Structural and Functional Interactions within the Neuronal ER/PM Junction

描述（由申请人提供）：内质网/质膜（ER/PM）连接在肌肉和免疫细胞中被最好地理解，它们介导收缩和淋巴细胞激活。尽管早期的电镜检测到神经元的亚表面池，但负责维持这种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K+通道在神经元ER/PM连接的形成中起着核心作用。该通道在靠近内质网膜的神经元体上形成高度稳定的细胞表面簇。这些局部通道大多是非导电的，并通过增强内质网/PM连接和显著增加连接表面积来发挥直接的结构作用，可能是通过结合未知的内质网膜蛋白。我们发表的数据表明，Kv2.1增强的ER/PM连接点是运输枢纽，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交界处。我们认为kv2.1稳定的ER/PM连接代表了一种大分子质膜复合物，其功能是膜运输和Ca2+信号传导的支架位点。鉴于该复合物受脑卒中相关的神经元损伤调节，因此需要进一步了解该细胞表面微域的组成、功能和动力学。该提案汇集了来自四个机构的跨学科团队，他们在细胞内Ca2+动力学，离子通道电生理学，分子和细胞生物学，nanoSIMS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方面具有综合专业知识。目的1旨在鉴定蛋白质和脂质

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.