A neuropsin-based optogenetic tool for precise control of Gq signaling

Abstract

G q -coupled receptors regulate numerous physiological processes by activating enzymes and inducing intracellular Ca 2+ signals.There is a strong need for an optogenetic tool that enables powerful experimental control over G q signaling. Here, we present chicken opsin 5 (cOpn5) as the long sought-after, single-component optogenetic tool that mediates ultra-sensitive optical control of intracellular G q signaling with high temporal and spatial resolution. Expressing cOpn5 in HEK 293T cells and primary mouse astrocytes enables blue light-triggered, G q -dependent Ca 2+ release from intracellular stores and protein kinase C activation.Strong Ca 2+ transients were evoked by brief light pulses of merely 10 ms duration and at 3 orders lower light intensity of that for common optogenetic tools. Photostimulation of cOpn5-expressing cells at the subcellular and single-cell levels generated fast intracellular Ca 2+ transition, thus demonstrating the high spatial precision of cOpn5 optogenetics. The cOpn5-mediated optogenetics could also be applied to activate neurons and control animal behavior in a circuit-dependent manner. cOpn5 optogenetics may find broad applications in studying the mechanisms and functional relevance of G q signaling in both non-excitable cells and excitable cells in all major organ systems.

Optogenetic manipulation of Gq- and Gi/o-coupled receptor signaling in neurons and heart muscle cells

“…The bistable rhodopsin tools that we designed are effective in zebrafish, but are also active in human HEK293S cells ( Table 1 ). Bistable rhodopsins were shown to be expressed in mammalian tissues and used to optogenetically manipulate GPCR signaling in vivo ( Copits et al, 2021 ; Dai et al, 2022 ; Mahn et al, 2021 ; Makowka et al, 2019 ; Rodgers et al, 2021 ; Wagdi et al, 2022 ). In this study, the expression plasmids for bistable rhodopsins were constructed to express tagged rhodopsin and P2A-TagCFP by the Gal4-UAS system in specific types of zebrafish cells.…”

“…MosOpn3 and lamprey parapinopsin (LamPP) were used to suppress neuronal activities in a light stimulation-dependent manner in mammals ( Copits et al, 2021 ; Mahn et al, 2021 ; Rodgers et al, 2021 ) and in Caenorhabditis elegans ( Koyanagi et al, 2022 ). In addition to these rhodopsins, a Gq-coupled rhodopsin, neuropsin (also known as Opn5), was used to induce the activation of neurons, the intestine, and heart ( Dai et al, 2022 ; Wagdi et al, 2022 ). Optogenetic activation of Gs-coupled rhodopsin jellyfish opsin (JellyOp) and Gi-coupled long wavelength-sensitive cone opsin (LWO) were shown to accelerate and suppress the excitation of cardiomyocytes, respectively ( Cokić et al, 2021 ; Makowka et al, 2019 ).…”

Cardiac optogenetics: shining light on signaling pathways

“…Besides rhodopsins that modulate the cAMP signaling pathway, melanopsin (OPN4) [ 98 ] and neuropsin (OPN5) [ 100 , 101 ], both activating the Gq pathway, have been expressed in cardiac cells, where optical activation of Gq signaling induced positive inotropic and chronotropic effects (Fig. 3 ).…”

Optogenetic activation of mGluR1 signaling in the cerebellum induces synaptic plasticity

“…For optogenetic modulations light-activated GPCRs such as vertebrate rod and cone opsins, parapinopsin, melanopsin and jellyfish opsin have been established to control the G i/o , G q/11 and G s pathways, respectively. 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 To achieve highly customized GPCR specific targeting and downstream signaling, chimeras of suitable opsins and GPCR domains of choice have been engineered by exchanging the intracellular domains between the light-activated and ligand-gated GPCRs. 39 , 40 , 44 , 45 , 46 …”