Primary cell-based high-throughput screening assay for identification of compounds that inhibit KCNQ2 potassium channels
Assay Implementation: Haibo Yu Ph.D., Beiyan Zou Ph.D., Shunyou Long M.S., Amy Scott M.S., Meng Wu Ph.D., Joseph Babcock, Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D. ..more
BioActive Compounds: 3405
Data Source: Johns Hopkins Ion Channel Center (JHICC_KCNQ2)
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 M.S., Meng Wu Ph.D., Joseph Babcock, Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D.
Name: Primary cell-based high-throughput screening assay for identification of compounds that inhibit KCNQ2 (Kv7.2) potassium channels
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. Voltage-gated K+ channels, exemplified by the Shaker K+ channel, share considerable sequence similarity. Isolation and characterization of bioactive chemical probes could form important pharmacological foundation, providing a great deal of insights into the structure and function.
The KCNQ (or also called Kv7) channel family includes five members, KCNQ1 to KCNQ5; KCNQ are members of Kv channel superfamily[1-6]. Different from other Kv channel classes, KCNQ channels commonly display a sub-threshold opening (i.e., at a voltage near resting potential) and are regulated by GPCR signaling, e.g., muscarinic receptors [7, 8]. KCNQ2-5 channels are primarily expressed in the brain, while KCNQ1 is abundantly expressed in cardiac tissue. Among congenital long QT patients, a disease which affects 1 in 2,500 live births, the most prevalent mutations are within the KCNQ1 allele. Mutations in KCNQ2 and KCNQ3 are known to cause benign familial neonatal convulsion (BFNC), a rare form of epilepsy .
Systemic compound screens for M-current have not been reported. Because of the rapid progress of molecular cloning and functional characterization, KCNQ2 has been validated as the key molecular target of M-current. It is therefore feasible to design non-biased high-throughput screens specifically targeting to KCNQ2 channels. This justifies use of the Tl+-based fluorescence assay in 384-well format by FDSS for the identification of modulatory compounds acting on KCNQ2 from a large compound library.
Principle of the assay
The Tl+ ion, which is permeable through potassium channels, serves as a surrogate for K+ flux . The thallium-sensitive dye is loaded into cells, and, in the absence of Tl+, exhibits very low basal fluorescence. Upon the addition of Tl+ onto cells expressing potassium channels, extracellular Tl+ flux into cells through open KCNQ2 channels, and the KCNQ2 potassium channel, when bound to a dye, produces a fluorescent signal that is monitored in real-time by a fluorescence imaging plate reader [11, 12].The binding to Tl+ causes increase in fluorescence. If activity of KCNQ2 is inhibited by a test compound, the fluorescent signal is suppressed.
KCNQ2, HTS assay, 384, primary, antagonist, inhibitor, blocker, FDSS, Thallium, fluorescence, Kinetic, FluxOR, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.
1. Charlier, C. et al. A pore mutation in a novel KQT-like potassium channel gene in an idiopathic epilepsy family. Nat. Genet. 1998.18, 53-55, PMID: 9425900
2. Gutman, G.A. et al. International Union of Pharmacology. XLI. Compendium of voltage-gated ion channels: potassium channels. Pharmacol. Rev. 2003. 55, 583-586, PMID: 14657415
3. Kubisch, C. et al. KCNQ4, a novel potassium channel expressed in sensory outer hair cells, is mutated in dominant deafness. Cell. 1999. 96, 437-446, PMID: 10025409
4. Schroeder, B.C., Hechenberger, M., Weinreich, F., Kubisch, C. & Jentsch, T.J. KCNQ5, a novel potassium channel broadly expressed in brain, mediates M-type currents. J. Biol. Chem. 2000, 275, 24089-24095,PMID: 10816588.
5. Singh, N.A. et al. A novel potassium channel gene, KCNQ2, is mutated in an inherited epilepsy of newborns. Nat. Genet. 1998.18, 25-29, PMID: 9425895.
6. Wang, Q. et al. Positional cloning of a novel potassium channel gene: KVLQT1 mutations cause cardiac arrhythmias. Nat. Genet. 1996, 12, 17-23, PMID: 8528244.
7. Brown, D.A. et al. Muscarinic suppression of a novel voltage-sensitive K+ current in a vertebrate neurone. Nature. 1980. 283, 673-676. PMID: 6965523.
8. Marrion, N.V. Control of M-current. Annu. Rev. Physiol.1997.59, 483-504, PMID: 9074774.
9. Maljevic, S. et al. Nervous system KV7 disorders: breakdown of a subthreshold brake. J Physiol. 2008. 586, 1791-1801. PMID: 18238816.
10. Delpire, E., et al., Small-molecule screen identifies inhibitors of the neuronal K-Cl cotransporter KCC2. Proc Natl Acad Sci U S A., 2009. 106(13),5383-5388. PMID: 19279215.
