Electrophysiology-Optical Workstation for Ion Channel Biophysics

描述（由申请人提供）：在过去的二十年中，我们见证了神经系统正常和病理功能知识的爆炸式增长。荧光成像和电生理学这两种方法的进步在很大程度上推动了这方面的进展。我们建议开始下一波的发现，通过结合活细胞的光学和电生理测量，实时探测它们的功能。神经元、分泌细胞和肌肉细胞的兴奋性受到大量蛋白质和第二信使的影响：转录因子决定离子通道和受体的数量；激酶、磷酸酶和离子决定通道和受体的活性；复杂的循环和降解途径决定了通道和受体在质膜上的停留时间。此外，构成质膜的磷脂在决定通道活性以及通道和受体内化率方面也起着积极的作用。我们将用共聚焦显微镜和TIRF显微镜对细胞内蛋白质和信号分子的运动进行成像，并使用电生理记录读出信号对细胞兴奋性的净效应。该提案要求为离子通道生物物理学的共聚焦-红外-电生理（CTE）工作站提供资金。在太平洋西北部没有这样的仪器。它将使我们能够同步细胞器的运动、蛋白质之间的相互作用、细胞内钙浓度的变化、分子内构象的变化和电活动，从而对细胞兴奋性是如何设定的、它是如何被细胞外信号干扰的、以及它是如何在信号事件后恢复的有一个有凝聚力的理解。使用这些方法，我们将提出以下问题：哪些钙激活信号调节脱敏？磷酸肌苷调节通道的机制是什么？增长因素如何调节渠道贩运？磷酸化如何调节通道活性和表达？门控背后的分子运动是什么？这些问题将在许多细胞类型中提出，以定量定义对人类健康至关重要的几个系统中的离子通道功能。公共卫生相关性：可兴奋细胞依赖离子通道蛋白传导电信号、调节力和介导分泌。事实上，超过一半的药物靶点是离子通道或直接调节离子通道的蛋白质。我们的工作旨在了解神经系统和血管系统中离子通道的正常和病理信号。

DESCRIPTION (provided by applicant): In the last two decades we have witnessed an explosion in knowledge of the normal and pathological function of the nervous system. Progress has been largely fueled by advances in two methodologies, fluorescence imaging and electrophysiology. We propose to begin the next wave of discovery, by combining optical and electrophysiological measurements of living cells, to probe their function in real time. The excitability of neurons, secretory cells, and muscle cells is influenced by a large number proteins and second messengers: transcription factors determine the number of ion channels and receptors that are produced; kinases, phosphatases, and ions determine the activity of channels and receptors; and complex recycling and degradation pathways determine the residence time of channels and receptors in the plasma membrane. As well, the phospholipids that compose the plasma membrane play an active role in determining channel activity and the rate of channel and receptor internalization. We will image the movement of proteins and signaling molecules within a cell with confocal and TIRF microscopy and read out the net effect of signaling on the cell's excitability using electrophysiological recording. This proposal requests funds for a Confocal-TIRF-Electrophysiological (CTE) workstation for ion channel biophysics. No such instrument exists in the Pacific Northwest. It would permit us to synchronize the movement of organelles, interactions between proteins, changes in intracellular calcium concentration, intra-molecular conformational changes, and electrical activity into a cohesive understanding of how cellular excitability is set, how it is perturbed by extracellular signals, and how it recovers after a signaling event. Using these methodologies, we will ask questions such as: Which calcium-activated signals regulate desensitization? What is the mechanism by which phosphoinositides regulate channels? How do growth factors regulate channel trafficking? How does phosphorylation regulate channel activity and expression? What are the molecular motions that underlie gating? These questions will be asked across a number of cell types, to define ion channel function quantitatively in several systems essential to human health. PUBLIC HEALTH RELEVANCE: Excitable cells depend on ion channel proteins to conduct electrical signals, regulate force, and mediate secretion. In fact, over half of all drug targets are ion channels or proteins that directly regulate ion channels. Our work is directed toward understanding the normal and pathological signaling by ion channels in the nervous system and vascular system.