Secondary automated electrophysiology assay of compounds that potentiate KCNQ1 potassium channels
Assay Implementation: Haibo Yu Ph.D., Kaiping Xu M.S., Zhihong Lin Ph.D., Xiaofang Huang M.S., Shunyou Long M.S., Owen McManus, Ph.D., Meng Wu Ph.D. ..more
BioActive Compounds: 26
Depositor Specified Assays
Data Source: Johns Hopkins Ion Channel Center (JHICC_KCNQ1_Act_IWS)
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: Haibo Yu Ph.D., Kaiping Xu M.S., Zhihong Lin Ph.D., Xiaofang Huang M.S., Shunyou Long M.S., Owen McManus, Ph.D., Meng Wu Ph.D.
Name: Secondary automated electrophysiology assay 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.
The purpose of this assay is to test the compounds generated from the primary screen and subsequent validation and secondary screens for KCNQ1 using automated population patch clamp electrophysiology system IonWorks (Molecular Devices).
KCNQ1, Automated electrophysiology, IonWorks, patch clamp, HTS assay, CHO-K1, 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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5. Peroz, D., Rodriguez, N., Choveau, F., et al. Kv7.1 (KCNQ1) properties and channelopathies. The Journal of Physiology 586(7), 1785-1789 (2008) PMID: 18174212
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10. Jespersen, T., Grunnet, M., and Olesen, S.-P. The KCNQ1 Potassium Channel: From Gene to Physiological Function. Physiology 20(6), 408-416 (2005) PMID: 16287990
11. Mackie, A. R., and Byron, K. L. Cardiovascular KCNQ (Kv7) Potassium Channels: Physiological Regulators and New Targets for Therapeutic Intervention. Mol Pharmacol 74(5), 1171-1179 (2008) PMID: 18684841
12. Maljevic, S., Wuttke, T. V., and Lerche, H. Nervous system KV7 disorders: breakdown of a subthreshold brake. The Journal of Physiology 586(7), 1791-1801 (2008) PMID: 18238816
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15. Delmas, P., and Brown, D. A. Pathways modulating neural KCNQ/M (Kv7) potassium channels. Nat Rev Neurosci 6(11), 850-862 (2005) PMID: 16261179
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18. Jonsson, A., Isomaa, B., Tuomi, T., et al. A variant in the KCNQ1 gene predicts future type 2 diabetes and mediates impaired insulin secretion. Diabetes, 58(10) 2409-13 (2009) PMID: 19584308
19. Schmitt, N., Schwarz, M., Peretz, A., et al. A recessive C-terminal Jervell and Lange-Nielsen mutation of the KCNQ1 channel impairs subunit assembly. EMBO J 19(3), 332-340 (2000) PMID: 10654932
20. OMIM. http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=607542 (2009)
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Protocol for automated patch clamp on KCNQ1-CHO cells with voltage clamp
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 by using 150mm dishes.
2. Split cells once they reach 80% to 90% confluence
2.1. Aspirate medium from culture, add 10 mL of PBS (without Ca2+ and Mg2+) to wash the cell monolayer.
2.2. Aspirate the PBS.
2.3. Add 5 mL of 0.05% Trypsin to the 150mm dish, leave dish undisturbed for 3~5 min at 37 to trypsinize the cells.
2.4. Add 20 mL of growth medium to neutralize the digestion of Trypsin.
2.5. Transfer cell suspension to 50 mL falcon tube and spin at 750 rpm for 4 min.
2.6. Remove supernatant and resuspend cells with 6 ml external solution, spin down at 450 rpm for 4 min.
2.7. Count the cells, adjust the cell density at 2x10;6 per ml.
3. Prepare 3x compound plates(30uM): test compounds are prepared using external solution, final concentration is 10uM;
4. Prepare Amphotericin B: dissolve 5 mg Amphotericin B with 180 uL DMSO, vortex for 1 min; transfer dissolved amphotericin B to 50 mL internal buffer, fill in the amphotericin B tube.
5. Fill the external solution in the buffer boat; fill the internal solution in the internal solution bottle.
6. Add the cells in the cell boat.
7. Load the protocol: The holding potential is -90 mV. To elicit the currents, cells were stimulated by 2,000 ms depolarizing step from -90 mV to +40 mV. Start the experiments.
8. Measure the currents at the steady state.
9. Calculate the percentage of current change for tested compounds with the following formula:
Percentage (%) =100* (Current (post-compound)-Current (pre-compound))/Current (pre-compound)
Percentage (%): Percentage of current potentiation observed after the application of the test compound.
Current (pre-compound): Current recorded before the test compound application at +40mV
Current (post-compound): Current recorded after the test compound application at +40mV
10. Outcome assignment
If the compound causes 3SD (of negative controls) or more activity increase (from mean of negative controls) in both duplicates, the compound is considered to be active (Value=2). Otherwise, it is designated as inactive (Value=1).
11. Score assignment
An inactive test compound is assigned the score of 0.
An active test compound is assigned the score of 100.
12. Internal buffer (40 mM KCl, 100 mM K-Gluconate,1 mM MgCl2, 2 mM CaCl2, 5 mM HEPES, pH 7.25)
13. External buffer (137 mM NaCl, 4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES and 10 mM Glucose, pH 7.4)
Possible artifacts of this assay may include, but are not limited to: unintended chemicals or dust in or under the wells of the microtiter plate. 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)