Endogenous Ion Channel Activity Tracers to Monitor the Involvement of Kv2 Channels During Ischemic Attack

项目摘要/摘要 内源性离子通道活性示踪剂监测Kv2通道在脑缺血发作中的作用 神经元电信号受多种离子通道亚型的共同作用。 不同组的离子通道加在一起，创造了神经元电兴奋性的显著差异。然而， 由于技术限制，剖析复杂的离子通道亚型的个体角色 生理学或病理生理学事件仍然困难。因此，我们对 单个内源性离子通道亚型的电动力学如何对全局信号做出贡献， 尤其是在完整的组织或活的动物体内。乔恩·萨克博士实验室开发的技术提供了一个机会 成像离子通道亚型在整个复杂组织中的活动。具体地说，这些 KV2活性示踪剂(KATS)报道内源性神经元钾电压门控离子通道的激活 亚型2。我们已经设计了这些KATS用于双光子成像，并证明KATS可以报告 激活脑片内源性Kv2离子通道。我建议测量一个特定的 模拟病理生理缺血应激的组织切片和活体动物的离子通道亚型 卒中。在大脑中，Kv2离子通道在大多数神经元(如果不是所有神经元)中都高度表达。KV2频道有 被认为是在包括缺血在内的许多应激期间抑制兴奋性毒性信号事件的关键 进攻。以往的研究表明，Kv2离子通道在缺血时变得非常活跃，受到抑制 电兴奋性，并提供神经保护免受兴奋性毒性信号。然而，也出现了 缺血期间钾离子通过Kv2通道过度外流可引发细胞凋亡的证据 导致神经元死亡的级联反应。这两个拟议的角色在数量上都出现了戏剧性的增长 活跃的Kv2通道，但这从未在复杂的组织中实时观察到。为了我的博士学位 研究I将探索大量激活的机制，以及模型中Kv2离子通道活动的图像变化 缺血性中风的症状。我的结果将提高我们对导致神经细胞的分子机制的理解 卒中后细胞死亡，以及Kv2通道是否为提高神经元存活的潜在药物靶点 中风后。此外，我的研究将是首次尝试使用荧光探针来测量 完整组织中特定离子通道的构象变化和潜在的蛋白质转化方式 激活是在生理环境中研究的。在整个项目中，赞助商/联合赞助商团队将 实施全面的培训计划，重点提高批判性思维、实验和分析能力 技能、演讲和公开演讲技能，以及帮助建立同事和合作者网络。

Project Summary/Abstract Endogenous Ion Channel Activity Tracers to Monitor the Involvement of Kv2 Channels During Ischemic Attack Neuronal electrical signals are governed by the combined action of many ion channel subtypes. Different sets of ion channels sum to create a remarkable diversity in neuronal electrical excitability. However, due to technological limitations, dissecting the individual role of an ion channel subtype during a complex physiological or pathophysiological event remains difficult. Consequently we have a limited understanding of how the electrical dynamics of individual endogenous ion channel subtypes contribute to global signals, especially in intact tissue or in live animals. Technology developed in Dr. Jon Sack’s lab offers an opportunity to image the activity of ion channel subtypes throughout a complex tissue. Specifically, these Kv2 Activity Tracers (KATs) report activation of endogenous neuronal potassium voltage-gated ion channels of subtype 2. We have engineered these KATs for 2-photon imaging, and demonstrated that KATs can report activation endogenous neuronal Kv2 ion channels in brain slices. I propose to measure activation of a specific ion channel subtype in tissue slices and live animals under pathophysiological ischemic stress that mimics stroke. In the brain, Kv2 ion channels are highly expressed in most, if not all neurons. Kv2 channels are proposed to be crucial to suppress excitotoxic signaling events during many stresses, including ischemic attack. Previous studies have suggested that Kv2 ion channels become very active during ischemia, suppress electrical excitability, and provide neural protection from excitotoxic signaling. However, there has also been evidence that excessive efflux of potassium through Kv2 channels during ischemia can trigger apoptotic cascades leading to neuronal death. Both of these proposed roles assume a dramatic increase in the number of active Kv2 channels, yet this has never been observed in real time in complex tissues. For my doctoral studies I will probe a mechanism for mass activation, and image changes in Kv2 ion channel activity in models of ischemic stroke. My results will improve our understanding of the molecular mechanisms leading to neuronal cells death following stroke, and whether Kv2 channels are a potential drug target to increase neuronal survival following stroke. Further, my research will be the first attempt at using fluorescent probes to measure conformational change of specific ion channels in intact tissue and potentially transform the way protein activation is studied in a physiological context. Throughout this project the sponsor/co-sponsor team, will implement a comprehensive training plan focused on improving critical thinking, experimental and analytical skills, presentation and public speaking skills, and aid in creating a network of colleagues and collaborators.