Name: Primary cell-based screen for identification of compounds that inhibit the two-pore domain potassium channel KCNK9
Data Source: Johns Hopkins Ion Channel Center (JHICC_KCNK9_Inh_Primary)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network

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: Meng Wu, Ph.D. , Johns Hopkins University, School of Medicine
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: R03 MH090849-01
Grant Proposal PI: Meng Wu, Ph.D. , Johns Hopkins University, School of Medicine
Assay Implementation: Melissa Miller, Joseph Babcock, Amy Scott M.S., Shunyou Long M.S., David Meyers Ph.D., Owen McManus Ph.D., and Meng Wu Ph.D.
HTS execution: Melissa Miller, Joseph Babcock, Amy Scott M.S., Shunyou Long M.S.


Description:

KCNK9 (also termed K2p9.1 or TASK-3) is a member of the two-pore domain family (KCNK) of potassium channels that contribute to resting membrane potential and the regulation of cell excitability by generating background currents [1-2].

In addition to its role in neurons where KCNK9 is most abundantly expressed, recent studies show that KCNK9 plays critical roles in a wide variety of human pathophysiologies. For example, KCNK9 is overexpressed in various human cancers and its exogenous overexpression promotes cell proliferation in culture and tumor development in animal models [3-4]. Moreover, KCNK9 deficiency causes a primary hyperaldosteronism in mouse [5]. A more recent study reports that KCNK9 mutation causes Birk Barel mental retardation dysmorphism syndrome in humans [6]. These findings suggest that KCNK9 may be a potential new therapeutic target for treating human diseases [7]. However, there are no suitable chemical probes to investigate the physiological roles of KCNK9 channels.

The objective of the current screen is to identify compounds that inhibit the flow of ions through KCNK9 using a HEK293 cell line that stably expresses this channel. Compounds selected as KCNK9 inhibitors will later be counter-screened for specificity.

Principle of the assay

Thallium based assays exploit the inherent permeability of potassium channels for another cation[8-9]. In the current work, we have used the FluxOR (Invitrogen) dye to detect changes in intracellular thallium levels. To assess potassium channel function, cells are initially loaded with FluxOR dye and incubated with test compounds prior to fluorescence signal recording. An extracellular solution containing both thallium and potassium is then added, which depolarizes the membrane and consequently causes activation of potassium channels. The electrochemical gradient drives the net inflow of thallium down its concentration gradient. The accumulation of intracellular thallium will increase the fluorescence of the FluxOR dye. In this way, the thallium signal is used as an indicator for the function of thallium permeable proteins, a method commonly used to reflect the activity of recombinantly expressed cation channels.

Keywords:

KCNK9, TASK3, Two-pore domain potassium channel 9, HTS assay, 384, primary, inhibitor, blocker, FDSS, Thallium, fluorescence, Kinetic, FluxOR, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.


