Figure 1

Figure 1 Activity of G-coupled bistable rhodopsins in HEK293S cells.

(A) Schematic of intracellular Ca²⁺ or cAMP measurements in HEK293S cells. Intracellular Ca²⁺ and cAMP concentrations in rhodopsin-expressing HEK293S cells were measured using the aequorin m2 assay or the GloSensor 20 F assay, respectively. (B) Comparison of optogenetic activities of Gq-coupled Spider Rh1 (SpiRh1) expressed using TagCFP fusion protein and the P2A-TagCFP system. HEK293S cells were transfected with an expression plasmid for the fusion protein of SpiRh1 and TagCFP (SpiRh1-TagCFP, left panel), or for that of Flag-tagged SpiRh1, porcine teschovirus 2 A peptide, and TagCFP (SpiRh1-P2A-TagCFP, right panel). Transfected cells were incubated with 11-cis retinal and stimulated by different intensities of 500 nm light (0.1%, 1%, or 10% of the light intensity, with 0.106 mW/mm² as 100%). Intracellular Ca²⁺ concentration was measured by using aequorin m2 and is indicated as a ratio to the unstimulated state in the graphs. (C) Comparison of activities of Flag-tagged SpiRh1 and SpiRh1 [S186F]. Transfected cells were stimulated by 500 nm (green arrow, 0.106 mW/mm²) or 410 nm (purple arrow, 0.0194 mW/mm²) light and intracellular Ca²⁺ concentration was measured. (D) Light-stimulus-dependent reduction of intracellular cAMP level by Gi/o-coupled mosquito Opn3 (MosOpn3), pufferfish TMT (PufTMT), lamprey PP (LamPP), and zebrafish PP1 (ZPP1). HEK293S cells were transfected with expression plasmids for flagged-tagged Gi/o rhodopsins. Transfected cells were incubated with 11-cis retinal and stimulated by 500 nm (green arrow) or 410 nm (purple arrow) light. Intracellular cAMP concentration was measured with GloSensor 20 F and is indicated as a ratio to the unstimulated state. (E) Effects of Gαq inhibitor YM254890 on SpiRh1. HEK293S cells transfected with an expression plasmid for SpiRh1 were incubated with 11-cis retinal alone (left panel) or with 11-cis retinal and YM254890 (right panel), and stimulated by 500 nm light. Intracellular Ca²⁺ concentration was measured.