Defining the Proteomic Composition of ER:Plasma Membrane Junctions in Brain Neurons

内质网（ER）和质膜（PM）之间的接触部位，称为ER-PM 连接或EPJ是存在于所有细胞中的专门的膜接触位点，并且在生理上 重要的Ca 2+信号传导事件、脂质交换、膜蛋白运输和其他重要的细胞生物学过程。 过程发生。在许多脑神经元，如海马锥体神经元（HPN）和纹状体介质中， 在多刺神经元（MSN）中，EPJ代表神经元的多刺区域中的主要Ca 2+信号传导微域。 神经蛋白质组学分析的大分子信号复合物在树突棘提供了 这些信息对于确定作为正常突触信号基础的特定分子事件至关重要， 在神经发育和成人神经和精神障碍中的失调。系统的解剖 在任何细胞类型中，在蛋白质组学水平上，EPJ中存在的大分子蛋白质复合物，尤其是在 大脑神经元，一直没有追求，由于缺乏适当的方法和合适的工具。缺乏 基本信息，从这些突出的蛋白质成分的分子目录开始， 突触外Ca 2+信号微区，代表了我们理解基本的 神经生理学和病理生理学。我们已经发现，一个丰富的和广泛表达的神经元， 电压门控K+通道Kv2.1特异性地定位于PM中的大簇，精确地定位于 EPJ形成。此外，最近的研究结果表明，Kv2.1积极促进形成和/或稳定的 EPJ通过与常驻ER蛋白直接相互作用。我们在这项探索性研究建议中提出， 利用Kv2.1与EPJ之间强大而广泛的联系， 神经蛋白质组学致力于鉴定HPN中这种Ca 2+信号微结构域的蛋白质成分， MSN。我们将免疫纯化和/或邻近标记这些含有Kv2.1的EPJ的蛋白质组分。 神经元，并通过串联质谱确定其鉴定。这些互补的神经蛋白质组学 分析将提供这些重要的Ca 2+信号传导的蛋白质成分的分子目录 HPN和MSN中的微域。这一信息将为今后的研究提供信息，以确定 这些Ca 2+信号事件中的成分塑造了这些神经元的生理学和可塑性。 最后，由于EPJ的蛋白质成分的失调可能有助于导致细胞内Ca 2+信号传导异常， 这些重要神经元的退化，例如阿尔茨海默病和中风后的HPN， MSNs在亨廷顿病中的作用，它们可能代表治疗调节的重要靶点。

Sites of contact between the endoplasmic reticulum (ER) and the plasma membrane (PM), termed ER-PM junctions or EPJs, are specialized membrane contact sites present in all cells, and at which physiologically important Ca2+ signaling events, lipid exchange, membrane protein trafficking, and other crucial cell biological processes occur. In many brain neurons, such as hippocampal pyramidal neurons (HPNs) and striatal medium spiny neurons (MSNs), EPJs represent the major Ca2+ signaling microdomain in aspiny regions of the neuron. Neuroproteomic analyses of the macromolecular signaling complexes at dendritic spines has provided information crucial to determining the specific molecular events that underlie normal synaptic signaling, and its dysregulation in neurodevelopmental and adult neurological and psychiatric disorders. A systematic dissection of the macromolecular protein complex present at EPJs at the proteomic level in any cell type, but especially in brain neurons, has not been pursued, due to the lack of appropriate methods and suitable tools. The lack of fundamental information, beginning with a molecular catalog of the protein constituents of these prominent extrasynaptic Ca2+ signaling microdomains, represents a major barrier to our understanding of basic neurophysiology and pathophysiology. We have found that an abundant and broadly expressed neuronal voltage-gated K+ channel, Kv2.1, is specifically localized to large clusters in the PM precisely at sites where EPJs form. Moreover, recent findings show that Kv2.1 actively promotes the formation and/or stabilization of EPJs through direct interaction with a resident ER protein. We propose in this exploratory research proposal to take advantage of the robust and widespread association of Kv2.1 with EPJs to undertake a concerted neuroproteomics effort to identify the protein constituents of this Ca2+ signaling microdomain in HPNs and MSNs. We will immunopurify and/or proximity label protein constituents of Kv2.1-containing EPJs in these neurons, and determine their identify by tandem mass spectrometry. These complementary neuroproteomics analyses will provide a molecular catalog of the protein constituents of these important Ca2+ signaling microdomains in HPNs and MSNs. This information will inform future studies to define the functional role of these constituents in the Ca2+ signaling events that shape the physiology and plasticity of these neurons. Lastly, as dysregulation of protein constituents of EPJs may contribute to the aberrant Ca2+ signaling that leads to degeneration of these important neurons, for example of HPNs in Alzheimer's disease and after stroke, and MSNs in Huntington's disease, they may represent important targets for therapeutic modulation.