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BioAssay: AID 2591

Manual electrophysiological patch clamp assay for SAR compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1

BioAssay Type: Electrophysiology, Patch Clamp, Orthogonal Assay, Specificity Screen, Multiple Concentration Activity in Multiplicates Observed ..more
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 Tested Compounds
 Tested Compounds
All(10)
 
 
Active(10)
 
 
 Tested Substances
 Tested Substances
All(10)
 
 
Active(10)
 
 
AID: 2591
Data Source: Johns Hopkins Ion Channel Center (JHICC_Kir2.1_inhibitors_ME_2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-03-17

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: inward rectifier potassium channel 2 [Mus musculus]
Description ..   
Protein Family: Inward rectifier potassium channel

Gene:KCNJ2     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 10
Related Experiments
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AIDNameTypeProbeComment
1672Primary cell-based high-throughput screening assay for identification of compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1Screening depositor-specified cross reference: Primary HTS of 305616 compounds where 2592 were identified as active.
1843Summary of probe development for inhibitors/blockers of inward-rectifying potassium ion channel Kir2.1Summary3 depositor-specified cross reference: Summary assay for Kir2.1 inhibitor assays.
504828Manual electrophysiological patch clamp assay for extended SAR compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1_1Confirmatory depositor-specified cross reference
2032Confirmatory screen for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1Screening same project related to Summary assay
2105Counter screen for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1Screening same project related to Summary assay
2236hERG counter screen for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1Screening same project related to Summary assay
2329KCNK9 specificity screen for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1Screening same project related to Summary assay
2345Specificity screen against Kir2.1 for compounds that potentiate KCNQ2Screening same project related to Summary assay
2404Manual electrophysiological patch clamp assay and ROMK specificity of compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1Confirmatory same project related to Summary assay
2581Automated electrophysiology assay of compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1Confirmatory same project related to Summary assay
2594Confirmation dose response assay for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1Confirmatory same project related to Summary assay
463252Secondary automated electrophysiology assay of compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1Other same project related to Summary assay
504555Dose response assay on hERG potassium channel for Kir2.1 inhibitors on automated electrophysiologyConfirmatory same project related to Summary assay
504557Confirmation dose response assay for SAR compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1.Confirmatory same project related to Summary assay
504559Dose response assay for compounds that inhibit inward-rectifying potassium channel Kir2.1 on automated electrophysiologyConfirmatory same project related to Summary assay
504693Dose response assay on hERG potassium channel for Kir2.1 inhibitors on automated electrophysiology (II)Confirmatory same project related to Summary assay
504694Dose response assay for compounds that inhibit inward-rectifying potassium channel Kir2.1 on automated electrophysiology (II)Confirmatory same project related to Summary assay
504695Confirmation dose response assay for SAR compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1 (II)Confirmatory same project related to Summary assay
Description:
Data Source: Johns Hopkins Ion Channel Center (JHICC_ Kir2.1_inhibitors_ME_2)
BioAssay Type: Electrophysiology, Patch Clamp, Orthogonal Assay, Specificity Screen, Multiple Concentration Activity in Multiplicates 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: Elena Makhina Ph.D., University of Pittsburgh
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 DA026212-01
Grant Proposal PI: Elena Makhina Ph.D., University of Pittsburgh
Assay Implementation: Hao-ran Wang Ph.D., Meng Wu Ph.D., Haibo Yu Ph.D., Bill Shi Ph.D., David Meyers Ph.D., and Jia Xu Ph.D.

Name: Manual electrophysiological patch clamp assay for SAR compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1

Description:

See the related essay (PubChem AID: 1672).
Protocol
Assay overview:
The purpose of this orthogonal assay is to test the compounds identified in the primary screen and subsequent validation and secondary screens for Kir2.1 (potassium inwardly-rectifying channel J2, KCNJ2) (Pubchem SAID 1843) using manual patch clamp assay for evaluation of SAR compounds.