11. Weaver, C.D., et al., A Thallium-Sensitive, Fluorescence-Based Assay for Detecting and Characterizing Potassium Channel Modulators in Mammalian Cells. J Biomol Screen, 2004. 9(8), 671-677. PMID: 15634793.
12. Niswender, C.M., et al., A Novel Assay of Gi/o-Linked G Protein-Coupled Receptor Coupling to Potassium Channels Provides New Insights into the Pharmacology of the Group III Metabotropic Glutamate Receptors. Mol Pharmacol, 2008. 73(4), 1213-1224. PMID: 18171729.
13. Zhang, J.-H., T.D.Y. Chung, and K.R. Oldenburg, A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen, 1999. 4(2),67-73. PMID: 10838414.
14. Malo, N., et al., Statistical practice in high-throughput screening data analysis. Nat Biotech, 2006. 24(2), 167-175. PMID: 16465162.
The purpose of this assay is to identify compounds that inhibit KCNQ2 potassium channels. This assay employs a CHO-K1 cell line that stably expresses the KCNQ2 potassium channel. The cells are treated with test compounds, followed by measurement of intracellular thallium, as monitored by a thallium-sensitive fluorescent dye, FluxOR. As designed, compound effects on the KCNQ2 channel were measured by thallium assay, using a commercial kit, the FluxOR detection kit. Those CHO-K1 cells stably expressing KCNQ2 channels were plated into 384-well plates. On the following day, cells were loaded with a thallium-sensitive dye, FluxOR, and then incubated with assay buffer, followed by the addition of compounds. Cells were incubated with 10 uM compound for 20 minutes, and detected upon the addition of stimulus solution (2.5 mM K2SO4 and 2.5 mM Tl2SO4).The fluorescence of FluxOR was measured on a Hamamatsu FDSS 6000 kinetic imaging plate reader. Compound effect was evaluated by the calculated FluxOR fluorescence ratio, normalized with negative controls. If the compound has a B-score of less than minus 3*SD of the average B score of the library compounds, the compound is then considered to be active as a inhibitor of the KCNQ2 channel.
Protocol for the KCNQ2 project:
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.
2. Cell plating: Add 50 ul/well of 120,000 cells/ml re-suspended in DMEM/F12 medium with 10% FBS.
3. Incubate overnight at 37C and 5% CO2.
4. Remove medium and add 25 ul /well of 1x FluxOR solution to cells.
5. Incubate 90 minutes at room temperature (RT) in darkness.
6. Prepare 7.5X compound plates and control plates on the Cybi-Well system: test compounds are prepared using assay buffer; controls are assay buffer (IC0), ICmax of XE-991 (all with DMSO concentrations matched to that of test compounds).
7. Remove FluxOR dye solution and add 20 ul /well of assay buffer to cells.
8. Add 4 ul of 7.5x compound stock into the cell plates via Cybi-Well system.
9. Incubate all cell plates for 20 minutes at RT in darkness.
10. Prepare 5x stimulus buffer containing 12.5 mM K2SO4 and 12.5 mM Tl2SO4.
11. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader.
12. Measure fluorescence for 10 seconds at 1Hz to establish baseline.
13. Depolarize cells with 6 ul/well of stimulus buffer and continue measuring fluorescence for 110 seconds.
14. Calculate ratio readout as F(max-min)/F0.
15. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors .
16. Calculate B scores  for test compounds using ratios calculated in Step 14.
17. Outcome assignment: If the B score of the test compound is more negative than -3 times the standard deviation (SD) of the B scores of ratios of the library compounds (<=-3*SD), AND the B score of initial fluorescence intensity is within 2 times the standard deviation of the B scores of the library compounds, the compound is designated in the Outcome as an active (Value=2) inhibitor of the KCNQ2 channels. Otherwise, it is designated as inactive (value=1).
18. Score assignment: An active test compound is assigned a score between 0 and 100 by calculation of INT(100*LOG(B Score inhibitor Ratio)), they are normalized to the smallest and largest LOG(B Score inhibitor Ratio), B Score inhibitor Ratio, as in the result definition.
List of reagents
1. KCNQ2-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)
9. HEPES (Sigma, Cat#H4034)
10.XE-991 (Tocris, Cat# 2000)
11. FluxOR detection kit (Invitrogen, Cat #F10017): FluxOR, assay buffer and stimulus buffer.
12. Triple-layer flask (VWR, Cat #62407-082)
13. BD Biocoat 384-well plates (BD, Cat# (35)4663 and Lot #7346273)
Possible artifacts of this assay may include, but are not limited to: unintended chemicals or dust in or on wells of the microtiter plate, compounds that non-specifically modulate the cell host or the targeted activity, compounds that induce K/Tl flux independent of KCNQ2, compounds that directly interact with the Tl sensitive dye molecule, and 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.
1. B score inhibitor Ratio (10 uM)
B score calculation based on the ratio of the test compound in the primary screen at the concentration of 10 uM.
2. B score inhibitor Intensity (10 uM)
B score calculation based on the initial fluorescence intensity of the test compound in the primary screen at the concentration of 10 uM.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)