Electrical transients are a fundamental currency of communication between, and within, all living cells. Dysregulated electrical handling is a hallmark of many diseases including neurological and cardiac diseases. Patch clamp, the gold standard technique for measuring and controlling cellular voltage, has been utilized by neuroscientists and cardiologists with many fantastic successes. Despite the power of the technique, patch clamp is limited by the nature of its technically challenging, slow, and serial experiments. Optical voltage sensing overcomes many limitations of patch clamp. Measurements can be obtained from hundreds of cells in parallel, enabling rapid electrophysiological tests across many conditions including chemical and genetic perturbations. Microbial rhodopsins were shown to be highly sensitive and fast genetically encoded, fluorescent voltage indicators. Combinations of these probes with channelrhodopsin yielded ?optopatch?, a platform for all-optical voltage actuation and sensing, permitting rapid automated single cell electrophysiology. The goal of this proposal is to generate an all-optical platform capable of measuring the contribution of every gene in the human genome to neuronal electrophysiology. This data will be the founding of electromics, the tie between electrophysiology and genetics. The platform will combine lenti-viral knockdown microarray chips, an automated optopatch microscope, human ES-derived neurons, and data processing will enable measurements at the necessary scale. Complete knockdown and expression libraries will be recorded. Electrophysiological perturbations arising from a model of ALS will also be tested on the platform to both (i) understand disease etiology, and (ii) search for protein knockdown targets to ameliorate symptoms. The proposed platform will vastly exceed the throughput of traditional measurements, and will generate revolutionary data for neuroscience. Combined with the burgeoning field of iPS derived disease models, our platform will allow us to investigate electromics in a variety of neurological and neurodegenerative diseases. However, other cell types will be accessible including cardiomyocytes (heart disease), T-cells (auto-immunity), and beta cells (diabetes). The ubiquity of voltage in biology multiplies the value of the instrument. Further studies will extend to any temporally encoded cellular signal including Ca++, transcription factor dynamics, or ATP. Ultimately, I envision a community-wide resource where investigators with interesting cellular models could utilize our platform to discover genetic influences on dynamic signaling.
Every thought, emotion, sensation, and movement arise from dynamic electrical transients in the brain, which in turn are controlled by the careful expression and organization of hundreds of proteins. Technical limitations have prohibited a complete model of cellular components that generate and modulate neuronal electrical signaling. This project will develop an instrument to quantify the electrophysiologic effect of every gene in the human genome, which will be of enormous benefit to researchers, pharmaceutical development, and clinicians.
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|Bruni, Giancarlo N; Weekley, R Andrew; Dodd, Benjamin J T et al. (2017) Voltage-gated calcium flux mediates Escherichia coli mechanosensation. Proc Natl Acad Sci U S A 114:9445-9450|