Protocol for manual patch clamp assay for Kir2.1:

1. Cell culture: Cells are routinely cultured in DMEM/F12 medium, supplemented with 10% Fetal Bovine Serum (FBS), 50 IU/ml penicillin, 50ug/ml streptomycin, and 500ug/ml G418.
2. The electrodes were pulled from borosilicate glass capillaries (World Precision Instruments, Sarasota, FL). Pipette resistance was around 3-4 megaohms.
3. Whole-cell currents of Kir2.1 were recorded by using an Axopatch 200B amplifier. The bath and pipette solution contained 140 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM Hepes (pH 8.5) and 140 mM KCl, 2 mM EDTA, 10 mM Hepes (pH 7.4), respectively. To apply the compound, 7-10ml of compound solution was injected by a 10ml syringe into recording chamber containing 0.5 ml bath solution. Excessive solution was removed by suction.
4. To record Kir2.1 current, voltage was stepped from 0 mV holding potential to -100mV (500 ms) with an interval of 30 seconds. Capacitance and access resistance were monitored and 75% compensated. Currents were filtered at 1 kHz, and data were acquired at 5 kHz with a Digidata 1322A computer interface and pClamp 9.2 software (Axon Instruments).
5. To check the quality of seal during recording, each voltage step was followed by a ramp protocol (500ms, from -100mV to 100 mV). Cells losing seal during recording were identified by a sudden increase of outward current in the ramp protocol, in which case, cell will be dropped.
6. Data were analyzed using pClamp 8 followed by Origin 6. The percentage of inhibition of the tested compounds was calculated with the following formula:
Percentage (%) = (Current(prior cpd)- Current(post cpd))/ Current(prior cpd)*100
Where:
Percentage (%): Percentage of current inhibition observed post the application of the test compound.
Current(prior cpd): Current recorded prior the test compound application at -100 mV
Current(post cpd): Current recorded post the test compound application at -100 mV
7. Outcome assignment:
If the test compound causes inhibition of the Kir2.1 current at -100 mV in any concentrations tested, the compound is considered to be active.
If the test compound does not cause inhibition of the Kir2.1 current at -100 mV in any concentrations tested, the compound is designated as inactive.

8. 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 ChEMBL:
Assay Type: Functional
Assay Cell Type: NULL
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition at 10nM (0.01μM**)% inhibition of Kir2.1 activity at the specified concentrationFloat
2SEM at 10nMStandard error of measurement for % inhibition at the specified concentrationFloat
3N at 10nMNumber of replicates at the specified concentrationInteger
4Inhibition at 100nM (0.1μM**)% inhibition of Kir2.1 activity at the specified concentrationFloat
5SEM at 100nMStandard error of measurement for % inhibition at the specified concentrationFloat
6N at 100nMNumber of replicates at the specified concentrationInteger
7Inhibition at 250nM (0.25μM**)% inhibition of Kir2.1 activity at the specified concentrationFloat
8SEM at 250nMStandard error of measurement for % inhibition at the specified concentrationFloat
9N at 250nMNumber of replicates at the specified concentrationInteger
10Inhibition at 0.5uM (0.5μM**)% inhibition of Kir2.1 activity at the specified concentrationFloat
11SEM at 0.5uMStandard error of measurement for % inhibition at the specified concentrationFloat
12N at 0.5uMNumber of replicates at the specified concentrationInteger
13Inhibition at 1uM (1μM**)% inhibition of Kir2.1 activity at the specified concentrationFloat
14SEM at 1uMStandard error of measurement for % inhibition at the specified concentrationFloat
15N at 1uMNumber of replicates at the specified concentrationInteger
16Inhibition at 3uM (3μM**)% inhibition of Kir2.1 activity at the specified concentrationFloat
17SEM at 3uMStandard error of measurement for % inhibition at the specified concentrationFloat
18N at 3uMNumber of replicates at the specified concentrationInteger
19Inhibition at 10uM (10μM**)% inhibition of Kir2.1 activity at the specified concentrationFloat
20SEM at 10uMStandard error of measurement for % inhibition at the specified concentrationFloat
21N at 10uMNumber of replicates at the specified concentrationInteger
22Inhibition at 30uM (30μM**)% inhibition of Kir2.1 activity at the specified concentrationFloat
23SEM at 30uMStandard error of measurement for % inhibition at the specified concentrationFloat
24N at 30uMNumber of replicates at the specified concentrationInteger
25IC50*IC50FloatμM

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 DA026212-01

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