Expanding the family of genetically encoded voltage indicators with a candidate Heliorhodopsin exhibiting near-infrared fluorescence

Abstract

Genetically encoded voltage indicators, particularly those based on microbial rhodopsins, are gaining traction in neuroscience as fluorescent sensors for imaging voltage dynamics with high-spatiotemporal precision. Here we establish a novel genetically encoded voltage indicator candidate based on the recently discovered subfamily of the microbial rhodopsin clade, termed heliorhodopsins. We discovered that upon excitation at 530 to 560 nm, wildtype heliorhodopsin exhibits near-infrared fluorescence, which is sensitive to membrane voltage. We characterized the fluorescence brightness, photostability, voltage sensitivity, and kinetics of wildtype heliorhodopsin in HEK293T cells and further examined the impact of mutating key residues near the retinal chromophore. The S237A mutation significantly improved the fluorescence response of heliorhodopsin by 76% providing a highly promising starting point for further protein evolution. Genetically encoded voltage indicators, particularly those based on microbial rhodopsins, are gaining traction in neuroscience as fluorescent sensors for imaging voltage dynamics with high-spatiotemporal precision. Here we establish a novel genetically encoded voltage indicator candidate based on the recently discovered subfamily of the microbial rhodopsin clade, termed heliorhodopsins. We discovered that upon excitation at 530 to 560 nm, wildtype heliorhodopsin exhibits near-infrared fluorescence, which is sensitive to membrane voltage. We characterized the fluorescence brightness, photostability, voltage sensitivity, and kinetics of wildtype heliorhodopsin in HEK293T cells and further examined the impact of mutating key residues near the retinal chromophore. The S237A mutation significantly improved the fluorescence response of heliorhodopsin by 76% providing a highly promising starting point for further protein evolution. Detailed studies of neural circuitry and computation are contingent upon resolving the electrical dynamics of several neurons in parallel with high spatiotemporal precision. Direct visualization of changes in neural membrane potential has been facilitated by engineering bright and sensitive probes of which the fluorescence is modulated by changes in membrane voltage. These engineered transmembrane proteins are termed genetically encoded voltage indicators (GEVIs) (1Mollinedo-Gajate I. Song C. Knöpfel T. Genetically encoded voltage indicators.Adv. Exp. Med. Biol. 2021; 1293: 209-224Crossref PubMed Scopus (10) Google Scholar). Various GEVI families have been optimized over the past years, and particularly GEVIs based on microbial rhodopsin proton pumps have enabled the recording of activity in an ensemble of neurons with submillisecond response time (2Gong Y. The evolving capabilities of rhodopsin-based genetically encoded voltage indicators.Curr. Opin. Chem. Biol. 2015; 27: 84-89Crossref PubMed Scopus (20) Google Scholar). The first rhodopsin-based GEVI was derived from the bacterial Proteorhodopsin, discovered due to the success of metagenomic sequencing efforts in Monterey Bay (3Kralj J.M. Hochbaum D.R. Douglass A.D. Cohen A.E. Electrical spiking in Escherichia coli probed with a fluorescent voltage-indicating protein.Science. 2011; 333: 345-348Crossref PubMed Scopus (259) Google Scholar). Another proton pump, Archaerhodopsin-3 (Arch) from the archaea Halorubrum sodomense, was found to be a better GEVI candidate for expression in mammalian cells (4Hochbaum D.R. Zhao Y. Farhi S.L. Klapoetke N. Werley C.A. Kapoor V. et al.All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins.Nat. Methods. 2014; 11: 825Crossref PubMed Google Scholar). The first Arch versions were very dim and required several iterations of molecular evolution (4Hochbaum D.R. Zhao Y. Farhi S.L. Klapoetke N. Werley C.A. Kapoor V. et al.All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins.Nat. Methods. 2014; 11: 825Crossref PubMed Google Scholar, 5McIsaa