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BioAssay: AID 2404

Manual electrophysiological patch clamp assay and ROMK specificity of 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
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Active(1)
 
 
 Tested Substances
 Tested Substances
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Active(1)
 
 
AID: 2404
Data Source: Johns Hopkins Ion Channel Center (JHICC_Kir2.1_inhibitors_ME)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-02-23
Modify Date: 2010-07-06

Data Table ( Complete ):           View Active Data    View All Data
Targets
Sequence: potassium inwardly-rectifying channel J2 [Mus musculus]
Description ..   
Protein Family: Inward rectifier potassium channel

Gene:KCNJ2     Related Protein 3D Structures     More BioActivity Data..


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BioActive Compound: 1
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 assay.
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
2581Automated electrophysiology assay 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
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
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
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 and ROMK specificity of compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1

Description:

See the related essay (PubChem AID: 1672).
Protocol
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 AID 1843) using manual patch clamp assay. ROMK (Kir1.1, the renal outer medullary potassium channel) was used as the specificity target.

Protocol for manual patch clamp assay for Kir2.1 and specificity target ROMK:

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 7.4) 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, and were 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.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
11 (0.01μM**)Kir2.1 %Block % inhibition at -100mV for Kir2.1 treated with 10nM of compound at pH8.5Float
22Kir2.1 SEM Standard Error of measure for % inhibition at -100mV for Kir2.1 treated with 10nM of compound at pH8.5Float
33Kir2.1 N Number of replicates for % Inhibition at -100mV for Kir2.1 treated with 10nM of compound at pH8.5Integer
44 (0.1μM**)Kir2.1 %Block: % inhibition at -100mV for Kir2.1 treated with 100nM of compound at pH8.5Float
55Kir2.1 SEM: Standard Error of measure for % inhibition at -100mV for Kir2.1 treated with 100nM of compound at pH8.5Float
66Kir2.1 N: Number of replicates for % Inhibition at -100mV for Kir2.1 treated with 100nM of compound at pH8.5Integer
77 (0.25μM**)Kir2.1 %Block: % inhibition at -100mV for Kir2.1 treated with 250nM of compound at pH8.5Float
88Kir2.1 SEM: Standard Error of measure for % inhibition at -100mV for Kir2.1 treated with 250nM of compound at pH8.5Float
99Kir2.1 N: Number of replicates for % Inhibition at -100mV for Kir2.1 treated with 250nM of compound at pH8.5Integer
1010 (0.5μM**)Kir2.1 %Block: % inhibition at -100mV for Kir2.1 treated with 500nM of compound at pH8.5Float
1111Kir2.1 SEM: Standard Error of measure for % inhibition at -100mV for Kir2.1 treated with 500nM of compound at pH8.5Float
1212Kir2.1 N: Number of replicates for % Inhibition at -100mV for Kir2.1 treated with 500nM of compound at pH8.5Integer
1313 (1μM**)Kir2.1 %Block: % inhibition at -100mV for Kir2.1 treated with 1uM of compound at pH8.5Float
1414Kir2.1 SEM: Standard Error of measure for % inhibition at -100mV for Kir2.1 treated with 1uM of compound at pH8.5Float
1515Kir2.1 N: Number of replicates for % Inhibition at -100mV for Kir2.1 treated with 1uM of compound at pH8.5Integer
1616 (3μM**)Kir2.1 %Block: % inhibition at -100mV for Kir2.1 treated with 3uM of compound at pH8.5Float
1717Kir2.1 SEM: Standard Error of measure for % inhibition at -100mV for Kir2.1 treated with 3uM of compound at pH8.5Float
1818Kir2.1 N: Number of replicates for % Inhibition at -100mV for Kir2.1 treated with 3uM of compound at pH8.5Integer
1919 (10μM**)Kir2.1 %Block: % inhibition at -100mV for Kir2.1 treated with 10uM of compound at pH8.5Float
2020Kir2.1 SEM: Standard Error of measure for % inhibition at -100mV for Kir2.1 treated with 10uM of compound at pH8.5Float
2121Kir2.1 N: Number of replicates for % Inhibition at -100mV for Kir2.1 treated with 10uM of compound at pH8.5Integer
2222 (30μM**)Kir2.1 %Block: % inhibition at -100mV for Kir2.1 treated with 30uM of compound at pH8.5Float
2323Kir2.1 SEM: Standard Error of measure for % inhibition at -100mV for Kir2.1 treated with 30uM of compound at pH8.5Float
2424Kir2.1 N: Number of replicates for % Inhibition at -100mV for Kir2.1 treated with 30uM of compound at pH8.5Integer
