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

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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.