Cover Feature: Design of an Ultrafast G Protein Switch Based on a Mouse Melanopsin Variant (ChemBioChem 14/2019)

Abstract

The primary goal of optogeneticsi st he light-controlled noninvasive and specific manipulation of various cellular processes. Herein, we present ah ybrid strategy for targeted protein engineering combining computational techniques with electrophysiologicala nd UV/visible spectroscopic experiments.W ev alidated our concept for channelrhodopsin-2 and applied it to modify the less-well-studied vertebrate opsin melanopsin. Melanopsin is ap romising optogenetic tool that functions as a selectivem olecular light switch forGprotein-coupled receptor pathways. Thus, we constructed am odel of the melanopsin G q protein complex and predicted an absorption maximum shift of the Y211F variant. This variant displays an arrow blue-shifted action spectrum and twofold faster deactivation kinetics compared to wild-type melanopsin on Gprotein-coupledi nward rectifying K + (GIRK) channels in HEK293c ells. Furthermore, we verifiedt he in vivo activity and optogenetic potential for the variant in mice. Thus, we proposet hat our developed concept will be generallyapplicable to designing optogenetic tools.

Lamprey Parapinopsin (“UVLamP”): a Bistable UV‐Sensitive Optogenetic Switch for Ultrafast Control of GPCR Pathways

“…We further characterized the properties of light‐induced G i/o signaling via UVLamP in detail in vitro and found that its light‐induced activation and deactivation time constants with 1.15 s for activation and 4.95 s for deactivation (Figure A). These time constants are even faster than those of wild‐type mouse melanopsin (≈1.4 and ≈8.5 s, respectively) and comparable with the recently engineered Y211F mutant . We next investigated the minimal light pulse duration for activation and deactivation of GIRK channels using UVLamP.…”

“…Recently, we characterized mouse and human homologues of melanopsin, another non‐visual opsin that can be found in intrinsically photosensitive retinal ganglion cells (ipRGCs) of the vertebrate retina and is involved in multiple physiological processes, for example, the circadian rhythm. We could show that melanopsin is a bistable/tristable opsin and a unique optogenetic tool that can be switched on/off with blue/green light pulses and can precisely control the G i/o and G q pathway in different expression systems like the mouse brain . Despite its advantageous characteristics, like high light sensitivity, bistable switching between sustained active and inactive states with short light pulses and strong expression in different systems, two drawbacks remained: First, melanopsin is activated/deactivated in the visible spectrum ranging from blue for activation to green/yellow for deactivation, therefore overlapping with most other optogenetic tools.…”

“…Here, we constructed in silico an atomistic model of membrane inserted solvated parapinopsin in complex with GDP bound G i/o protein, reflecting the dark state structure. To this end, we employed the modeling concept we recently developed to build and validate an atomic model of melanopsin as outlined under Model construction and Model validation in the Supporting Information. The X‐ray structure of bovine rhodopsin (PDB ID: 1U19) served as a basis to build parapinopsin.…”

“…Because GPCRs coupling to the G i/o pathway are important drug targets for various diseases including anxiety, depression, epilepsy and pain and importantly contribute to synaptic plasticity and neuronal development, our next goals are to engineer UVLamP variants for altered trafficking into disease relevant GPCR domains in the mammalian brain. Because of the unique, blue‐shifted, narrow bandwidth features of UVLamP it will now be possible, in combination with other optogenetic tools and genetically encoded sensors, to shed light onto important, unresolved questions in neurobiology, that is, how differently shaped GPCR signals such as fast, transient, long lasting or sustained signals contribute to brain function and change during disease states …”

The roles of an extended N-terminal region and ETD motif in a pump-like cation channelrhodopsin discovered in a lake microbiome

“…Importantly, in the case of Cr ChR2, substituting alanine for serine at this position causes a red shift in the absorption spectrum. [32] Nevertheless, the action spectrum of the photocurrents of the HulaCCR C303A mutant showed no significant red shift compared to the WT (Fig. S2c).…”