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BioAssay: AID 588673

SAR analysis for compounds that activate KCNQ1 potassium channels in the KCNQ1 expressing cells on automated patch clamp

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 [3]. 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 more ..
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 Tested Compounds
 Tested Compounds
All(25)
 
 
Active(17)
 
 
Inactive(8)
 
 
 Tested Substances
 Tested Substances
All(25)
 
 
Active(17)
 
 
Inactive(8)
 
 
AID: 588673
Data Source: Johns Hopkins Ion Channel Center (JHICC_KCNQ1_Act_SAR_IWS)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-10-25
Hold-until Date: 2012-10-23
Modify Date: 2012-10-23

Data Table ( Complete ):           Active    All
Target
Sequence: potassium voltage-gated channel subfamily KQT member 1 [Homo sapiens]
Description ..   
Protein Family: KCNQ voltage-gated potassium channel

Gene:KCNQ1     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 17
Depositor Specified Assays
AIDNameTypeComment
2648Primary cell-based high-throughput screening assay for identification of compounds that potentiate/activate KCNQ1 potassium channelsscreeningPrimary cell-based high-throughput screening assay for identification of compounds that potentiate/activate KCNQ1 potassium channels
493007Validation assay for identification of compounds that potentiate KCNQ1 potassium channelsscreeningValidation assay for identification of compounds that potentiate KCNQ1 potassium channels
2699Summary of assays for compounds that potentiate/activate KCNQ1 potassium channelssummarySummary of assays for compounds that potentiate/activate KCNQ1 potassium channels
Description:
Data Source: Johns Hopkins Ion Channel Center (JHICC_KCNQ1_Act_SAR_IWS)
BioAssay Type: Confirmatory, Concentration-Response Relationship 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.
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., Owen McManus, Ph.D., Meng Wu Ph.D.

Description:

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 [3]. 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 [4]. 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 [16], 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 [10]. 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 [18], Romano-Ward syndrome, Jervell and Lange-Nielsen syndrome [19] and familial atrial fibrillation [20], 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 [17].

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 [17]. 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.

Principle of the assay
Patch clamp recording provides a high resolution, linear measure of channel activities. The purpose of this assay is to test the SAR compounds for KCNQ1 activators using automated population patch clamp electrophysiology system IonWorks (Molecular Devices). This assay employs automated patch clamp (Ionworks Quattro) to investigate the current responses of KCNQ1-CHO elicited by voltage clamp protocols in the presence or absence of test compounds. Compounds were tested in quadruplicates at varying concentrations

Keywords:

KCNQ1, activator, agonist, Concentration Response Curve, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.

