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BioAssay: AID 588635

SAR-Confirmatory assay to confirm a potent KCNQ2 inhibitor using manual electrophysiology

Assay Implementation: Haibo Yu Ph.D., Kaiping Xu, Shunyou Long M.S, David Meyers Ph.D., Meng Wu Ph.D., Owen McManus Ph.D. ..more
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Active(1)
 
 
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Active(1)
 
 
AID: 588635
Data Source: Johns Hopkins Ion Channel Center (JHICC_KCNQ2_Inh_ManualEphys_1)
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-10-24
Hold-until Date: 2012-10-21
Modify Date: 2012-10-21

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 Compound: 1
Related Experiments
Show more
AIDNameTypeComment
2156Primary cell-based high-throughput screening assay for identification of compounds that inhibit KCNQ2 potassium channelsScreeningdepositor-specified cross reference: Primary cell-based high-throughput screening assay for identification of compounds that inhibit KCNQ
2262Summary of probe development for inhibitors of KCNQ2 potassium channelSummarydepositor-specified cross reference: Summary of probe development for inhibitors of KCNQ2 potassium channel
2239Primary cell-based high-throughput screening assay for identification of compounds that potentiate KCNQ2 potassium channelsScreeningsame project related to Summary assay
2258Summary of probe development for potentiators of KCNQ2 potassium channelsSummarysame project related to Summary assay
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
2287Confirmatory screen 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
493038Mode of action assay-Dose response assay for compounds that activate KCNQ2 potassium channels on automated patch clampConfirmatorysame 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
493113Mode of action assay-SAR analysis for compounds that activate KCNQ2 potassium channels on automated patch clampConfirmatorysame 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
493025Confirmatory screen for compounds that inhibit KCNQ2 potassium channelsScreeningsame project related to Summary assay
493026Specificity screen against KCNQ1 for compounds that inhibit KCNQ2 potassium channelsScreeningsame project related to Summary assay
493029Counter screen against parental CHO cells for compounds that inhibit KCNQ2 potassium channelsScreeningsame project related to Summary assay
504837SAR analysis for compounds that inhibit KCNQ2 potassium channels on automated electrophysiological assayConfirmatorysame project related to Summary assay
504839Dose response assay for compounds that inhibit KCNQ2 potassium channels on automated electrophysiological assayConfirmatorysame project related to Summary assay
588425Dose response assay of SAR compounds for the identification of selective inhibitors of KCNQ2 potassium channels in the KCNQ1 expressing cells on automated patch clampConfirmatorysame project related to Summary assay
588426SAR analysis for compounds that inhibit KCNQ2 potassium channels on automated electrophysiological assay, CRC2Confirmatorysame project related to Summary assay
588531Confirmatory screen for compounds that inhibit KCNQ2 potassium channels using automated patch clampOthersame project related to Summary assay
588637Dose response assay for compounds that inhibit KCNQ2 potassium channels on automated electrophysiological assay IIConfirmatorysame project related to Summary assay
602271SAR analysis for compounds that inhibit KCNQ2 potassium channels in an automated electrophysiological assayConfirmatorysame project related to Summary assay
Description:
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, Shunyou Long M.S, David Meyers Ph.D., Meng Wu Ph.D., Owen McManus Ph.D.
Name: SAR-Confirmatory assay to confirm a potent KCNQ2 inhibitor using manual electrophysiology

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 the human genome encoding different but homologous potassium channels. Isolation and characterization of bioactive chemical probes could enable pharmacological experiments to study channel function provides tools to analyze structure and function.

The M-type channels are unique voltage-gated and ligand-regulated K+ channels with distinct physiological and pharmacological characteristics. They are activated at a voltages near the threshold for action potential initiation and thus regulate membrane excitability. KCNQ (or also called Kv7) channels, members of Kv channel superfamily, include five members, KCNQ1 to KCNQ5. Among them, homodimers and/or heterodimers of KCNQ2 and KCNQ3 are believed components of the M-current channel. Modulators of KCNQ2 may play important roles regulating neuronal function, and KCNQ2 openers have demonstrated efficacy in treating epilepsy and may have further uses for treating pain and anxiety. In vivo studies have supported a role for KCNQ2 inhibitors as cognition enhancers.

Molecular and functional studies of M current have indicated that KCNQ2 is a key molecular component of the M-current. It is therefore feasible to design robust, high-throughput screens specifically targeting KCNQ2 channels.

Principle of the assay
Patch clamp is gold standard to measure channel activities. The purpose of the assay is to validate a compound identified as a potent inhibitor in the KCNQ2 SAR analysis on the KCNQ2 potassium channel. This assay employs manual patch clamp to investigate the current response of KCNQ2-CHO elicited by voltage clamp protocols in the presence or absence of test compound. Compounds were tested in quadruplicates at single concentration 0.1 microM.

Keywords:

KCNQ2,inhibitor, blocker, Concentration Response Curve, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Protocol
1. Cell culture
KCNQ2-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 3, CaCl2 2, MgCl2 1.5, 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 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
4. Outcome assignment
If the test compound causes inhibition effect on KCNQ2 in the tested concentration and repeatable, the compound is considered to be active.
If the test compound does not cause inhibition effect on KCNQ2 in 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.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: CHO
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition (0.1μM**)Inhibition (%): Percentage of current inhibition observed after the application of the test compound.Float
2SDStandard deviation of Inhibition (%)Float

** 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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