Article Figures & Data
Figures
- Fig. 1.
Truncation and removal of the ER retention signal from KV11.1-G601S improves Tl+ flux assay results. (A) Treatment layout used for 384-well plates with HEK-293 cells expressing KV11.1-G601S or the truncated KV11.1-G601S-G965*X variant. Every other well was treated with vehicle (black) or positive (red) control in a checkerboard layout for 24 hours prior to experiments. (B) A fluorescent trace from an individual well plated with a monolayer of HEK-293 cells expressing KV11.1 in 384-well plates. Fluorescent signal was normalized to baseline using the average fluorescence of the first five time points to generate a static ratio (F/F0). Blue Box Inset: Timepoints used to calculate slope (slope = average of segments 1-5 or 15-20 second). Black arrow indicates Tl+ (4.5 mM) addition. (C) Fluorescent traces for vehicle and positive control–treated wells with HEK-293 cells expressing KV11.1-G601S or KV11.1-G601S-G965*X. Black arrow represents Tl+ (4.5 mM) addition. All wells were washed before experiments and replaced with assay buffer. (D) Scatter plot of fluorescent slope (ΔF/s) for vehicle and positive control wells. For all experiments, vehicle (0.1% DMSO) and positive control (10 µM E-4031), n = 96 wells/treatment condition/plate. Data for cell lines expressing each KV11.1 variant are separate plates on the same experimental day.
- Fig. 2.
Tl+ flux assay detects reduced KV11.1 current in HEK-293 cells expressing KV11.1-G601S-G965*X. (A) Representative current traces (left) and current-voltage (I-V) relationship (right) from whole cell patch-clamp recordings of HEK-293 cells expressing KV11.1 (blue) or KV11.1-G601S-G965*X (black, n = 6 cells/group). Data are mean ± S.D. (B) Fluorescent traces from monolayers of HEK-293 cells expressing KV11.1 or KV11.1-G601S-G965*X in 384-well plates. Black arrow indicates Tl+ (4.5 mM) addition. (C) Slope (ΔF/s) values calculated from individual wells of cells expressing KV11.1 or KV11.1-G601S-G965*X (n = 96 wells/group). Bars show the mean of each group. Data compared with Student’s t test. * = P < 0.05.
- Fig. 3.
Tl+ flux assay detects increased channel trafficking of KV11.1-G601S-G965*X with high sensitivity. (A) Example traces (left) and bar graph (right) of peak tail current density of KV11.1 current at −120 mV step in whole cell patch clamp. Data show KV11.1-G601S-G965*X channel current in absence (n = 7 cells) and presence (n = 8 cells) of 10 µM E-4031 for 24 hours. Inset shows enlarged current at −120 mV voltage step. Washout was performed before the start of experiments. Horizontal bar in scatter plot shows mean current in each treatment condition. (B) Representative fluorescent traces (Tl+ flux) of concentration response after 24-hour incubation with E-4031 and washout before experiments. Traces are normalized to the first five fluorescent values to generate a static ratio, (F/F0), then average fluorescence of 60 representative control wells were subtracted from normalized traces (F/F0 – Fcontrol). Black arrow represents Tl+ (4.5 mM) addition. (C) Concentration response curve for HEK-293 cells expressing KV11.1-G601S-G965*X and treated with E-4031 for 24 hours with washout before recording. The graph is normalized by dividing slope (ΔF/s) in E-4031 treated wells by slope (ΔF/s) in vehicle control (0.1% DMSO)–treated wells and subtracting 1. (n = 13 wells for each concentration, data plotted as mean ± S.D.). * = P < 0.05.
- Fig. 4.
Nonoptimized Tl+ flux assay not suitable for HTS. (A) Nonoptimized, average fluorescent traces for vehicle (black) and positive (red) control wells during Tl+ flux experiments. Black arrow represents Tl+ (4.5 mM) addition. All wells were washed out before experiments. (B) Nonoptimized scatter plot showing fluorescent slope (ΔF/s) of vehicle and positive control wells in the Tl+ flux assay. Red lines indicate mean for vehicle or positive control. Dotted black lines indicate ± 3 S.D. from the mean of each control group. For all experiments, vehicle (0.1% DMSO) or positive (10 µM E-4031) controls were used (n = 96 wells/treatment).
- Fig. 5.
KV11.1 channel activator (VU0405601) increases separation of KV11.1-G601S-G965*X steady-state current between vehicle and E-4031 treatment. (A) Example current traces from HEK-293 cells expressing KV11.1-G601S-G965*X in absence (black) or presence (red) of E-4031 for 24 hours with wash. For vehicle and E-4031 treatment, KV11.1 current was measured in absence or presence of 30 µM VU0405601. Black dotted line represents 0 current for each trace. (B) Current-voltage (I-V) plot of KV11.1-G601S-G965*X treated for 24 hours with vehicle or E-4031 with washout (n = 8 cells/treatment) in absence of VU0405601. (C) I-V plot of KV11.1-G601S-G965*X treated for 24 hours with vehicle or E-4031 with washout in presence (n = 6 cells) and absence (n = 8 cells) of 30 µM VU0405601 in bath solution during experiments. All data plotted as mean ± S.D. For all experiments, vehicle (0.1% DMSO) or positive (10 µM E-4031) control were used.
