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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

基本信息

批准号：
10403588
负责人：
Manu Ben Johny
金额：
$ 20.25万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
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/10403588
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 induced pluripotent stem cell derived cardiomyocytes molecular modeling neuropsychiatric disorder neuropsychiatry next generation novel novel strategies optical switch optogenetics prevent programs small molecule spatiotemporal stem tool voltage

项目摘要

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.

项目总结