteomic diversity. Aim1 will demonstrate that electrical synapse proteins can be identified using genome engineered zebrafish that express electrical synapse proteins tagged with TurboID. TurboID is an evolved E.coli protein that allows for in vivo, proximity-depending labeling of proteins with biotin. Such biotinylated proteins can then be efficiently isolated from the animal and analyzed using mass spectrometry. Aim2 will then assess the biochemical interactions and cellular localization of the identified proteins using expression systems for protein-protein interactions and in vivo immunohistochemistry in zebrafish. If successful, this grant will fundamentally shift the understanding of electrical synapses, revealing proteins involved in trafficking pathways, synaptic structure, and functional regulation. The proposed studies will provide novel insight into the molecular complexes of the electrical synapse in a model vertebrate, providing a foundation for the identification of targets for therapy of neurodevelopmental disorders.