SAR analysis for compounds that inhibit KCNQ2 potassium channels on automated electrophysiological assay, CRC2
Assay Implementation: Haibo Yu Ph.D., Kaiping Xu, Shunyou Long M.S, David Meyers Ph.D., Meng Wu Ph.D., Owen McManus Ph.D. ..more
BioActive Compounds: 50
Data Source: Johns Hopkins Ion Channel Center (KCNQ2_Inh_IWS_SAR_CRC2)
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., Kaiping Xu, Shunyou Long M.S, David Meyers Ph.D., Meng Wu Ph.D., Owen McManus Ph.D.
Name: SAR analysis for compounds that inhibit KCNQ2 potassium channels on automated electrophysiological assay, CRC2
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 the human genome encoding different but homologous potassium channels. Isolation and characterization of bioactive chemical probes could enable pharmacological experiments to study channel function provides tools to analyze structure and function.
The M-type channels are unique voltage-gated and ligand-regulated K+ channels with distinct physiological and pharmacological characteristics. They are activated at a voltages near the threshold for action potential initiation and thus regulate membrane excitability. KCNQ (or also called Kv7) channels, members of Kv channel superfamily, include five members, KCNQ1 to KCNQ5. Among them, homodimers and/or heterodimers of KCNQ2 and KCNQ3 are believed components of the M-current channel. Modulators of KCNQ2 may play important roles regulating neuronal function, and KCNQ2 openers have demonstrated efficacy in treating epilepsy and may have further uses for treating pain and anxiety. In vivo studies have supported a role for KCNQ2 inhibitors as cognition enhancers.
Results of large scale compound screens for M-current modulators have not been reported. Molecular and functional studies have indicated that KCNQ2 is a key molecular component of the M-current. It is therefore feasible to design robust, high-throughput screens specifically targeting KCNQ2 channels. This assay describes a Tl+-based fluorescence assay in 384-well format used to identify of KCNQ2 modulatory compounds from a large compound library.
Principle of the assay
Patch clamp recording provides a high resolution, linear measure of channel activities. The purpose of the assay is to validate KCNQ2 SAR compounds derived from hit compounds identified in a high throughput thallium flux assay (PubChem AID: 2156) using electrophysiological recordings from KCNQ2 channels. This assay employs automated patch clamp (Ionworks Quattro) to investigate the current responses 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,inhibitor, blocker, Concentration Response Curve, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol for automated patch clamp on KCNQ2-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 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 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 -10 mV and +40 mV. Start the experiments.
8.#Measure the currents at the steady state.
-10mV was used to evaluate activators if activators were detected among the SAR compounds.
+40mV was used to evaluate inhibitors.
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 inhibition 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 inhibition effect on KCNQ2 in any concentrations tested and the dose response is generated, the compound is considered to be active.
If the test compound does not cause inhibition effect on KCNQ2 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)
* Activity Concentration. ** Test Concentration.
Data Table (Concise)