SAR analysis for the identification of selective inhibitors of the two-pore domain potassium channel (KCNK9) -selectivity assay against KCNK3: Automated Electrophysiology
The purpose of this assay is to evaluate dose dependent compound inhibition of TASK1 (KCNK3) two pore domain potassium channels using automated electrophysiology voltage clamp technique. The inhibitory effects of test compounds on TASK1 channels were evaluated in electrophysiological assays using a QPatch16 automated electrophysiology instrument. Whole cell recordings were made from CHO cells more ..
BioActive Compounds: 5
Principle of the assay
The purpose of this assay is to evaluate dose dependent compound inhibition of TASK1 (KCNK3) two pore domain potassium channels using automated electrophysiology voltage clamp technique. The inhibitory effects of test compounds on TASK1 channels were evaluated in electrophysiological assays using a QPatch16 automated electrophysiology instrument. Whole cell recordings were made from CHO cells stably expressing TASK1 channels. For each experiment, channel function was tested first in 4mM potassium solution. After stabilization, external buffer was switched to one containing 140mM potassium. Following external buffer switch, the effect of test compounds was measured in the presence of 140mM potassium. Treatment of 2 mM Ba2+ in pH5.8 solution was used as reference for 100% inhibition for each cell tested.
Stable CHO cells with inducible TASK1 expression were seeded in a 15 cm dish three days before testing on QPatch16 automated patch clamp system. After one day culture at 37 degrees C, channel expression was induced (1ug/ml Tetracycline) for 24 hours at 37 degrees C and for another 24 hours at 30 degrees C.
Cells were detached into single cell suspension and allowed to recover for at least 1hr before running in single-hole mode on QPatch16 instrument. Whole cell currents were filtered at 1 kHz (4-pole Bessel) and sampled at 5 kHz. The series resistance was compensated by 80% with a cut-off frequency of 0.8 kHz. TASK1 currents were elicited by voltage ramp protocols consisting of 50ms at -80 mV, 50ms at -100 mV, followed by a 750 ms ramp to +50 mV, 5 ms at +50 mV, and then back to -80 mV for 40 ms. The voltage protocol was applied every 5 sec from a holding potential of -80 mV. After a 3 minute stabilization period in external bath solution containing 4 mM potassium, the bath solution was exchanged to the 140 mM potassium solution. After a 3 minute baseline period in 140 mM potassium external solution (Imax), cumulative dose response experiments were performed by applying compounds in a dual addition mode in which external solution was fully exchanged twice at each compound concentration. Compound effects were determined after a 5 minute incubation period at each concentration (Itest). A reference 140 mM potassium solution containing 2 mM barium with pH 5.8 was then applied to fully block TASK1 channels and determine maximal possible block (Ibaseline).
Current amplitudes were measured at -30 mV. Fractional channel block at each compound concentration was calculated as ((Imax-Itest)/(Imax-Ibaseline)). IC50 values were determined for each cell by fitting fractional block values versus compound concentration with a Hill equation with the maximal and minimal values fixed to one and zero, respectively, and the slope fixed to one.
Internal solution (in mM): 140 KCl, 2 MgCl2, 10 EGTA, 5 MgATP, and 10 HEPES, pH 7.30 with KOH
4K External solution (in mM): 150 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, and 10 HEPES, pH 7.40, with NaOH
140K external solution (in mM): 14 NaCl, 140 KCl, 1.8 CaCl2, 1 MgCl2, and 10 HEPES, pH 7.40, with NaOH
1. Outcome assignment
If the test compound causes greater than 50% inhibition of TASK1 at 10 microM, the compound is considered to be active (Outcome=2). Otherwise, the compound is designated as inactive (Outcome=1).
2. Score assignment
An inactive test compound is assigned the score of 0. An active test compound is assigned a score between 1 and 100 according to the following criteria: 25 for compounds with 10 microM
B. Number of cells included in each determination.
List of reagents:
1. Stable CHO cell line with inducible TASK1 expression
2. PBS: pH7.4 (Invitrogen Cat#10010023)
3. Medium: Ham's F-12 Nutrient Mix (Invitrogen, Cat#11765)
4. Fetal Bovine Serum (Gibco, Cat#26140)
5. 100x Penicillin-Streptomycin (Mediatech, Cat#30-001-CI)
6. Detachin (Genlantis, Cat#T100110 )
7. CHO-S-SFMII (Invitrogen Cat#12052)
8. Tetracycline Hydrochloride (Sigma,Cat#T7660)
9. QPlate16 (Sophion Bioscience, Ballerup, Denmark)
10. Blasticidin S (Research Products International Corp., Cat#B12150)
11. Hygromycin (Mediatech, Cat#30-240-CR)
* Activity Concentration.
Data Table (Concise)