Validation assay for identification of compounds that potentiate KCNQ1 potassium channels
Assay Implementation: Zhihong Lin Ph.D., Xiaofang Huang M.S., Shunyou Long M.S., Owen McManus, Ph.D., Meng Wu Ph.D. ..more
BioActive Compounds: 1024
Data Source: Johns Hopkins Ion Channel Center (JHICC, JHICC_KCNQ1_Act_Vali)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed, Duplicate
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.
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH090837-01
Grant Proposal PI: Meng Wu, Ph.D., Johns Hopkins University School of Medicine
Assay Implementation: Zhihong Lin Ph.D., Xiaofang Huang M.S., Shunyou Long M.S., Owen McManus, Ph.D., Meng Wu Ph.D.
Name: Validation assay for identification of compounds that potentiate KCNQ1 potassium channels
Voltage-gated potassium channels [1,2] are tetrameric membrane proteins that selectively conduct K+ across cellular membranes, thus open, close, and inactivate in response to changes in transmembrane voltage . Individual subtypes of these potassium channels often have a unique expression pattern allowing cells to "fine-tune" membrane potentials and excitability according to their respective physiological functions . Dysfunctions of these electrical excitability controlling proteins, either congenital or acquired, are attributed to a variety of diseases [5,6], such as cardiac arrhythmias, diabetes, hypertension, and epilepsy. Specific modulation of individual potassium channel types therefore represents an enormous potential for the development of physiological tool compounds and new drugs [7-9].
KCNQ1 (Kv7.1, KvLQT) [10,11] is an alpha-subunit subtype of voltage-gated KCNQ potassium channel family, which is composed of five members of KCNQ1-KCNQ5. They share between 30% and 65% amino acid identity. A classical KCNQ alpha-subunit is composed of six transmembrane segments, including a voltage-sensor segment and a pore domain [12-15]. Unique from other members of KCNQ family , KCNQ1 has been generally absent from neuronal tissues, mainly expressed in heart, kidney, small intestine, pancreas, prostate and other non-excitable epithelial tissues. Also contrast to other members of KCNQ family which form both alpha-subunit homo- and heterotetrameric channels, KCNQ1 channels only form alpha-subunit homotetramers . They commonly co-assemble with beta-subunit KCNE proteins to give rise to functional variations in different tissues.
These molecular assemblies have afforded KCNQ1 with two important physiological functions: 1) repolarization of the cardiac tissue following an action potential and 2) water and salt transport in epithelial tissues. Mutations in this gene are associated with hereditary long QT syndrome, diabetics , Romano-Ward syndrome, Jervell and Lange-Nielsen syndrome  and familial atrial fibrillation , as well as impairment of cyclic AMP-stimulated intestinal secretion of chloride ions related to cystic fibrosis [21,22] and pathological forms of secretary diarrhea [23-25]. Furthermore, drug-induced acquired KCNQ1 and KCNQ1/KCNE dysfunctions also raise concerns of KCNQ1/KCNE as potential hERG-like drug safety issue in pharmaceutical development .
For their pharmacological responses, KCNQ1/KCNE heteromultimers function differently from KCNQ1 alone. Initial discovery of KCNQ1 modulators is focused on the KCNQ1 (and KCNQ1/KCNE1 IKs) inhibitors . In contrast to KCNQ1 channel blockers, only until recently have KCNQ1 channel activators/ potentiators been generating a lot of interests partially due to KCNQ1/KCNE activators might be useful agents to counteract the loss of delayed rectifier function in LQT syndromes, as well as counter target of other KCNQ family members for potential drugs for the treatment of epilepsy and neuropathic pain. Overall there are a very limited number of KCNQ1 activators/ potentiators, a further limited number of KCNQ1/E1 heteromultimer-specific modulators, and no reported KCNQ1/E2 or KCNQ1/E3 heteromultimer-specific modulators. This has hindered a more systematic study to understand the roles of on beta-subunits. Therefore it justifies the necessity of primary high throughput screen of KCNQ1 with the MLSMR library of >300,000-500,000 compounds covering large chemical space. Here the assay, Tl+-based fluorescence assay in 384 format by FDSS, therefore, was used for the identification of activating/potentiating compounds acting on KCNQ1 from the large MLSMR 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, in this case, KCNQ1 potassium channel, extracellular Tl+ flux into cells through open KCNQ1 channels, and when bound to the dye, produce a fluorescent signal that is monitored in real-time by a fluorescence imaging plate reader [27, 28].The binding to Tl+ causes increase in fluorescence. If the activity of KCNQ1 is potentiated by a test compound, the fluorescent signal increase is enhanced.
To validate the hit compounds that potentiate KCNQ1 potassium channels from the primary screen, a CHO-K1 cell line that stably expresses KCNQ1 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, FluxOR. Compound effect was evaluated by the calculated FluxOR fluorescence ratio, normalized with negative controls, from the duplicates.
KCNQ1, HTS assay, 384, primary, agonist, activator, potentiator, allosteric, FDSS, Thallium, fluorescence, Kinetic, FluxOR, JHICC, Johns Hopkins, MLSMR, Molecular Libraries Probe Production Centers Network, MLPCN.
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Protocol for the KCNQ1 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 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), activator control R-L3 and inhibitor control XE991 (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 the dark
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 the percentage of tested compounds with the following formula: Percentage (%)=100* (Ratio(cmpd)- AvgRatio(Buffer))/(AvgRatio(R-L3)-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(R-L3): Ratio of R-L3.
17. Outcome assignment: If the compound (the average of the duplicates of the Percentage (%, avPercent) as readout) causes more than those of negative controls (Buffer) plus 3SD 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 Int(((Log(ABS(avPercent))-1.16)*60.13, avPercent, as in the result definition. The inactive test compounds are assigned a score of 0.
List of reagents
1. KCNQ1-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. R-L3 (Tocris Bioscience)
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 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)