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BioAssay: AID 493038

Mode of action assay-Dose response assay for compounds that activate KCNQ2 potassium channels on automated patch clamp

Name: Mode of action assay-Dose response assay for compounds that activate KCNQ2 potassium channels on automated patch clamp ..more
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 Tested Compounds
 Tested Compounds
All(5)
 
 
Active(5)
 
 
 Tested Substances
 Tested Substances
All(7)
 
 
Active(7)
 
 
AID: 493038
Data Source: Johns Hopkins Ion Channel Center (KCNQ2_Act_IWS_CRC)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-01-18
Hold-until Date: 2011-12-07
Modify Date: 2011-12-07

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: potassium voltage-gated channel, KQT-like subfamily, member 2 [Rattus norvegicus]
Description ..   
Protein Family: KCNQ voltage-gated potassium channel

Gene:KCNQ2     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 5
Related Experiments
Show more
AIDNameTypeComment
2239Primary cell-based high-throughput screening assay for identification of compounds that potentiate KCNQ2 potassium channelsScreeningdepositor-specified cross reference: Primary HTS assay for KCNQ2 potentiators with 305606 compounds tested, and 1644 are found to be acti
2258Summary of probe development for potentiators of KCNQ2 potassium channelsSummarydepositor-specified cross reference: Summary assay for the KCNQ2 potentiators.
2287Confirmatory screen for compounds that potentiate KCNQ2 potassium channelsScreeningdepositor-specified cross reference: Confirmatory screen in duplicates for 1189 compounds and 804 of them are found active.
493113Mode of action assay-SAR analysis for compounds that activate KCNQ2 potassium channels on automated patch clampConfirmatorydepositor-specified cross reference
2282Counter screen for compounds that potentiate KCNQ2 potassium channelsScreeningsame project related to Summary assay
2283Specificity screen against KCNQ1 for compounds that potentiate KCNQ2 potassium channelsScreeningsame project related to Summary assay
2345Specificity screen against Kir2.1 for compounds that potentiate KCNQ2Screeningsame project related to Summary assay
2408Mode of action - current amplitude concentration response for ztz240, a potentiator of KCNQ2 potassium channelsConfirmatorysame project related to Summary assay
2415Mode of Action - subtype specificity assay for ztz240, a potentiator of KCNQ2 potassium channelsScreeningsame project related to Summary assay
2432Mode of action assay - molecular determinants for ztz240, a potentiator of KCNQ2 potassium channelsScreeningsame project related to Summary assay
2443Mode of action - deactivation constant concentration response for ztz240, a potentiator of KCNQ2 potassium channelsConfirmatorysame project related to Summary assay
2548Mode of action assay - current amplitude concentration response for derivatives of ZTZ240, a potentiator of KCNQ2 potassium channelsConfirmatorysame project related to Summary assay
2558Mode of action - Automated patch clamp assay for KCNQ2 potentiators on Retigabine insensitive KCNQ2 Mutant W236L cell lineScreeningsame project related to Summary assay
2603Mode of action assay-Automated electrophysiology assay of compounds that potentiate KCNQ2 potassium channelConfirmatorysame project related to Summary assay
2654Dose response of Retigabine-insensitive compounds that potentiate KCNQ2 potassium channelConfirmatorysame project related to Summary assay
493037Mode of action assay-Confirmatory dose response assay for compounds that activate KCNQ2 potassium channelsConfirmatorysame project related to Summary assay
493039Mode of action assay-Dose response assay for KCNQ2 activators in the KCNQ2/KCNQ3 co-expressing cells on automated patch clampConfirmatorysame project related to Summary assay
493042Mode of action assay-Dose response assay for the identification of selective activators of KCNQ2 potassium channels in the KCNQ1/KCNE1 expressing cells on automated patch clampConfirmatorysame project related to Summary assay
493043Mode of action assay-Dose response assay for the identification of selective activators of KCNQ2 potassium channels in the KCNQ4 expressing cells on automated patch clampConfirmatorysame project related to Summary assay
493044Mode of action assay-Dose response assay for the identification of selective activators of KCNQ2 potassium channels in the KCNQ1 expressing cells on automated patch clampConfirmatorysame project related to Summary assay
493046Mode of action assay-Specificity test for the KCNQ2 activators in the KCNQ5 expressing cellsConfirmatorysame project related to Summary assay
493047Mode of action assay-Specificity test for the KCNQ2 activators in the KCNQ3 expressing cellsConfirmatorysame project related to Summary assay
504416Mode of action assay- Specificity dose response assay for the KCNQ2 activators in the KCNQ4 expressing cells by the Tl fluxConfirmatorysame project related to Summary assay
504417Mode of action assay-Specificity dose response assay for the KCNQ2 activators in the KCNQ1 expressing cells by the Tl flux assayConfirmatorysame project related to Summary assay
504418Mode of action assay-Specificity dose response assay for the KCNQ2 activators in the KCNQ1/E1 expressing cells by the Tl fluxConfirmatorysame project related to Summary assay
Description:
Data Source: Johns Hopkins Ion Channel Center (KCNQ2_Act_5_CRC_IWS)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Name: Mode of action assay-Dose response assay for compounds that activate KCNQ2 potassium channels on automated patch clamp

