(A) Optical assay to detect perturbations to Kir2.1, CheRiff, or QuasAr2 in NaV1.7-OS HEK cells. NaV1.7 was blocked with 1 μM TTX. Cells were stimulated with increasing intensities of blue light (500 ms, 3.2, 12, 27, 41, 56 mW/cm2), and QuasAr2 fluorescence was monitored with 635 nm excitation, 400 W/cm2. Fluorescence with a test compound (10 μM amitriptyline, 100 μM carbamazepine, 10 μM trifluoperazine, 1 μM PF-04856264, 200 μM lidocaine, 10 μM isradipine, 10 μM iloperidone) was compared to TTX alone. The fluorescence changes were normalized to that of TTX only treated cells under 56 mW/cm2 stimulation. (B) Mean fluorescence changes in (A) during the blue stimuli as a function of stimulus intensity. Error bars represent s.e.m. of n = 9–10 wells. (C) Voltage clamp protocol to test for drug effects on CheRiff photocurrent. Cells were held at −80 mV and then stepped to −60 mV to +40 mV in 20 mV increments. During each step depolarization, a blue light pulse (100 ms, 0.5 W/cm2) was applied to activate CheRiff current. Control trace and trace after carbamazepine treatment are shown as an example. (D) I-V relationship of CheRiff current under control condition (before drug treatment) and with test compounds at the same concentrations as in (A). To control for cell-to-cell variations in CheRiff expression, the current amplitudes were normalized to that of control at −80 mV. Error bars represent s.e.m., n = 29 cells for control, n = 3–4 cells for each compound. (E) Voltage-clamp measurements to test for drug effects on QuasAr2 voltage sensitivity. QuasAr2 fluorescence was monitored with 640 nm excitation, 400 W/cm2 while membrane voltage was modulated as shown. Single cell recordings were performed before and after addition of drug or buffer control. Example traces show fluorescence before and after addition of buffer, lidocaine or carbamazepine. (F) Mean QuasAr2 fluorescence as a function of voltage in the presence of test compounds at the same concentrations as in (A). To control for cell-to-cell variation in QuasAr2 expression, the fluorescence changes were normalized to the pre-drug fluorescence at +40 mV. Error bars represent s.e.m., n = 30 cells for control, n = 3–4 cells for each compound.