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

内源性离子通道活动示踪剂监测缺血性发作期间 Kv2 通道的参与情况

• 批准号: 9761043
• 负责人: Rebecka Jane Sepela
• 金额: $ 3.72万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-02 至 2022-04-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9761043
• 关键词: Affect; Animals; Apoptosis; Apoptotic; Biochemical Process; Brain; Cephalic; Cerebral Thrombosis; Complex; Critical Thinking; Dendrites; Dependence; Development; Diffuse; Diffusion; Drug Targeting; Electrophysiology (science); Engineering; Event; Fluorescent Probes; Gated Ion Channel; Genetic Models; Genetic Techniques; Health; Image; Individual; Ion Channel; Ion Channel Gating; Ischemia; Ischemic Stroke; Knowledge; Location; Measures; Mediating; Membrane; Middle Cerebral Artery Occlusion; Modeling; Molecular; Molecular Conformation; Molecular Probes; Monitor; Morphology; Neurons; Pattern; Pharmacology; Physiological; Play; Potassium; Potassium Channel; Procedures; Public Speaking; Refractory; Reporting; Research; Resolution; Role; Signal Transduction; Slice; Specificity; Stress; Stroke; Sum; System; Techniques; Technology; Testing; Time; Tissues; Tracer; Training; Transfection; Work; base; excitotoxicity; experimental study; fluorescence imaging; human tissue; imaging approach; improved; in vivo; neuron loss; neuronal cell body; neuronal survival; neurotransmission; preservation; protein activation; relating to nervous system; response; skills; spatiotemporal; spectrograph; targeted treatment; two-photon; voltage; voltage clamp

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.

项目摘要/摘要 内源离子通道活性示踪剂以监测缺血性发作期间KV2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以创造出神经元电气刺激性的显着多样性。然而， 由于技术局限性，在复合物过程中剖析离子通道亚型 生理或病理生理事件仍然很困难。因此，我们对 单个内源离子通道亚型的电气动力学如何促进全局信号， 特别是在完整​​的组织或活动物中。乔恩·萨克（Jon Sack）博士实验室开发的技术提供了机会 在整个复杂组织中成像离子通道亚型的活性。具体来说，这些 KV2活性示踪剂（KATS）报告激活内源性神经元钾电压门控离子通道 亚型2。我们已经设计了这些Kats进行2光子成像，并证明Kats可以报告 激活内源性神经元KV2离子通道中的脑切片。我建议测量特定的激活 在病理生理缺血性压力下，组织切片和活体动物中的离子通道亚型模仿 中风。在大脑中，KV2离子通道在大多数（如果不是全部神经元）中高度表达。 KV2通道是 建议在许多应力中抑制兴奋性信号事件至关重要，包括缺血 攻击。先前的研究表明，在缺血期间，KV2离子通道变得非常活跃，抑制 电兴奋性，并提供神经保护免受兴奋性信号的传导。但是，也有 证据表明，缺血期间通过KV2通道过量钾会引发凋亡 级联导致神经元死亡。这两个提出的角色都假设数量急剧增加 活跃的KV2通道中，在复杂组织中从未实时观察到这一点。为我的博士 研究我将探测一种质量激活的机制，并在模型中的KV2离子通道活动中的图像变化 缺血性中风。我的结果将提高我们对导致神经元的分子机制的理解 中风后的细胞死亡，以及KV2通道是否是增加神经元存活的潜在药物靶标 下风。此外，我的研究将是首次尝试使用荧光问题进行测量的尝试 特定离子通道完整组织中特定离子通道的构象变化，并有可能改变蛋白质的方式 在物理环境中研究了激活。通过这个项目，赞助商/共同赞助商团队将 实施一项旨在改善批判性思维，实验和分析的全面培训计划 技能，演示和公开演讲技巧，并有助于建立同事和合作者网络。