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: Min Li, Ph.D.
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 DA027716-01
Grant Proposal PI: Min Li, Ph.D., Johns Hopkins University School of Medicine
Assay Implementation: Haibo Yu Ph.D., Kaiping Xu M.S., Shunyou Long M.S., Meng Wu Ph.D., David Meyers Ph.D., Owen Mcmanus Ph.D.



Description:
Voltage-gated potassium (K) channels are critical for neuronal function in excitable tissues such as brain and heart. They are also found in non-excitable tissues important for other functions such as hormone secretion, oxygen-sensing and immune responses. There are more than 100 genes in human genome encoding different but homologous potassium channels. Isolation and characterization of bioactive chemical probes could form important pharmacological foundation, providing a great deal of insights into the structure and function.

The M-type channels are unique voltage-gated and ligand-regulated K+ channels with their distinct physiological and pharmacological characteristics. They are activated at a voltage near the threshold for action potential initiation and regulate membrane excitability. The KCNQ (or also called Kv7), members of Kv channel superfamily, includes five members, KCNQ1 to KCNQ5. Among them homodimer and/or heterodimer of KCNQ2 and KCNQ3 are believed components of the M-channel. The modulators of KCNQ2 are playing an important role in the neuronal function regulation. As the therapeutic targets, the KCNQ2 openers have great potential used to treat epilepsy, pain and anxiety et al.



Principle of the assay
Patch clamp is gold standard to measure channel activities. The purpose of the assay is to validate the compounds identified as active in the primary screen (PubChem AID: 2239) and examine the potentiation effects on the KCNQ2 potassium channel. This assay employs automated patch clamp (Ionworks Quattro) to investigate the current response of KCNQ2-CHO elicited by voltage clamp protocols in the presence or absence of test compounds. Compounds were tested in quadruplicates at varying concentrations.

Keywords:

KCNQ2,agonist, activator, potentiator, IonWorks, Automated patch clamp, Concentration Response Curve, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Protocol for automated patch clamp on KCNQ2-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 -90 mV. To elicit the currents, cells were stimulated by 2,000 ms depolarizing step from -90 mV to -10 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
Current (post-compound): Current recorded after the test compound application
10.Outcome assignment
If the test compound causes potentiation effect on KCNQ2 in any concentrations tested and the dose response is generated, the compound is considered to be active.
If the test compound does not cause potentiation effect on KCNQ2 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 m 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)
Comment
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, or 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.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: CHO
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1potentiation at 14nM (0.014μM**)% potentiation for KCNQ2 at the specified concentrationFloat
2SD at 14nMStandard Deviation of measurement for % potentiation at the specified concentrationFloat
3potentiation at 41nM (0.041μM**)% potentiation for KCNQ2 at the specified concentrationFloat
4SD at 41nMStandard Deviation of measurement for % potentiation at the specified concentrationFloat
5Potentiation at 120nM (0.12μM**)% potentiation for KCNQ2 at the specified concentrationFloat
6SD at 120nMStandard Deviation of measurement for % potentiation at the specified concentrationFloat
7Potentiation at 370nM (0.37μM**)% potentiation for KCNQ2 at the specified concentrationFloat
8SD at 370nMStandard Deviation of measurement for % potentiation at the specified concentrationFloat
9Potentiation at 1.1uM (1.1μM**)% potentiation for KCNQ2 at the specified concentrationFloat
10SD at 1.1uMStandard Deviation of measurement for % potentiation at the specified concentrationFloat
11Potentiation at 3.3uM (3.3μM**)% potentiation for KCNQ2 at the specified concentrationFloat
12SD at 3.3uMStandard Deviation of measurement for % potentiation at the specified concentrationFloat
13Potentiation at 10uM (10μM**)% potentiation for KCNQ2 at the specified concentrationFloat
14SD at 10uMStandard Deviation of measurement for % potentiation at the specified concentrationFloat
15Potentiation at 30uM (30μM**)% potentiation for KCNQ2 at the specified concentrationFloat
16SD at 30uMStandard Deviation of measurement for % potentiation at the specified concentrationFloat
17EC50*mean EC50 of compound potentiation of on KCNQ2FloatμM
18EC50 SDStandard deviation of mean of EC50FloatμM
19Nnumber of repeats for EC50 of compound potentiationInteger
20HillHill constantFloat
21Hill SDStandard deviation of Hill constantFloat

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 DA027716-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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