Topology and Mechanism of Broadband and Fast Multi‐Characteristic Opsin for Neuromodulation

Abstract Optogenetics has enabled targeted manipulation of neural circuits, offering insights into the intricacies of brain function and paving the way for potential therapeutic applications in neurological disorders. Multi‐characteristic Opsin (MCO) is an optogenetics therapy candidate that is undergoing multiple clinical trials, including randomized control trials. However, its cellular topography and mechanism of action have remained unknown. Here, we reveal the biophysical characteristics of the MCO molecule, including its unique structure and function, using a combination of predictive protein modeling, western blotting‐based protease protection assay, and electrophysiology. MCO’s first extracellular subunit forms a cation‐channel pore, while the second subunit is partially embedded in the membrane, extending to the third intracellular cytoplasmic subunit. Functional studies using whole‐cell recordings showed that MCO is activated by broadband visible light with fast on‐off kinetics with high photosensitivity, and large dynamic range across the visible spectrum, enabling best‐in‐class light‐activation of MCO‐expressing cells. Further, multi‐electrode array recording confirmed multi‐color light‐activation of MCO‐expressing neurons in the retina. The unique topological and functional activation features of fused MCO protein represent novel findings linking the in‐vitro and in‐vivo efficacy of this opsin, which is particularly important for neuromodulation leading to vision restoration in retinal degenerative diseases and other neurological therapies.