2525 (0.01μM**)Kir2.1 %Block: % inhibition at -100mV for Kir2.1 treated with 10nM of compound at pH7.4Float
2626Kir2.1 SEM: Standard Error of measure for % inhibition at -100mV for Kir2.1 treated with 10nM of compound at pH7.4Float
2727Kir2.1 N: Number of replicates for % Inhibition at -100mV for Kir2.1 treated with 10nM of compound at pH7.4Integer
2828 (0.1μM**)Kir2.1 %Block: % inhibition at -100mV for Kir2.1 treated with 100nM of compound at pH7.4Float
2929Kir2.1 SEM: Standard Error of measure for % inhibition at -100mV for Kir2.1 treated with 100nM of compound at pH7.4Float
3030Kir2.1 N: Number of replicates for % Inhibition at -100mV for Kir2.1 treated with 100nM of compound at pH7.4Integer
3131 (0.5μM**)Kir2.1 %Block: % inhibition at -100mV for Kir2.1 treated with 500nM of compound at pH7.4Float
3232Kir2.1 SEM: Standard Error of measure for % inhibition at -100mV for Kir2.1 treated with 500nM of compound at pH7.4Float
3333Kir2.1 N: Number of replicates for % Inhibition at -100mV for Kir2.1 treated with 500nM of compound at pH7.4Integer
3434 (3μM**)Kir2.1 %Block: % inhibition at -100mV for Kir2.1 treated with 3uM of compound at pH7.4Float
3535Kir2.1 SEM: Standard Error of measure for % inhibition at -100mV for Kir2.1 treated with 3uM of compound at pH7.4Float
3636Kir2.1 N: Number of replicates for % Inhibition at -100mV for Kir2.1 treated with 3uM of compound at pH7.4Integer
3737 (10μM**)Kir2.1 %Block: % inhibition at -100mV for Kir2.1 treated with 10uM of compound at pH7.4Float
3838Kir2.1 SEM: Standard Error of measure for % inhibition at -100mV for Kir2.1 treated with 10uM of compound at pH7.4Float
3939Kir2.1 N: Number of replicates for % Inhibition at -100mV for Kir2.1 treated with 10uM of compound at pH7.4Integer
4040 (30μM**)Kir2.1 %Block: % inhibition at -100mV for Kir2.1 treated with 30uM of compound at pH7.4Float
4141Kir2.1 SEM: Standard Error of measure for % inhibition at -100mV for Kir2.1 treated with 30uM of compound at pH7.4Float
4242Kir2.1 N: Number of replicates for % Inhibition at -100mV for Kir2.1 treated with 30uM of compound at pH7.4Integer
4343 (50μM**)Kir2.1 %Block: % inhibition at -100mV for Kir2.1 treated with 50uM of compound at pH7.4Float
4444Kir2.1 SEM: Standard Error of measure for % inhibition at -100mV for Kir2.1 treated with 50uM of compound at pH7.4Float
4545Kir2.1 N: Number of replicates for % Inhibition at -100mV for Kir2.1 treated with 50uM of compound at pH7.4Integer
4646 (30μM**)ROMK %Block: % inhibition at -100mV for ROMK treated with 30uM of compound at pH7.4Float
4747ROMK SEM: Standard Error of measure for % inhibition at -100mV for ROMK treated with 30uM of compound at pH7.4Float
4848ROMK N: Number of replicates for % Inhibition at -100mV for ROMK treated with 30uM of compound at pH7.4Integer
4949 (100μM**)ROMK %Block: % inhibition at -100mV for ROMK treated with 100uM of compound at pH7.4Float
5050ROMK SEM: Standard Error of measure for % inhibition at -100mV for ROMK treated with 100uM of compound at pH7.4Float
5151ROMK N: Number of replicates for % Inhibition at -100mV for ROMK treated with 100uM of compound at pH7.4Integer
5252 (300μM**)ROMK %Block: % inhibition at -100mV for ROMK treated with 300uM of compound at pH7.4Float
5353ROMK SEM: Standard Error of measure for % inhibition at -100mV for ROMK treated with 300uM of compound at pH7.4Float
5454ROMK N: Number of replicates for % Inhibition at -100mV for ROMK treated with 300uM of compound at pH7.4Integer
5555 (10μM**)ROMK %Block: % inhibition at -100mV for ROMK treated with 10uM of compound at pH8.5Float
5656ROMK SEM: Standard Error of measure for % inhibition at -100mV for ROMK treated with 10uM of compound at pH8.5Float
5757ROMK N: Number of replicates for % Inhibition at -100mV for ROMK treated with 10uM of compound at pH8.5Integer
5858 (30μM**)ROMK %Block: % inhibition at -100mV for ROMK treated with 30uM of compound at pH8.5Float
5959ROMK SEM: Standard Error of measure for % inhibition at -100mV for ROMK treated with 30uM of compound at pH8.5Float
6060ROMK N: Number of replicates for % Inhibition at -100mV for ROMK treated with 30uM of compound at pH8.5Integer
6161 (100μM**)ROMK %Block: % inhibition at -100mV for ROMK treated with 100uM of compound at pH8.5Float
6262ROMK SEM: Standard Error of measure for % inhibition at -100mV for ROMK treated with 100uM of compound at pH8.5Float
6363OMK N: Number of replicates for % Inhibition at -100mV for ROMK treated with 100uM of compound at pH8.5Integer
6464*IC50 for Kir2.1 at pH8.5FloatμM

* 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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