Specificity screen against Kir2.1 for compounds that modulate the two-pore domain potassium channel (KCNK9)
Assay Implementation: Melissa Miller, Joseph Babcock, Shunyou Long M.S., David Meyers Ph.D., Owen Mcmanus, Ph.D., Meng Wu, Ph.D. ..more
BioActive Compounds: 197
Data Source: Johns Hopkins Ion Channel Center
BioAssay Type: Confirmatory, Counter Screen, Duplicate, 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: Meng Wu, Ph.D. , Johns Hopkins University, School of Medicine
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1R03MH090849-01
Grant Proposal PI: Meng Wu, Ph.D. , Johns Hopkins University, School of Medicine
Assay Implementation: Melissa Miller, Joseph Babcock, Shunyou Long M.S., David Meyers Ph.D., Owen Mcmanus, Ph.D., Meng Wu, Ph.D.
Name: Specificity screen against Kir2.1 for compounds that modulate the two-pore domain potassium channel (KCNK9)
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 . A more recent study reports that KCNK9 mutation causes Birk Barel mental retardation dysmorphism syndrome in humans . These findings suggest that KCNK9 may be a potential new therapeutic target for treating human diseases . 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.
The purpose of this specific assay is to counter screen those compounds with KCNK9 activity against the Kir2.1 channel. This counter screen will eliminate any compounds that interact with Kir2.1 expressing HEK293 cells, rather than KCNK9 expressing HEK293 cells. The protocol as in AID 488922 is employed for this assay, except Kir2.1 expressing HEK293 cells are used. Compounds were tested in duplicate using a thallium sensitive dye (FluxOR, Invitrogen) and were evaluated by calculation of the fluorescence ratio normalized to control wells. If the compound caused a decrease of the thallium signal greater than 5SD of the average IC0 control wells' signal it is termed active. Those compounds active in both duplicate plates are considered non-specific modulators of Kir2.1 expressing HEK293 cells. Such compounds are therefore NOT specific to KCNK9 channels.
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.
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
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9. Hille, B., Potassium channels in myelinated nerve. Selective permeability to small cations. J Gen Physiol, 1973. 61(6): p. 669-86.PMID: 4541077
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1. Cell culture: Cells are routinely cultured in DMEM/F12 medium, supplemented with 10% Fetal Bovine Serum (FBS), 50 IU/ml penicillin, 50ug/ml streptomycin, and 500ug/ml G418.
2. Cell plating: Add 50 ul/well of 300,000 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 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 (IC0), and IC100 of Chlorpromazine
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
16. Calculate percent inhibition by normalizing fluorescent ratios to EC0 controls
17. Outcome assignment: If the compound caused a decrease of the thallium signal greater than 5SD of the average EC0 control it is termed active. Those compounds active in both duplicate plates are considered non-specific modulators of Kir2.1 expressing HEK293 cells. (Value=2). Otherwise, it is designated as inactive (Value=1).
18. Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of Int((Lg(Abs([avPercentInhibition]))-1.40)*168.1), avPercentInhibition, as in the result definition. Inactive test compounds are assigned a score of 0.
List of reagents
1. Kir2.1 HEK293 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. G418 (Geneticin): (Gibco, Cat#11811-031)
9. HEPES (Sigma, Cat#H4034)
10. Chlorpromazine hydrochloride (Sigma, C8138)
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)6663 and Lot #8163495)
14. 10x HBSS (Gibco, Cat#14065)14. Triple-layer flask (VWR, Cat #62407-082)
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. The condition is optimal for screening for compounds that modulate KCNK9 channels, NOT for the assay of Kir2.1 modulators. Normalization is to this set of data and cannot be used for comparison with other counter screens.
** Test Concentration.
Data Table (Concise)