- Fig. 6.
KV11.1 channel activator (VU0405601) increases Z’ value of Tl+ flux assay. (A) Representative fluorescent traces from HEK-293 cell monolayers expressing KV11.1-G601S-G965*X and checkerboard-treated plates with vehicle (black) or positive (red) control for 24 hours with washout. After 24-hour treatment and wash, plates were treated acutely with vehicle (n = 192), VU0405601 (10 µM, n = 96), or VU0405601 (30 µM, n = 96) for 10–15 minutes before experiments and left in solution. Black arrow indicates Tl+ (4.5 mM) addition. (B) Scatter plot showing fluorescent slope values (ΔF/s) for every well and each respective acute treatment condition from A. Red lines indicate the mean of slope (ΔF/s) for all wells after 24-hour treatment with positive or vehicle control. Black dotted lines indicate ± 3 S.D. from the mean slope (ΔF/s) of each 24-hour treatment group. Light blue boxes highlight area of separation (Z’) for 24-hour treatment conditions: (mean of positive control − 3 S.D.s) and (mean of vehicle control + 3 S.D.s). All data from the same the day of experiments. For all experiments, vehicle (0.1% DMSO) or positive (10 µM E-4031) control were used.
- Fig. 7.
Combination of cesium (20 mM) with KV11.1 channel activator (30 µM VU0405601) increases Z’ value of Tl+ flux assay. (A) Representative fluorescent traces from HEK-293 cell monolayers expressing KV11.1-G601S-G965*X and checkerboard treated with positive (n = 64 wells) or vehicle (n = 64 wells) control for 24 hours with washout. In addition to 24-hour checkerboard treatment, wells were treated with 20 mM cesium, 30 µM VU0405601, or both (n = 96 wells/treatment) for 10-15 minutes before experiments. Black arrows indicate Tl+ (4.5 mM) addition. (B) Scatter plot showing slope values (ΔF/s) for every well and each condition from A. Red lines indicate the mean of slope (ΔF/s) for all wells after 24-hour treatment with positive or vehicle control. Black dotted lines indicate ± 3 S.D. from the mean slope (ΔF/s) of each 24-hour treatment group. Light blue boxes highlight area of separation (Z’) for 24-hour treatment conditions: (mean of positive control − 3 S.D.s) and (mean of vehicle control + 3 SDs). All data from the same day of experiments. For all experiments, vehicle (0.1% DMSO) or positive (10 µM E-4031) control were used.
- Fig. 8.
Optimized Tl+ flux assay detected increased KV11.1-G601S-G965*X trafficking with three known KV11.1 inhibitors. (A) Bar graph showing Tl+ flux Z scores after 24-hour treatment with E-4031. 56 wells were selected from central rows of the plate as representative of control. Dotted black line indicates threshold for “hit” selection set to Z score ≥ 3. (B) Western blot showing increased fully glycosylated protein (mature), representative of increased KV11.1 protein trafficking after 24-hour treatment with 10 µM azelastine, azaperone, or ibutilide. All wells loaded with 20 µg of protein. (C) Western blot showing increased KV11.1 protein trafficking after 24 hours of treatment with 10 µM azelastine, azaperone, or ibutilide in HEK-293 cells expressing KV11.1-N470D and KV11.1-G601S. All wells loaded with 20 µg of protein. (D) Whole cell current recordings at −120 mV step. Representative current traces (left) and bar graphs (right) depicting KV11.1-G601S-G965*X current after 24-hour incubation with 10 µM drug and washout before experiments. Inset shows enlarged current traces at −120 mV voltage step. N = 8 for E-4031 treatment and n = 7 for all other treatments. Treated samples compared with vehicle control (0.1% DMSO) by one-way ANOVA with post hoc Dunnett’s t test. Bars on scatter plots represent mean of each group. * = P < 0.05. Veh. = 0.1% DMSO, E-4. = E-4031, Azel. = azelastine, Azap. = azaperone, and Ibu. = ibutilide.
Additional Files
Data Supplement
- Supplemental Data -
Table S1. Oligonucleotide primers used for identification of 3’ KV11.1 stop codon.
Figure S1. Western blot and sequencing reveal KV11.1 truncation at glycine residue at amino acid 965 (G965).
Figure S2. KV11.1-G601S-G965*X variant performs better than KV11.1-G601S in optimized Tl+-flux assay.
Figure S3. KV11.1 channel activator (VU0405601) increases steady-state activation current in wild-type KV11.1.
Figure S4. KV11.1 channel activator (VU0405601) decreases KV11.1 channel inactivation in wild-type KV11.1.
Figure S5. Western blot from HEK-293 cells expressing KV11.1-G601S showing increased fully glycosylated protein, representative of increased protein trafficking after 24-hour treatment with 10 µM of azelastine, azaperone, or ibutilide. All wells loaded with 20 µg of protein. Vehicle = 0.1% DMSO.
Figure S6. Drugs that increase KV11.1-G601-G965*X trafficking do not increase KV11.1 (wildtype) trafficking or function.
- Supplemental Data -
