Abstract
Optogenetics is a powerful research approach that allows localized optical modulation of selected cells within an animal via the expression of genetically encoded photo-excitable ion channels. Commonly used optogenetic techniques rely on the expression of microbial opsin variants, which have many excellent features but suffer from various degrees of blue spectral overlap and limited channel conductance. Here, we expand the optogenetics toolbox in the form of a tunable, high-conductance vertebrate cation channel, zTrpa1b, coupled with photo-activated channel ligands, such as optovin and 4g6. Our results demonstrate that zTrpa1b/ligand pairing offers high light sensitivity, millisecond-scale response latency in vivo, as well as adjustable channel off latency. Exogenous in vivo expression of zTrpa1b in sensory neurons allowed subcellular photo-activation, enabling light-dependent motor control. zTrpa1b/ligand was also suitable for cardiomyocyte pacing, as shown in experiments performed on zebrafish hearts in vivo as well as in human stem cell-derived cardiomyocytes in vitro. Therefore, zTrpa1b/optovin represents a novel tool for flexible, high-conductance optogenetics.
Highlights
Over the last decade, optogenetics as a versatile optical interrogation tool has proven to be a transformative approach to various fields of basic research
The zebrafish Trpa1b (zTrpa1b) cation channel was identified as the endogenous target, as trpa1b mutants failed to respond to optovin/ light activation (Fig. 1c,d,e)
We found that trpa1b mutants expressing human TRPA1 (hTRPA1) were less sensitive to optovin/light activation compared to mutants expressing zTrpa1b (p = 0.0571) (Fig. 2b)
Summary
Optogenetics as a versatile optical interrogation tool has proven to be a transformative approach to various fields of basic research. Chemo-optogenetic channels (optopharmacology or optogenetic pharmacology tools) have been under rapid development (see review ref.[28]) High conductance channels, such as nicotinic acetylcholine receptors LinAChRs or light-gated glutamate receptors LiGluRs, have been engineered to be photo-controllable[29,30,31,32]. We show that exogenous expression of zTrpa1b in neurons and cardiomyocytes enables photo-dependent cell depolarization that activates physiological cell functions in vitro and in vivo These data suggest that zTrpa1b/ligand pairing, as a robust chemo-optogenetic actuator, can be used in both neuronal and non-neuronal excitable cells. Features such as its violet light activation, high unitary conductance, high light sensitivity, Ca2+ selectivity, and adjustable channel off rate make it a complementary alternative to the existing chemo-optogenetic tools
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