Confirmation assay for identification of compounds that inhibit the two-pore domain potassium channel KCNK3 [Primary Screening]
Assay Implementation: Zhihong Lin Ph.D., Kaiping Xu M.S., Alison Neal B.S., Owen McManus Ph.D., and Meng Wu Ph.D. ..more
BioActive Compounds: 2372
Data Source: Johns Hopkins Ion Channel Center (JHICC)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed, Triplicate
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: Zhihong Lin Ph.D., Kaiping Xu M.S., Alison Neal B.S., Owen McManus Ph.D., and Meng Wu Ph.D.
Name: Confirmation assay for identification of compounds that inhibit the two-pore domain potassium channel KCNK3
Two-pore domain potassium channels (K2P channels) are essential for the background or "leak" potassium conductances that contribute to generating the negative membrane potentials in some excitable and non-excitable cells. Their functions go beyond acting as simply leak channels to set resting membrane potentials, and also involve complex regulation of physiological and pathological functions, and are subject to regulation by neurotransmitters, local pH changes, free fatty acids and lysopophospholipids, mechanical forces of the cell membrane, temperature and inhalant aesthetics. TASK1 (KCNK3), as one of the K2P family members, is a pH-dependent, voltage-insensitive, background potassium channel protein, and is in the same TASK channel family as TASK3 (KCNK9) [1, 2]. It is widely expressed in multiple organs, with highest mRNA expression in heart, adrenal gland, placenta, adult and fetal lung, pancreatic islet, whole brain and cerebellum. Recent studies on these channels have revealed their molecular identities, voltage independent gating characteristics, and roles as mediators for a variety of important modulators, including volatile anesthetics, neurotransmitters and hormones. Recent data suggest a role for TASK channels in nocturnal activity and they have been hypothesized to contribute to obstructive sleep apneas[3-5]. Different from TASK3, TASK1 is strongly expressed in the heart where it may contribute to noradrenergic regulation of cardiomyocyte electrophysiology. In addition, specific roles for TASK1 have been described in regulation of breathing by hypoxemia, ischemic neuroprotection and in an experimental autoimmune model of multiple sclerosis. Consequently, TASK1 has been considered as a possible drug target. However, few TASK specific inhibitors are available as molecular tools for further mechanistic elucidation of TASK1 channel functions, and much less as potential therapeutic leads for the TASK1 related diseases. Thus, the lack of specific modulators has hindered definitive identification of these channels in native tissues, which is especially critical for mechanistic studies and determining the therapeutic potential for targeting these channels.
The objective of the current screen is to confirm KCNK3 inhibitor activity for compounds identified in a high throughtput screen using a CHO cell line stably expressing KCNK3 channels.
Principle of the assay
Thallium based assays exploit the inherent permeability of potassium channels for another cations [6-7]. In the current work, we have used the FluxORtrade mark (Invitrogen) dye to detect changes in intracellular thallium levels. To assess potassium channel function, cells are initially loaded with FluxORtrade mark 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 some 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 FluxORtrade mark 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.
To confirm the activity of KCNK3 inhibitor compounds identified in a high throughput primary screen (AID: 602410), a CHO cell line stably expressing KCNK3 potassium channel is employed. The cells are treated with test compounds, followed by measurement of intracellular thallium, as monitored by a commercially available thallium-sensitive fluorescent dye, FluxORtrade mark. Compound effect was evaluated by the calculated FluxORtrade mark fluorescence ratio, normalized with negative controls, from the triplicates.
KCNK3, TASK1, Two-pore domain potassium channel 9, HTS assay, confirmation assay, primary, inhibitor, blocker, FDSS, Thallium, fluorescence, Kinetic, FluxORtrade mark, 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.
2. Mu, D., et al., Genomic amplification and oncogenic properties of the KCNK9 potassium channel gene. Cancer Cell, 2003. 3(3): p. 297-302.
3. 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.
4. 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.
5. 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.
6. 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.
7. Hille, B., Potassium channels in myelinated nerve. Selective permeability to small cations. J Gen Physiol, 1973. 61(6): p. 669-86.
8. 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.
Protocol for the KCNK3 project:
1. Cell culture: Cells are routinely cultured in Ham's F12 medium with 10% Fetal Bovine Serum, 50 IU/ml penicillin, 50 ug/mL streptomycin, 10 ug/mL Blasticidin S and 400 ug/mL hygromycin.
2. Cell plating: Add 50 ul/well of 160,000 cells/ml in Ham's F12 medium with 10% Fetal Bovine Serum 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 carvedilol.
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 12.5 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 180 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 the percentage of tested compounds with the following formula: Percentage (%)=100* (Ratio(cmpd)- AvgRatio(Buffer))/(AvgRatio(Carvedilol)-AvgRatio(Buffer)); Percentage(%): percentage change of compound readout over those of negative controls (Buffer), Ratio(cmpd): Ratio of the test compound. AvgRatio(Buffer): Ratio average of the negative controls with Buffer, Ratio(Carvedilol): Ratio of carvedilol.
17. Outcome assignment: If the signal in compound (the average of the triplicates of the Percentage (%, RatioPercentage) as readout) is greater than those of negative controls (Buffer) plus 5SD of negative controls (Buffer), the compound is considered to be active (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 Integer((log10([RatioPercent])-1.69897)/0.0057+5), RatioPercent, as in the result definition. The inactive test compounds are assigned a score of 0.
List of reagents
1. KCNK3-CHO Cells (provided by Douglas A. Bayliss, PhD, University of Virginia Health System)
2. Ham's F-12 Nutrient Mix (Invitrogen, Cat #11765-062)
3. Fetal Bovine Serum (Gibco Cat #26140)
4. 100x Penicillin-Streptomycin (Mediatech, Cat #30-001-CI)
5. Trypsin-EDTA (Invitrogen Cat #25300054)
6. Blasticidin (Sigma Cat #R21001)
7. Hygromycin (Mediatech, Cat #30-240-CR)
8. HEPES (Sigma, Cat #H4034)
9. 10XHBSS (Invitrogen, Cat #14065056)
10. Tetracycline (Sigma, Cat #T7660)
11. Carvedilol (Sigma, Cat #C3993)
12. FluxOR detection kit (Invitrogen, Cat #F10017)
13. Triple-layer flask (VWR, Cat #62407-082)
14. BD Biocoat 384-well plates (BD, Cat #354663 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.
** Test Concentration.
Data Table (Concise)