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. 2016 May 24;5:e15202. doi: 10.7554/eLife.15202

Figure 5. High throughput screening of a FDA-approved drug library in NaV1.7-OS HEK cells.

(A) QuasAr2 fluorescence from positive (amitriptyline) and negative (DMSO) control wells. Cells were stimulated with eight pulses of blue light (20 ms, 50 mW/cm2) at 10 Hz, and QuasAr2 fluorescence was monitored with 635 nm excitation, 400 W/cm2. (B) Screen results. The response of each well was parameterized by its use dependence index and standard deviation in spike amplitude. Positive controls (red) and negative controls (green) were well separated. Selected hits were chosen for further analysis. Inset: structures of amitriptyline and doxepin. (C) QuasAr2 fluorescence traces of doxepin, trifluoperazine, isradipine and iloperidone recorded in the screen. (D) Validation of select hits by manual electrophysiology. Nav1.7-Optopatch cells were held at −100 mV. A 200 ms prepulse to 0 mV allowed drug binding. Recovery times at −100 mV ranged from 1 ms to 256 ms. A test pulse to 0 mV, 100 ms duration, probed the degree of channel recovery. Blue: NaV1.7 current during prepulse. Red: NaV1.7 current during test pulse. (Prepulse and test pulse currents have been time-shifted and overlaid for easy comparison). Each compound was tested at 10 μM. (E) Quantification of compound effects on NaV1.7 recovery from inactivation. The plots show ratio of current amplitude at test pulse to prepulse, as a function of recovery period (n = 3 cells for each compound, n = 12 cells for control). (F) Characterization of select hits from (B) in NaV1.5-OS HEK cells. Cells were stimulated with eight pulses of blue light (20 ms, 50 mW/cm2) at 4 Hz. The 4 Hz stimulus was selected because action potential width of Nav1.5-OS cells lasted longer than 200 ms under control conditions. Data analyzed and plotted as in (B) (n = 4–6 wells per drug). Drug concentrations were TTX: 1 μM, PF-04856264: 1 μM, amitriptyline: 10 μM, trifluoperazine: 10 μM, isradipine: 10 μM, iloperidone: 30 μM).

DOI: http://dx.doi.org/10.7554/eLife.15202.012

Figure 5—source data 1. Spreadsheet containing compound names and screening results.
DOI: http://dx.doi.org/10.7554/eLife.15202.013
elife-15202-fig5-data1.xlsx (49.6KB, xlsx)
DOI: 10.7554/eLife.15202.013

Figure 5.

Figure 5—figure supplement 1. Fluorescence traces from NaV1.5-OS HEK cells with different drugs.

Figure 5—figure supplement 1.

NaV1.5-OS HEK cells were stimulated with eight pulses of blue light (20 ms, 50 mW/cm2) at 4 Hz, and QuasAr2 fluorescence was monitored with 635 nm excitation, 400 W/cm2. TTX and PF-04856264 had little effect on channel function. Amitriptyline and trifluoperazine showed strong use-dependent block. Isradipine and iloperidone showed use-dependent block with fast recovery, leading to alternating response amplitudes.
DOI: http://dx.doi.org/10.7554/eLife.15202.014
Figure 5—figure supplement 2. Characterization of off-target effects via optical and manual patch assays.

Figure 5—figure supplement 2.

(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.
DOI: http://dx.doi.org/10.7554/eLife.15202.015