Voltage imaging provides unparalleled spatial and temporal resolution of the brain’s electrical signaling at the cellular and circuit levels. A longstanding challenge has been to develop genetically encoded voltage sensors to track membrane voltage from multiple neurons in behaving animals. However, brightness and signal to noise ratio have limited the utility of existing voltage sensors, especially in vivo. In the first part of my talk, I will describe a ‘chemigenetic,’ or hybrid protein–small molecule, voltage sensor scaffold that we call Voltron, which irreversibly binds synthetic fluorophore dyes. Voltage is reported as a fluorescence change that arises from energy transfer (FRET) between the dye emission and the rhodopsin voltage sensor domain absorption. The chemigenetic sensor platform is significantly brighter and more photostable than existing voltage sensors, extending both productive imaging time and number of neurons imaged by more than 10 times. This enabled, for the first time, the precise correlation of spike timing with behavior in model organisms. In the second part of my talk, I will describe detailed mechanistic insights into the rhodopsin protein’s response to voltage changes that we discover using site-directed mutagenesis and electrophysiology recordings. These mechanistic insights allow rational control of the sensor’s fluorescence response to membrane voltage, laying the groundwork for further sensor development. Overall, my talk will demonstrate how proteins can be engineered into useful optogenetic tools for functional analysis of the brain.
