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

项目总结 派：Manu Ben-Johny，博士。 CaV1通道(CaV1.2/1.3)基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功能障碍与 神经精神障碍包括双相情感障碍(BD)、精神分裂症和自闭症 精神障碍(ASD)。阐明CaV1.2/1.3在脑和脑中的正常生理功能 确定潜在的病理生理机制已成为一项高度优先的任务。在这方面， CaV1通道由普遍存在的钙结合蛋白钙调蛋白(CaM)有效地调节 导致钙离子反馈抑制(CDI)。在内心深处，这种形式的渠道监管已经出现 作为钙离子动态平衡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)和功能方面的作用在很大程度上仍然存在 根据简化系统中的生物物理分析，尽管被认为是重要的，但仍未确定。在……里面 部分地，理解上的差距源于缺乏动态操纵凸轮的工具- 在完整的生理细胞中的反馈。遗传方法，如显性-负性表达 CaM很难解释，因为CaM本身在调节一群细胞蛋白质方面是杂乱无章的。 同样，小分子药理学只能间接影响钙离子的调节。相应地，小说 高选择性和增强的空间分辨率调节CaV1钙离子反馈的方法 迫切需要开发一种新的CaV1神经生物学和病理生理学框架。这个 这项高风险高回报提案的总体目标是开发能够 在神经元环境中调整依赖于钙的反馈。为此，我们利用最近的一次 鉴定了名为SH3的CaV1调节子和半胱氨酸富含结构域(STAC)蛋白 最小的结构域(称为U-结构域)，它自然地对抗钙离子/CaM反馈。在这里，我们 利用燕麦的光敏Lov2结构域设计STACU结构域 在原生设置中可逆地光调制其与CaV1.2/1.3频道的交互。跟随 对这种新型的CaV1执行器LovU-CaV1进行了优化，并对其进行了功能分析 CaV1 CDI在海马神经元AP中的作用总而言之，小说的发展 光遗传CaV1执行器开辟了新的前沿，明确定义了 钙离子--在自然环境中的反馈，并在此过程中，概述长期寻求的病理生理 将CaV1错误调节与复杂的神经精神表型联系起来的机制。

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.