Bookmark and Share
BioAssay: AID 588425

Dose response assay of SAR compounds for the identification of selective inhibitors of KCNQ2 potassium channels in the KCNQ1 expressing cells on automated patch clamp

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
_
   
 Tested Compounds
 Tested Compounds
All(21)
 
 
Active(14)
 
 
Inactive(7)
 
 
 Tested Substances
 Tested Substances
All(21)
 
 
Active(14)
 
 
Inactive(7)
 
 
AID: 588425
Data Source: Johns Hopkins Ion Channel Center (KCNQ2_Inh_IWS_SAR_Q1_CRC)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-09-30
Hold-until Date: 2012-09-26
Modify Date: 2012-09-26

Data Table ( Complete ):           View Active Data    View All Data
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: 14
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
493025Confirmatory screen for compounds that inhibit KCNQ2 potassium channelsScreeningdepositor-specified cross reference: Confirmatory screen for compounds that inhibit KCNQ2 potassium channels
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
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
588635SAR-Confirmatory assay to confirm a potent KCNQ2 inhibitor using manual electrophysiologyOthersame 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:
Data Source: Johns Hopkins Ion Channel Center (KCNQ2_Inh_IWS_SAR_Q1_CRC)
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: 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: Dose response assay of SAR compounds for the identification of selective inhibitors of KCNQ2 potassium channels in the KCNQ1 expressing cells on automated patch clamp

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.

Results of large scale compound screens for M-current modulators have not been reported. Molecular and functional studies 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. This assay describes a Tl+-based fluorescence assay in 384-well format used to identify of KCNQ2 modulatory compounds from a large compound library.
Protocol
Principle of the assay
Patch clamp recording provides a high resolution, linear measure of channel activities. The purpose of the assay is to examine the non-specific effects of KCNQ2 inhibitors on the KCNQ1 expressing cells. 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:

KCNQ2,KCNQ1, inhibitor, blocker, Concentration Response Curve, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.

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 -790 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 inhibition 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 inhibition 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
Show more
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 DA027716-01

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