SAR-Confirmatory assay to confirm a potent KCNQ2 inhibitor using manual electrophysiology
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 Compound: 1
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-Confirmatory assay to confirm a potent KCNQ2 inhibitor using manual electrophysiology
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.
Molecular and functional studies of M current 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.
Principle of the assay
Patch clamp is gold standard to measure channel activities. The purpose of the assay is to validate a compound identified as a potent inhibitor in the KCNQ2 SAR analysis on the KCNQ2 potassium channel. This assay employs manual patch clamp to investigate the current response of KCNQ2-CHO elicited by voltage clamp protocols in the presence or absence of test compound. Compounds were tested in quadruplicates at single concentration 0.1 microM.
KCNQ2,inhibitor, blocker, Concentration Response Curve, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.
1. Cell culture
KCNQ2-CHO cells were grown in 50/50 DMEM/F12 (Cellgro, Manassas, VA) with 10% fetal bovine serum (FBS), and 2 mM L-glutamine (Gibco, Carlsbad, CA). Before the recording, cells were split and re-plated onto coverslips coated with poly-L-lysine (Sigma-Aldrich, St. Louis, MO).
2. Electrophysiological recording in CHO cells
Whole-cell voltage clamp recording was carried out, using cultured CHO cells, at room temperature, by an Axopatch-200B amplifier (Molecular Devices, Sunnyvale, CA). The electrodes were pulled from borosilicate glass capillaries (World Precision Instruments, Sarasota, FL). When filled with the intracellular solution, the electrodes have resistances of 3-5 megaohms. Pipette solution contained (mM): KCl 145, MgCl2 1, EGTA 5, HEPES 10, MgATP 5 (pH=7.3 with KOH). During the recording, constant perfusion of extracellular solution was maintained using a BPS perfusion system (ALA scientific Instruments, Westburg, NY). Extracellular solution contained (mM): NaCl 140, KCl 3, CaCl2 2, MgCl2 1.5, HEPES 10, glucose 10 (pH=7.4 with NaOH). Signals were filtered at 1KHz, and digitized using a DigiData 1322A, with pClamp 9.2 software (Molecular Devices, Sunnyvale, CA). Series resistance was compensated by 60-80%. The holding potential was set at -80 mV. To elicit the currents, cells were stimulated by a 2,000 ms depolarizing step to 50 mV and the steady state currents were measured.
3. 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
4. Outcome assignment
If the test compound causes inhibition effect on KCNQ2 in the tested concentration and repeatable, the compound is considered to be active.
If the test compound does not cause inhibition effect on KCNQ2 in the tested concentration, 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.
** Test Concentration.
Data Table (Concise)