References:
1. Gutman, G. A., Chandy, K. G., Grissmer, S., et al. International Union of Pharmacology. LIII. Nomenclature and Molecular Relationships of Voltage-Gated Potassium Channels. Pharmacol Rev 57(4), 473-508 (2005) PMID: 16382104
2. Dai, S., Hall, D. D., and Hell, J. W. Supramolecular Assemblies and Localized Regulation of Voltage-Gated Ion Channels. Physiol. Rev. 89(2), 411-452 (2009) PMID: 19342611
3. Borjesson, S., and Elinder, F. Structure, Function, and Modification of the Voltage Sensor in Voltage-Gated Ion Channels. Cell Biochemistry and Biophysics 52(3), 149-174 (2008) PMID: 18989792
4. Pischalnikova, A., and Sokolova, O. The Domain and Conformational Organization in Potassium Voltage-Gated Ion Channels. Journal of Neuroimmune Pharmacology 4(1), 71-82 (2009) PMID: 18836841
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
6. Cannon, S. C. Physiologic Principles Underlying Ion Channelopathies. Neurotherapeutics 4(2), 174-183 (2007) PMID: 17395127
7. Ahern, C. A., and Kobertz, W. R. Chemical Tools for K+ Channel Biology. Biochemistry 48(3), 517-526 (2008) PMID: 19113860
8. Wulff, H., and Zhorov, B. S. K+ Channel Modulators for the Treatment of Neurological Disorders and Autoimmune Diseases. Chemical Reviews 108(5), 1744-1773 (2008) PMID: 18476673
9. Wickenden, A. D. K+ channels as therapeutic drug targets. Pharmacology & Therapeutics 94(1-2), 157-182 (2002) PMID: 12191600
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
13. Robbins, J. KCNQ potassium channels: physiology, pathophysiology, and pharmacology. Pharmacology & Therapeutics 90(1), 1-19 (2001) PMID: 11448722
14. Hernandez, C. C., Zaika, O., Tolstykh, G. P., et al. Regulation of neural KCNQ channels: signalling pathways, structural motifs and functional implications. The Journal of Physiology 586(7), 1811-1821 (2008) PMID: 18238808
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
16. Brown, D. A., and Passmore, G. M. Neural KCNQ (Kv7) channels. British Journal of Pharmacology 156(8), 1185-1195 (2009) PMID: 19298256
17. Towart, R., Linders, J. T. M., Hermans, A. N., et al. Blockade of the IKs potassium channel: An overlooked cardiovascular liability in drug safety screening? Journal of Pharmacological and Toxicological Methods 60(1), 1-10 (2009) PMID: 19439185
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)
21. Namkung, W., Song, Y., Mills, A. D., et al. In Situ Measurement of Airway Surface Liquid [K+] Using a Ratioable K+-sensitive Fluorescent Dye. J. Biol. Chem. 284(23), 15916-15926 (2009) PMID: 19364771
22. Moser, S., Harron, S., Crack, J., et al. Multiple KCNQ Potassium Channel Subtypes Mediate Basal Anion Secretion from the Human Airway Epithelial Cell Line Calu-3. Journal of Membrane Biology 221(3), 153-163 (2008) PMID: 18264812
23. Schroeder, B. C., Waldegger, S., Fehr, S., et al. A constitutively open potassium channel formed by KCNQ1 and KCNE3. Nature 403(6766), 196-199 (2000) PMID: 10646604
24. Greenwood, I., Yeung, S., Hettiarachi, S., et al. KCNQ-encoded channels regulate Na+ transport across H441 lung epithelial cells. Pflugers Archiv European Journal of Physiology 457(4), 785-794 (2009) PMID: 18663467
25. Matos, J. E., Sausbier, M., Beranek, G., et al. Role of cholinergic-activated K Ca 1.1 (BK), K Ca 3.1 (SK4) and K V 7.1 (KCNQ1) channels in mouse colonic Cl - secretion. Acta Physiologica 189(3), 251-258 (2007) PMID: 17305705
Protocol
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: test compounds are prepared using external solution;
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 -70 mV. To elicit the currents, cells were stimulated by 2,000 ms depolarizing step from -70 mV to +40 mV. Start the experiments.
8. Measure the currents at the steady state.
+40mV was used to evaluate compounds.
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 inhibition observed after the application of the test compound.
Current (pre-compound): Current recorded before the test compound application
Current (post-compound): Current recorded after the test compound application
10. Outcome assignment
If the test compound causes activation effect on KCNQ1 in any concentrations tested and the dose response is generated, the compound is considered to be active.
If the test compound does not cause activation effect on KCNQ1 in any concentrations tested or a dose response is not generated, the compound is designated as inactive.
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)

Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
10.014uM-Percentage% (0.014μM**)% Percentage for KCNQ2 at the specified concentrationFloat
20.014uM-SDStandard Deviation of measurement for % Percentage at the specified concentrationFloat
30.041uM-Percentage% (0.041μM**)% Percentage for KCNQ2 at the specified concentrationFloat
40.041uM-SDStandard Deviation of measurement for % Percentage at the specified concentrationFloat
50.12uM-Percentage% (0.12μM**)% Percentage for KCNQ2 at the specified concentrationFloat
60.12uM-SDStandard Deviation of measurement for % Percentage at the specified concentrationFloat
70.37uM-Percentage% (0.37μM**)% Percentage for KCNQ2 at the specified concentrationFloat
80.37uM-SDStandard Deviation of measurement for % Percentage at the specified concentrationFloat
91.11uM-Percentage% (1.1μM**)% Percentage for KCNQ2 at the specified concentrationFloat
101.11 uM-SDStandard Deviation of measurement for % Percentage at the specified concentrationFloat
113.33uM-Percentage% (3.3μM**)% Percentage for KCNQ2 at the specified concentrationFloat
123.33 uM-SDStandard Deviation of measurement for % Percentage at the specified concentrationFloat
1310uM-Percentage% (10μM**)% Percentage for KCNQ2 at the specified concentrationFloat
1410 uM-(SD)Standard Deviation of measurement for % Percentage at the specified concentrationFloat
1530uM-Percentage% (30μM**)% Percentage for KCNQ2 at the specified concentrationFloat
1630 uM-SDStandard Deviation of measurement for % Percentage at the specified concentrationFloat
17IC50*mean IC50 of compound inhibition of on KCNQ2FloatμM
18IC50 SDStandard deviation of mean of IC50FloatμM
19Nnumber of repeats for IC50 of compound inhibitionInteger
20Hill constantHill constantFloat
21Hill constant SDStandard deviation of Hill constantFloat

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH090837-01

Data Table (Concise)
Classification
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