Mode of action - Automated patch clamp assay for KCNQ2 potentiators on Retigabine insensitive KCNQ2 Mutant W236L cell line
Assay Implementation: Haibo Yu Ph.D., Beiyan Zou Ph.D., Shunyou Long M.S., Amy Scott, Meng Wu Ph.D., Joseph Babcock, Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D. ..more
BioActive Compounds: 91
Data Source: Johns Hopkins Ion Channel Center
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Source (MLPCN Center Name): Johns Hopkins Ion Channel Center (JHICC)
Center Affiliation: Johns Hopkins University, School of Medicine
Screening Center PI: Min Li, Ph.D.
Assay Provider: Min Li, Ph.D.
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 DA027716-01
Grant Proposal PI: Min Li, Ph.D., Johns Hopkins University School of Medicine
Assay Implementation: Haibo Yu Ph.D., Beiyan Zou Ph.D., Shunyou Long M.S., Amy Scott, Meng Wu Ph.D., Joseph Babcock, Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D.
Name: Mode of action - Automated patch clamp assay for KCNQ2 potentiators on Retigabine insensitive Mutant KCNQ2 W236L cell line
See the related assay (PubChem AID:2239).
Principle of the assay
Patch clamp is the gold standard to measure channel activities. The purpose of the assay is to identify compounds that are dissimilar in structure and function to Retigabine. The tested compounds have been identified as active in primary screen assay (PubChem AID 2239) and confirmatory screen assay (PubChem AID 2287).KCNQ2 W236L-CHO stable cell line is insensitive to Retigabine modulation. This assay employs automated patch clamp (Ionworks Quattro) to investigate the current response of KCNQ2-W236L-CHO in the presence or absence of test compounds.
Compounds were tested in duplicate at 25 uM and their effects were evaluated by the calculated current change in percentage, normalized with negative control. If the compound causes 3SD (of negative controls) or more current increase (of negative controls) in both duplicates, the compound is considered to be active.
KCNQ2, mutant, W236L, agonist, activator, potentiator, Retigabine, Retabine-insensitive, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol for automated patch clamp and W236L-CHO test with voltage clamp
1. Cell culture: Cells are routinely cultured in DMEM/F12 medium, supplemented with 10% Fetal Bovine Serum (FBS), 50 IU/ml penicillin, 50 ug/ml streptomycin, and 500 ug/ml G418 by using 150mm dishes.
2. Split cells once they reach 80% to 90% confluence
2.1. Aspirate medium from culture, add 10 mL of PBS (without Ca2+ and Mg2+) to wash the cell monolayer.
2.2. Aspirate the PBS.
2.3. Add 5 mL of 0.05% Trypsin to the 150mm dish, leave dish undisturbed for 3~5 min at 37 C to trypsinize the cells.
2.4. Add 20 mL of growth medium to neutralize the digestion of Trypsin.
2.5. Transfer cell suspension to 50 mL falcon tube and spin at 750 rpm for 4 min.
2.6. Remove supernatant and resuspend cells with 6 ml external solution, spin down at 450 rpm for 4 min.
2.7. Count the cells, adjust the cell density at 2x10^6 per ml.
3. Prepare 3x compound plates: test compounds are prepared using external solution; negative control (the external solution) and ZTZ240( ECmax) are all with DMSO concentrations matched to that of test compounds.
4. Prepare Amphotericin B: dissolve 5 mg Amphotericin B with 180 uL DMSO, vortex for 1 min; transfer dissolved amphotericin B to 50 mL internal buffer, fill in the amphotericin B tube.
5. Fill the external solution in the buffer boat; fill the internal solution in the internal solution bottle.
6. Add the cells in the cell boat.
7. Load the protocol: The holding potential is -90 mV. To elicit the currents, cells were stimulated by 2,000 ms depolarizing step from -90 mV to -35 mV. Start the experiments.
8. Measure the currents at the steady state.
9. Calculate the percentage of current change for tested compounds with the following formula:
Percentage (%) =100* (Current (post-compound)-Current (pre-compound))/Current (pre-compound)
Percentage (%): Percentage of current potentiation observed after the application of the test compound.
Current (pre-compound): Current recorded before the test compound application
Current (post-compound): Current recorded after the test compound application
10. Outcome assignment
If the compound causes 3SD (of negative controls) or more Percentage (%) increase (of negative controls) in both duplicates, the compound is confirmed as active (Value=2). Otherwise, it is designated as inactive (Value=1).
11. Score assignment
An inactive test compound is assigned the score of 0.
An active test compound is assigned a score between 0 and 100 by calculation of INT (Normalized Percentage (%)).
Percentage (%) is the percentage average of the duplicates of the test compound over the 3SD of negative controls. They are normalized to the smallest (0%) and the largest (100%).
List of reagents
1. KCNQ2-W236L-CHO cell lines (provided by JHICC)
2. PBS: pH7.4 (Gibco, Cat#10010)
3. Medium: DMEM/F12 50/50 (Mediatech, Cat#15-090-CV)
4. Fetal Bovine Serum (Gemini, Cat# 100-106)
5. 200 mM L-Glutamine (Gibco, Cat#25030)
6. 100x Penicillin-Streptomycin (Mediatech, Cat#30-001-CI)
7. 0.05% Trypsin-EDTA (Gibco, Cat#25300)
8. Geneticin: (Gibco, Cat#11811-031)
11. Amphotericin B( Sigma, Cat# A4888)
12. Internal buffer (40 mM KCl, 100 mM K-Gluconate,1 mM MgCl2, 2 mM CaCl2, 5 mM HEPES, pH 7.25)
13. External buffer (137 mM NaCl, 4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES and 10 mM Glucose, pH 7.4)
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)