Manual patch clamp assay to confirm a potent KCNQ1 activator
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 more ..
BioActive Compound: 1
Depositor Specified Assays
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., Owen McManus, Ph.D., Meng Wu Ph.D.
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.
KCNQ1, activator, agonist, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.
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
Principle of the assay
Patch clamp recording provides a high resolution, linear measure of channel activities. The purpose of the assay is to validate a compound identified as a potent activator in the KCNQ1 SAR analysis on the KCNQ1 potassium channel. This assay employs manual patch clamp to investigate the current response of KCNQ1-CHO elicited by voltage clamp protocols in the presence or absence of test compound. Compounds were tested at single concentration 0.3microM.
Protocol for manual patch clamp on KCNQ1-CHO cells with voltage clamp
1. Cell culture
KCNQ1-CHO cells were grown in 50/50 DMEM/F12 (Cellgro, Manassas, VA) with 10% fetal bovine serum (FBS), and 2 mM L-glutamine (Gibco, Carlsbad, CA). Before the recording, cells were split and re-plated onto coverslips coated with poly-L-lysine (Sigma-Aldrich, St. Louis, MO).
2. Electrophysiological recording in CHO cells
Whole-cell voltage clamp recording was carried out, using cultured CHO cells, at room temperature, by an Axopatch-200B amplifier (Molecular Devices, Sunnyvale, CA). The electrodes were pulled from borosilicate glass capillaries (World Precision Instruments, Sarasota, FL). When filled with the intracellular solution, the electrodes have resistances of 3-5 megaohms. Pipette solution contained (mM): KCl 145, MgCl2 1, EGTA 5, HEPES 10, MgATP 5 (pH=7.3 with KOH). During the recording, constant perfusion of extracellular solution was maintained using a BPS perfusion system (ALA scientific Instruments, Westburg, NY). Extracellular solution contained (mM): NaCl 140, KCl 5, CaCl2 2, MgCl2 1, HEPES 10, glucose 10 (pH=7.4 with NaOH). Signals were filtered at 1KHz, and digitized using a DigiData 1322A, with pClamp 9.2 software (Molecular Devices, Sunnyvale, CA). Series resistance was compensated by 60-80%. The holding potential was set at -80 mV. To elicit the currents, cells were stimulated by a 2,000 ms depolarizing step to +50 mV and the steady state currents were measured.
3. Calculate the current change for tested compounds with the following formula:
Activation (xFold) = Current (post-compound)/Current (pre-compound)
Current (pre-compound): Current recorded before the test compound application
Current (post-compound): Current recorded after the test compound application
4. Outcome assignment
If the test compound causes activation effect on KCNQ1 at the tested concentration and repeatable, the compound is considered to be active.
If the test compound does not cause activation effect on KCNQ1 at the tested concentration, the compound is designated as inactive.
5. Score assignment
An inactive test compound is assigned the score of 0.
An active test compound is assigned the score of 100.
** Test Concentration.
Data Table (Concise)