Fig. 4.

mML23Cm6, hML23Cm6, and RL23Cm6 exhibit little or no light response. a HEK293T cells treated with pertussis toxin and transfected with Glo22F cAMP reporter, +/- opsin, and Gso, Gsi, or Gst were stimulated with 470 nm light. Normalized light responses were calculated as above. For details, see “Methods.” None of these opsins exhibited light responses that satisfied our statistical criteria. b HEK293T cells were transfected with Glo22F and opsin, as indicated, and treated with 2 μM forskolin for 30 mins prior to experiment to elevate basal cAMP, in an assay for opsin coupling to endogenous Gi. Cells were stimulated with 470 nm light and the minimum GloSensor cAMP level post-flash was recorded for each trial. Only RL23Cm6 exhibited statistically significant activity, and its fitted response curve is shown. a, b Graphs show mean cAMP response +/- SEM (n = 3) at varying irradiance. Symbols for mML23Cm6 and RL23Cm6 have been offset slightly in the x-axis to aid visualization. Error bars smaller than symbols are not shown. hML23Cm6 human melanopsin/mGluR6 (mouse) triple-fragment chimera, mML23Cm6 mouse melanopsin/mGluR6 (mouse) triple-fragment chimera, PTX pertussis toxin, RL23Cm6 RHO/mGluR6 three-fragment chimera, SEM standard error of the mean