Mode of action assay-Dose response assay for the identification of selective activators of KCNQ2 potassium channels in the KCNQ1/KCNE1 expressing cells on automated patch clamp
Name: Mode of action assay-Dose response assay for the identification of selective activators of KCNQ2 potassium channels in the KCNQ1/KCNE1 expressing cells on automated patch clamp ..more
BioActive Compounds: 2
Data Source: Johns Hopkins Ion Channel Center (KCNQ1E1_Act_IWS_CRC)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Name: Mode of action assay-Dose response assay for the identification of selective activators of KCNQ2 potassium channels in the KCNQ1/KCNE1 expressing cells on automated patch clamp
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., Kaiping Xu M.S., Shunyou Long M.S., Meng Wu Ph.D., David Meyers Ph.D., Owen Mcmanus Ph.D.
Voltage-gated potassium (K) channels are critical for neuronal function in excitable tissues such as brain and heart. They are also found in non-excitable tissues important for other functions such as hormone secretion, oxygen-sensing and immune responses. There are more than 100 genes in human genome encoding different but homologous potassium channels. Isolation and characterization of bioactive chemical probes could form important pharmacological foundation, providing a great deal of insights into the structure and function.
The M-type channels are unique voltage-gated and ligand-regulated K+ channels with their distinct physiological and pharmacological characteristics. They are activated at a voltage near the threshold for action potential initiation and regulate membrane excitability. The KCNQ (or also called Kv7), members of Kv channel superfamily, includes five members, KCNQ1 to KCNQ5. Among them homodimer and/or heterodimer of KCNQ2 and KCNQ3 are believed components of the M-channel. The modulators of KCNQ2 are playing an important role in the neuronal function regulation. As the therapeutic targets, the KCNQ2 openers have great potential used to treat epilepsy, pain and anxiety et al.
Principle of the assay
Patch clamp is gold standard to measure channel activities. The purpose of the assay is to examine the non-specific effects of KCNQ2 activators on the KCNQ1/KCNE1 expressing cells. This assay employs automated patch clamp (Ionworks Quattro) to investigate the current response of KCNQ1/KCNE1-CHO elicited by voltage clamp protocols in the presence or absence of test compounds. Compounds were tested in quadruplicates at varying concentrations.
KCNQ2, KCNQ1/KCNE1, agonist, activator, potentiator, Concentration Response Curve, IonWorks, automated patch clamp, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol for automated patch clamp on KCNQ1/KCNE1-CHO cells 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 200 ug/ml Zeocin 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 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 -70 mV. To elicit the currents, cells were stimulated by 3,000 ms depolarizing step from -70 mV to +40 mV, followed by hyperpolarization to -20 mV for 500 ms. Start the experiments.
8.Measure the currents at the steady state of +40mV voltage step.
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 change 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
If the test compound causes potentiation or inhibition effect on KCNQ1/KCNE1 in any concentrations tested and the dose response is generated, the compound is considered to be active.
If the test compound does not cause potentiation or inhibition effect on KCNQ1/KCNE1 in any concentrations tested or a dose response is not generated, the compound is designated as inactive.
An inactive test compound is assigned the score of 0.
An active test compound is assigned the score of 100.
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)
Possible artifacts of this assay may include, but are not limited to: unintended chemicals or dust in or under the wells of the microtiter plate, or compounds that quench or emit light or fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR. The screened target(s) in this assay is for human but not rat. It is because of the availability of the cells/plasmids. It can not be excluded that the difference of the species will cause the artifact of the results.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)