Mode of action assay - current amplitude concentration response for derivatives of ZTZ240, a potentiator of KCNQ2 potassium channels
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: 4
Depositor Specified Assays
Data Source: Johns Hopkins Ion Channel Center
BioAssay Type: Confirmatory, Concentration-Response Relationship 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 assay- current amplitude concentration response for derivatives of ZTZ240, a potentiator of KCNQ2 potassium channels
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 assess the ZTZ240 (IUPAC name: N-(6-chloropyridin-3-yl)-4-fluorobenzamide; PubChem SID: 87350301) derivatives for their effects to potentiate KCNQ2 potassium channel. This assay employs automated patch clamp (Ionworks Quattro) to investigate the current response of KCNQ2-CHO elicited by voltage clamp protocols in the presence or absence of test compounds. Compounds were tested in quadruplicates at varying concentrations.
KCNQ2, agonist, activator, potentiator, ZTZ240, derivative, Concentration Response Curve, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol for automated patch 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;
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
11. If the test compound causes potentiation of the KCNQ2 current at -35 mV in any concentrations tested, the compound is considered to be active.
If the test compound does not cause potentiation of the KCNQ2 current at -35 mV in any concentrations tested, the compound is designated as inactive.
12. Score assignment
An inactive test compound is assigned the score of 0.
An active test compound is assigned the score of 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)
* Activity Concentration. ** Test Concentration.
Data Table (Concise)