References:
1. Chapman, CG, et al., Cloning, localisation and functional expression of a novel human, cerebellum specific, two pore domain potassium channel. Brain Res Mol Brain Res, 2000. 82(1-2):p. 74-83. PMID: 11042359
2. Goldstein, S. A. N., D. A. Bayliss, et al. (2005). International Union of Pharmacology. LV. Nomenclature and Molecular Relationships of Two-P Potassium Channels. Pharmacol Rev 57(4): 527-540.PMID: 16382106
3. Mu, D., et al., Genomic amplification and oncogenic properties of the KCNK9 potassium channel gene. Cancer Cell, 2003. 3(3): p. 297-302. PMID: 12676587
4. Pei, L., et al., Oncogenic potential of TASK3 (Kcnk9) depends on K+ channel function. Proc Natl Acad Sci U S A, 2003. 100(13): p. 7803-7. PMID: 12782791
5. Davies LA , et al., TASK channel deletion in mice causes primary hyperaldosteronism. Proc Natl Acad Sci U S A. 2008. 105(6):p. 2203-8. PMID: 18250325
6. Barel O , et al., Maternally inherited Birk Barel mental retardation dysmorphism syndrome caused by a mutation in the genomically imprinted potassium channel KCNK9. Am J Hum Genet. 2008. 83(2):p. 193-9.PMID: 18678320
7. Bayliss, D. A. and P. Q. Barrett (2008). Emerging roles for two-pore-domain potassium channels and their potential therapeutic impact. Trends in Pharmacological Sciences 29(11): 566-575.PMID: 18823665
8. 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): p. 671-7.PMID: 15634793
9. Hille, B., Potassium channels in myelinated nerve. Selective permeability to small cations. J Gen Physiol, 1973. 61(6): p. 669-86.PMID: 4541077
10. 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), p. 67-73.PMID: 10838414
11. Malo, N., et al., Statistical practice in high-throughput screening data analysis. Nat Biotech, 2006. 24(2), p. 167-175.PMID: 16465162
12. Christine Brideau, Bert Gunter, Bill Pikounis and Andy Liaw, Improved Statistical Methods for Hit Selection in High-Throughput Screening. J Biomol Screen 2003. 8: p. 634 PMID: 14711389

Protocol for the KCNK9 project:

1. Cell culture: Cells are routinely cultured in DMEM/high glucose medium, supplemented with 10% Fetal Bovine Serum (HiFBS), 50 IU/ml penicillin, 50 ug/mL streptomycin, 15 ug/mL Blasticidin S and 400 ug/mL hygromycin
2. Cell plating: Add 50 ul/well of 300,000 cells/ml re-suspended in DMEM/high glucose medium with 10% FBS and 1ug/ul Tetracycline.
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 the dark
6. Prepare 7.5X compound plates and control plates on Cybi-Well system: test compounds are prepared using assay buffer; controls are assay buffer (EC0), and ECmax of SID:17386958
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 the dark
10. Prepare 5x stimulus buffer containing 25 mM K2SO4 and 7 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. Add 6 ul/well of stimulus buffer onto cells 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 [10]
16. Calculate B scores [11-12] for test compounds using ratios calculated in Step 14
17. Outcome assignment: If the B score of the test compound is less than the mean minus 3 times the standard deviation (SD) of the B scores of ratios from all library compounds (Bscore_Ratio 18. Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of Int((Log10(abs([Bscore_Ratio]))-0.78)*154); they are normalized to the smallest and largest LOG10(Bscore_Ratio), Bscore_Ratio, as in the result definition. The inactive test compounds are assigned a score of 0.

List of reagents

1. KCNK9-expressing HEK293 Cells (provided by Sojin Shikano, PhD, DVM, University of Illinois at Chicago)
2. Dulbecco's Modified Eagle Medium (D-MEM) (1X), liquid (high glucose) w/L-Glut (Mediatech, Cat#10-013-CV)
3. Fetal Bovine Serum (Gibco, Cat#26140)
4. L-Glutamine (Invitrogen, Cat#25030081)
5. 100x Penicillin-Streptomycin (Mediatech, Cat#30-001-CI)
6. CellStripper (Mediatech, Cat#25-056-Cl)
7. Blasticidin S (Research Products Internationl Corp., Cat#B12150)
8. Hygromycin (Mediatech, Cat#30-240-CR)
9. HEPES (Sigma, Cat#H4034)
10. 10XHBSS (Invitrogen, Cat#14065056)
11. Tetracycline (SIGMA, Cat#T7660)
12. SID 17386958 (Chembridge, Cat#8926102)
13. FluxOR detection kit (Invitrogen, Cat#F10017)
14. Triple-layer flask (VWR, Cat#62407-082)
15. BD Biocoat 384-well plates (BD, Cat#(35)4663 and Lot #7346273)

Possible artifacts of this assay can include, but are not limited to: non-intended chemicals, or dust in or on wells of the microtiter plate, compounds that non-specifically modulate the cell host or the targeted activity, 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.

Grant Number: 1 R03 MH090849-01