Design of an Ultrafast G Protein Switch Based on a Mouse Melanopsin Variant

Abstract

The primary goal of optogenetics is the light‐controlled noninvasive and specific manipulation of various cellular processes. Herein, we present a hybrid strategy for targeted protein engineering combining computational techniques with electrophysiological and UV/visible spectroscopic experiments. We validated our concept for channelrhodopsin‐2 and applied it to modify the less‐well‐studied vertebrate opsin melanopsin. Melanopsin is a promising optogenetic tool that functions as a selective molecular light switch for G protein‐coupled receptor pathways. Thus, we constructed a model of the melanopsin Gq protein complex and predicted an absorption maximum shift of the Y211F variant. This variant displays a narrow blue‐shifted action spectrum and twofold faster deactivation kinetics compared to wild‐type melanopsin on G protein‐coupled inward rectifying K+ (GIRK) channels in HEK293 cells. Furthermore, we verified the in vivo activity and optogenetic potential for the variant in mice. Thus, we propose that our developed concept will be generally applicable to designing optogenetic tools.

Melanopsin and the Intrinsically Photosensitive Retinal Ganglion Cells: Biophysics to Behavior

“…At the same time, light of a particular composition can be used to suppress melanopsin activity, providing a measure of temporal control (Emanuel and Do, 2015;Spoida et al, 2016). The time course of activation also can be tuned by using different variants of melanopsin (Spoida et al, 2016;Tennigkeit et al, 2019;Tsukamoto et al, 2015). Given the large number of uncharacterized variants and advances in protein engineering, the artificial uses of melanopsin may expand in coming years.…”

Selective optogenetic control of Gq signaling using human Neuropsin

“…In order to prove that UV illumination of hOPN5/eYFP is not activating G i signaling, we took advantage of the GIRK channel assay with its high level of sensitivity to reveal even small G i protein activation and which had already been used to show the promiscuous nature of Melanopsin activating also G i signaling 19 , 47 . Specifically it was shown that illumination of Melanopsin in HEK cells overexpressing GIRK channels 1 and 2 leads to increased GIRK currents by the G i βγ subunit complex 19 , 47 . In contrast, specific activation of the G q signaling cascade only would inhibit GIRK channels via PLC-dependent PIP2 depletion as well as by protein kinase Cδ mediated phosphorylation 48 .…”

Reverse optogenetics of G protein signaling by zebrafish non-visual opsin Opn7b for synchronization of neuronal networks

“…G protein-coupled receptors (GPCRs) couple to four main G protein pathways, i.e., G i/o , G s , G q/11 , and G 12/13 . In recent years various light-activated GPCRs have been used to specifically control these signaling pathways, including vertebrate rod and cone opsins as well as parapinopsin (UVLamp) for the control of the G i/o pathway 2,4,5 , vertebrate melanopsin and invertebrate rhodopsin for the control of the G q/11 pathway [6][7][8] and jellyfish opsin for the control of the G s pathway 9 .…”