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BioAssay: AID 2581

Automated electrophysiology assay of compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1

BioAssay Type: Automated Electrophysiology, Patch Clamp, Multiple Concentration Activity in Multiplicates Observed ..more
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 Tested Compounds
 Tested Compounds
All(49)
 
 
Active(31)
 
 
Inactive(18)
 
 
 Tested Substances
 Tested Substances
All(49)
 
 
Active(31)
 
 
Inactive(18)
 
 
AID: 2581
Data Source: Johns Hopkins Ion Channel Center (JHICC_Kir2.1_inhibitors_IWS_VU)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-03-16
Modify Date: 2010-09-21

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: 31
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.
463252Secondary automated electrophysiology assay of compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1Other depositor-specified cross reference
504555Dose response assay on hERG potassium channel for Kir2.1 inhibitors on automated electrophysiologyConfirmatory depositor-specified cross reference
504557Confirmation dose response assay for SAR compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1.Confirmatory depositor-specified cross reference
504559Dose response assay for compounds that inhibit inward-rectifying potassium channel Kir2.1 on automated electrophysiologyConfirmatory depositor-specified cross reference
504693Dose response assay on hERG potassium channel for Kir2.1 inhibitors on automated electrophysiology (II)Confirmatory depositor-specified cross reference
504694Dose response assay for compounds that inhibit inward-rectifying potassium channel Kir2.1 on automated electrophysiology (II)Confirmatory depositor-specified cross reference
504695Confirmation dose response assay for SAR compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1 (II)Confirmatory 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
2591Manual electrophysiological patch clamp assay for SAR 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
504828Manual electrophysiological patch clamp assay for extended SAR compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1_1Confirmatory same project related to Summary assay
Description:
Data Source: Johns Hopkins Ion Channel Center (JHICC)
BioAssay Type: Automated Electrophysiology, Patch Clamp, 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: Haibo Yu Ph.D., Meng Wu Ph.D., Hao-ran Wang Ph.D., Bill Shi Ph.D., David Meyers Ph.D., and Jia Xu Ph.D.

Name: Automated electrophysiology assay of compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1

Description:

See the related assay (PubChem AID: 1672).
Protocol
Assay overview:
The purpose of this assay is to test the compounds generated from the primary screen and subsequent validation and secondary screens for Kir2.1 (potassium inwardly-rectifying channel J2, KCNJ2) (Pubchem SAID 1843) using automated population patch clamp electrophysiology system IonWorks (Molecular Devices).
Protocol for automated electrophysiology 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. Prepare 3X compound plates: test compounds are prepared using external buffer (140 mM NaCl, 4 mM KCl, 1.8 mM CaCl2,1 mM MgCl2, 10 mM HEPES, 10 mM Glucose, pH 8.5); controls are external buffer (both with DMSO concentrations matched to that of test compounds)
3. Cell preparation: Cells are dislodged using 0.125 % Trypsin/EDTA and resuspended at 2X10E6 /mL in external buffer.
4. Patchplate hole test on Ionworks: External buffer is added to each well of a 384-well PPC plate, 3.5 uL/well, and hole resistance of each well is measured.
5. Cell plating on Ionworks: Cells are dispensed to the 384-well PPC plate on Ionworks, 3.5 uL/well.
6. Membrane permeablization: Cells are exposed to 0.1mg/mL amphotericin B in internal buffer (140 mM KCl, 2 mM MgCl2, 0.5 mM EGTA, 10 mM HEPES, pH 7.2).
7. Currents are measured using the following voltage protocol: Hold cells at +10 mV, depolarize to -100 mV for 800 ms, back to +10 mV for 600 ms and run a ramp from -100 mV to +100 mV in 800 ms before back to +10 mV.
8. Dispense 3.5 ul /well of compounds to each well on Ionworks and incubate for 4 minutes.
9. Measure currents using the same protocol described in 7.
10. Calculate Kir2.1 Channel Activity (%) = Ipostcompound/Iprecompound*100
11. 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.
12. Score assignment:
An inactive test compound is assigned the score of 0.
An active test compound is assigned the score of 100.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1activity at 14nM (0.014μM**)Kir2.1 Channel Activity (%) at the specified concentration Float
2SD at 14nMStandard Deviation of measurement of Kir2.1 channel activity (%) at the specified concentrationFloat
3activity at 41nM (0.041μM**)Kir2.1 Channel Activity (%) at the specified concentrationFloat
4SD at 41nMStandard Deviation of measurement of Kir2.1 channel activity (%) at the specified concentrationFloat
5activity at 123nM (0.123μM**)Kir2.1 Channel Activity (%) at the specified concentrationFloat
6SD at 123nMStandard Deviation of measurement of Kir2.1 channel activity (%) at the specified concentrationFloat
7activity at 370nM (0.37μM**)Kir2.1 Channel Activity (%) at the specified concentrationFloat
8SD at 370nMStandard Deviation of measurement of Kir2.1 channel activity (%) at the specified concentrationFloat
9activity at 1.1uM (1.1μM**)Kir2.1 Channel Activity (%) at the specified concentrationFloat
10SD at 1.1uMStandard Deviation of measurement of Kir2.1 channel activity (%) at the specified concentrationFloat
11activity at 3.3uM (3.3μM**)Kir2.1 Channel Activity (%) at the specified concentrationFloat
12SD at 3.3uMStandard Deviation of measurement of Kir2.1 channel activity (%) at the specified concentrationFloat
13activity at 10uM (10μM**)Kir2.1 Channel Activity (%) at the specified concentrationFloat
14SD at 10uMStandard Deviation of measurement of Kir2.1 channel activity (%) at the specified concentrationFloat
15activity at 30uM (30μM**)Kir2.1 Channel Activity (%) at the specified concentrationFloat
16SD at 30uMStandard Deviation of measurement of Kir2.1 channel activity (%) at the specified concentrationFloat
17NNumber of replicates for Kir2.1 Channel Activity (%) measurementsInteger
18IC50 *IC50 (uM)FloatμM
19IC50 SDStandard deviation of IC50FloatμM
20HillHill constant of fittingFloat
21Hill SDStandard deviation of Hill constantFloat

* 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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