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

项目总结/摘要 内源性离子通道活性示踪剂监测缺血发作时Kv 2通道的参与 神经元电信号由许多离子通道亚型的联合作用控制。 不同的离子通道集合在神经元电兴奋性中产生显著的多样性。然而，在这方面， 由于技术的限制，剖析离子通道亚型在复杂过程中的个体作用， 生理或病理生理事件仍然是困难的。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， 特别是在完整的组织或活的动物中。乔恩·萨克博士实验室开发的技术提供了一个机会 来对整个复杂组织中离子通道亚型的活动进行成像。具体来说，这些 Kv 2活性示踪剂（KAT）报告了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KAT用于双光子成像，并证明KAT可以报告 激活脑切片中的内源性神经元Kv 2离子通道。我建议测量一个特定的 在病理生理缺血应激下组织切片和活体动物中离子通道亚型 中风在大脑中，Kv 2离子通道在大多数（如果不是所有）神经元中高度表达。Kv 2频道 在许多应激过程中，包括缺血性脑损伤， 攻击以前的研究表明，Kv 2离子通道在缺血期间变得非常活跃，抑制了Kv 2离子通道。 电兴奋性，并提供神经保护免受兴奋毒性信号。然而， 有证据表明，缺血期间通过Kv 2通道的钾过度流出可引发细胞凋亡 导致神经元死亡的级联反应。这两个提议的角色都假设数量急剧增加， 然而，这在复杂组织中从未在真实的时间内观察到。为我的博士 研究我将探索大规模激活的机制，并在模型中Kv 2离子通道活性的图像变化 缺血性中风我的研究结果将提高我们对导致神经元损伤的分子机制的理解。 中风后细胞死亡，以及Kv 2通道是否是增加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，并可能改变蛋白质 在生理学背景下研究激活。在整个项目中，赞助商/共同赞助商团队将 实施全面的培训计划，重点是提高批判性思维、实验和分析能力， 技能，演讲和公开演讲技能，并帮助建立同事和合作者的网络。

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.