Structural and Functional Interactions within the Neuronal ER/PM Junction

描述(申请人提供)：内质网/质膜(ER/PM)连接在肌肉和免疫细胞中被最好地理解，在那里它们介导收缩和淋巴细胞激活。尽管早期在电子显微镜下检测到神经元中的亚表面池，但负责维持这一膜连接的功能和分子相互作用却知之甚少。我们的初步数据表明，Kv2.1延迟整流钾通道在神经元ER/PM连接的形成中起核心作用。这种通道在神经元胞体上形成高度稳定的细胞表面簇，这些胞体与内质网膜紧密结合。这些局部通道大多是非导电的，通过增强ER/PM连接和显著增加连接表面积而发挥直接的结构作用，可能是通过结合未知的ER膜蛋白。我们公布的数据表明，Kv2.1增强型ER/PM连接是运输中心，为多种类型的膜蛋白提供传递和检索平台。此外，我们的初步数据表明，钙信号蛋白定位于神经元Kv2.1/ER/PM连接。我们认为Kv2.1稳定的ER/PM连接代表了一个大分子质膜复合体，它作为膜转运和钙信号转导的支架位置。鉴于这种复合体受中风相关神经元损伤的调节，需要更好地理解这个细胞表面微域中的成分、功能和动力学。这项提议汇集了一个来自四个机构的跨学科团队，他们拥有细胞内钙动力学、离子通道电生理学、分子和细胞生物学、纳米SIMS细胞成像技术、高分辨率实时成像、光学和单分子扩散定量分析等方面的综合专业知识。目标1试图确定蛋白质和脂类 在Kv2.1/ER/PM复合体中，Aim 2研究了Kv2.1/ER/PM连接处的L型钙通道功能，Aim 3研究了该区域内钙通道/β2肾上腺素能受体的动力学。

DESCRIPTION (provided by applicant): Endoplasmic reticulum/plasma membrane (ER/PM) junctions are best understood in muscle and immune cells where they mediate contraction and lymphocyte activation. Despite the early electron microscopic detection of sub-surface cisterns in neurons the function and molecular interactions responsible for the maintenance of this membrane junction are poorly understood. Our preliminary data demonstrate that the Kv2.1 delayed rectifier K+ channel plays a central role in the formation of neuronal ER/PM junctions. This channel forms highly stable cell surface clusters on the neuronal soma that reside in close apposition the ER membrane. Most of these localized channels are non-conducting and play a direct structural role by enhancing the ER/PM junctions and dramatically increasing the junction surface area, likely by binding unknown ER membrane proteins. Our published data indicate that the Kv2.1 enhanced ER/PM junctions are trafficking hubs, providing platforms for delivery and retrieval for multiple types of membrane proteins. In addition, our preliminary data indicate that calcium signaling proteins localize to the neuronal Kv2.1/ER/PM junction. We propose the Kv2.1-stabilized ER/PM junctions represent a macromolecular plasma membrane complex that functions as a scaffolding site for both membrane trafficking and Ca2+ signaling. Given that this complex is regulated by stroke-related neuronal insults, an improved understanding of the components, function and dynamics within this cell surface microdomain is needed. This proposal assembles an interdisciplinary team from four institutions with combined expertise in intracellular Ca2+ dynamics, ion channel electrophysiology, molecular and cell biology, nanoSIMS cellular imaging technology, high-resolution real time imaging, optics, and quantitative analysis of single molecule diffusion. Aim 1 seeks to identify the proteins and lipids involved in the Kv2.1/ER/PM complex, Aim 2 examines L-type calcium channel function at the Kv2.1/ER/PM junction and Aim 3 studies calcium channel/beta2 adrenergic receptor dynamics within this domain.