|Validation for compounds that inhibit KCNQ1 potassium channels on automated electrophysiology assay - BioAssay Summary
Name: Validation for compounds that inhibit KCNQ1 potassium channels on automated electrophysiology assay ..more
BioActive Compounds: 87
Depositor Specified Assays
Depositor Category: NIH Molecular Libraries Probe Production Network
Data Source: Johns Hopkins Ion Channel Center (JHICC_KCNQ1_Inh_IWS)
BioAssay Type: Other
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.
Name: Validation for compounds that inhibit KCNQ1 potassium channels on automated electrophysiology assay
Voltage-gated potassium channels [1,2] are tetrameric membrane proteins that selectively conduct K+ across cellular membranes, and also 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 potential 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], including 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 the voltage-gated KCNQ potassium channel family, which is composed of five members, KCNQ1-KCNQ5. These subtypes 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. In 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 .
The pharmacological responses of KCNQ1/KCNE heteromultimers differ from KCNQ1 alone. Initial discovery of KCNQ1 modulators was focused on KCNQ1 and KCNQ1/KCNE1 (IKs) inhibitors , including Chromonal 293B, linopirdine and XE991 and newer potent inhibitors, i.e. Merck-IKs (IC50 ~0.08 nM), JNJ 303(IC50 0.064 uM) and JNJ282 (IC50 0.001 uM).
The purpose of this assay is to validate the compounds identified as inhibitors in the primary KCNQ1 screen and confirmatory assay using an orthogonal assay. This assay employs automated patch clamp (IonWorks Quattro) to investigate the current response of KCNQ1 expressed in CHO cells elicited by voltage clamp protocols in the presence or absence of test compounds. Compounds were tested in duplicates at 10 micromolar.
KCNQ1, inhibitor, blocker, automated electrophysiology, IonWorks, patch clamp, HTS assay, CHO-K1, 384, primary, JHICC, Johns Hopkins, MLSMR, 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
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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 cell digestion by 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 cells to the cell boat.
7. Load the protocol: The holding potential is -80 mV. To elicit the currents, cells were stimulated by 2,000 ms depolarizing step from -80 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 a decrease of current amplitudes greater that 3SD of negative controls in both duplicate tests, 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)