Fig. 2

Opsins tested in this study. a Schematic of opsins tested (and mGluR6), illustrating intracellular regions exchanged in chimeras. RHO indicates wild-type human rod opsin, SWS indicates wild-type human short-wave-sensitive cone opsin, and LWS marks wild-type human long-wave-sensitive cone opsin. mGluR6 was not tested, but is presented to illustrate chimeric opsin construction. mML23Cm6 was previously published as Opto-mGluR6. All of the opsin constructs were tagged with the C-terminal nine amino acids of rod opsin, which is the epitope for the 1D4 monoclonal antibody. b Amino-acid sequences of the transmembrane helix (TM)–intracellular fragment–TM splice junctions of the opsin chimeras. c,d HEK293T cells were transfected with opsins, fixed, stained with 1D4 anti-rod opsin antibody and fluorescent secondary antibody, and imaged on a wide-field fluorescent microscope at 20× magnification. c Representative images of cells expressing each opsin and control. d Quantification of integrated fluorescence intensity (minus background) for ten randomly selected fields from each sample, normalized to mean of no-opsin control, with mean and SEM. AmphiOp1 amphioxus opsin 1, AU arbitrary units, hML23Cm6 human melanopsin/mGluR6 (mouse) triple-fragment chimera, LWS long-wave sensitive, mGluR6 metabotropic glutamate receptor 6, mML23Cm6 mouse melanopsin/mGluR6 (mouse) triple-fragment chimera, RHO human rod opsin, RL23Cm6 RHO/mGluR6 three-fragment chimera, RL3Am RHO loop 3/AmphiOp1 loop 3 chimera, RL3m6L2 RHO loop 3/mGluR6 loop 2 chimera, RL3Sc RHO loop 3/ScallOp2 loop 3 chimera, ScallOp2 scallop opsin 2, SEM standard error of the mean, SWS short-wave sensitive, TM transmembrane helix