Next-generation Light-programmable Actuators of Voltage-gated Ca2+ channels

电压门控 Ca2 通道的下一代光可编程执行器

• 批准号: 10287793
• 负责人: Manu Ben Johny
• 金额: $ 24.3万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-15 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10287793
• 关键词: Action Potentials; Address; Affect; Affinity; Amino Acids; Avena sativa; Benchmarking; Binding; Biological Assay; Biomimetics; Bipolar Disorder; Brain; Calmodulin; Calmodulin-Binding Proteins; Cardiac; Cardiac Myocytes; Cells; Closure by clamp; Complex; Cysteine-Rich Domain; Data; Development; Disease; Doctor of Philosophy; Dominant-Negative Mutation; Electrophysiology (science); Engineering; Exhibits; Feedback; Fluorescence Resonance Energy Transfer; Functional disorder; Genetic Transcription; Goals; Heart; Hippocampus (Brain); Homeostasis; Human; Hybrids; Light; Lighting; Link; Location; Mediating; Muscle; Names; Neurobiology; Neurons; Optics; Pharmacology; Phenotype; Physiological; Process; Property; Proteins; Regulation; Resolution; Role; Schizophrenia; System; autism spectrum disorder; base; biophysical analysis; cell type; design; fluorescence imaging; frontier; genetic approach; heart rhythm; high reward; high risk; induced pluripotent stem cell; molecular modeling; neuropsychiatric disorder; neuropsychiatry; next generation; novel; novel strategies; optical switch; optogenetics; prevent; programs; small molecule; spatiotemporal; stem; tool; voltage

PROJECT SUMMARY PI: Manu Ben-Johny, Ph.D. CaV1 channels (CaV1.2/1.3) are fundamentally involved in the normal function and pathophysiology of the brain. In humans, dysfunction of CaV1.2/1.3 has been linked to neuropsychiatric disorders including bipolar disorder (BD), schizophrenia, and autism spectrum disorders (ASD). Elucidating the normal physiological functions of CaV1.2/1.3 in the brain and identifying potential pathophysiological mechanisms has emerged as a high priority. In this regard, CaV1 channels are potently tune by the ubiquitous Ca2+-binding protein calmodulin (CaM) that results in Ca2+- feedback inhibition (CDI). In the heart, this form of channel regulation has emerged as a dominant factor for Ca2+ homeostasis and for maintaining cardiac rhythm. Yet, in the brain, the role for CaV1 CDI in tuning neuronal action potential (AP) and function remains largely unidentified, though considered important based on biophysical analysis in reduced systems. In part, this gap in understanding stems from the absence of tools to dynamically manipulate CaM- feedback in intact physiological cells. Genetic approaches such as expressing dominant-negative CaM are challenging to interpret, as CaM itself is promiscuous in tuning a bevy of cellular proteins. Similarly, small molecule pharmacology only indirectly affects Ca2+-regulation. Accordingly, novel approaches that tune CaV1 Ca2+-feedback with high selectivity and enhanced spatial resolution are highly-desired to develop a new framework of CaV1 neurobiology and pathophysiology. The overall goal of this high-risk high-reward proposal is to develop optogenetic CaV1 actuators that tune Ca2+-dependent feedback in neuronal settings. To do so, we take advantage of a recently identified CaV1 modulator called SH3 and cysteine rich domain (stac) protein that contains a minimal domain (called U-domain) that naturally antagonizes Ca2+/CaM-feedback. Here, we engineer the stac U-domain with a light-sensitive Lov2 domain from the Avena sativa phototropin1 to reversibly photo-modulate its interaction with CaV1.2/1.3 channels in native settings. Following optimizing of this novel CaV1 actuator, name LovU-CaV1, we deploy it to dissect the functional contribution of CaV1 CDI for APs in hippocampal neurons. In all, the development of novel optogenetic CaV1 actuators open new frontiers to unambiguously define the functional impact of Ca2+-feedback in native settings, and in so doing, outline long-sought pathophysiological mechanisms that link CaV1 mis-regulation to complex neuropsychiatric phenotypes.

项目概要 PI：Manu Ben-Johny 博士 CaV1 通道 (CaV1.2/1.3) 从根本上参与正常功能和 大脑的病理生理学。在人类中，CaV1.2/1.3 的功能障碍与 神经精神疾病，包括双相情感障碍 (BD)、精神分裂症和自闭症谱系 障碍（ASD）。阐明CaV1.2/1.3在大脑中的正常生理功能 确定潜在的病理生理机制已成为当务之急。对此， CaV1 通道可通过普遍存在的 Ca2+ 结合蛋白钙调蛋白 (CaM) 进行有效调节，该蛋白 导致 Ca2+- 反馈抑制 (CDI)。在心脏里，已经出现了这种形式的通道调节 作为 Ca2+ 稳态和维持心律的主导因素。然而，在大脑中， CaV1 CDI 在调节神经元动作电位 (AP) 和功能方面的作用仍然很大程度上 尚未确定，但根据简化系统中的生物物理分析认为很重要。在 在某种程度上，这种理解上的差距源于缺乏动态操纵 CaM 的工具。 完整生理细胞的反馈。遗传方法，例如表达显性失活 CaM 的解释具有挑战性，因为 CaM 本身在调节一系列细胞蛋白质方面是混杂的。 同样，小分子药理学仅间接影响 Ca2+ 调节。据此，新颖的 具有高选择性和增强空间分辨率的调整 CaV1 Ca2+ 反馈的方法 非常希望开发 CaV1 神经生物学和病理生理学的新框架。这 这项高风险高回报提案的总体目标是开发光遗传学 CaV1 执行器 调节神经元环境中 Ca2+ 依赖性反馈。为此，我们利用最近 鉴定出称为 SH3 的 CaV1 调节剂和富含半胱氨酸结构域 (stac) 的蛋白，该蛋白含有 天然拮抗 Ca2+/CaM 反馈的最小结构域（称为 U 结构域）。在这里，我们 使用来自燕麦趋光素1的光敏 Lov2 结构域设计 stac U 结构域 在天然环境中可逆地光调制其与 CaV1.2/1.3 通道的相互作用。下列的 对这种新颖的 CaV1 执行器（名为 LovU-CaV1）的优化，我们部署它来剖析功能 CaV1 CDI 对海马神经元 AP 的贡献。总而言之，小说的发展 光遗传学 CaV1 执行器开辟了新的领域，明确定义了功能影响 原生环境中的 Ca2+ 反馈，并在此过程中概述了长期寻求的病理生理学 将 CaV1 失调与复杂的神经精神表型联系起来的机制。