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BioAssay: AID 623898

SAR analysis for the identification of selective inhibitors of the two-pore domain potassium channel (KCNK9): Automated Electrophysiology

The purpose of this assay is to evaluate dose dependent compound inhibition of KCNK9 two pore domain potassium channels using an automated electrophysiology voltage clamp technique. The inhibitory effects of test compounds on KCNK9 channels were evaluated in electrophysiologcal assays using a QPatch16 automated electrophysiology instrument. Whole cell recordings were made from HEK293 cells stably more ..
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 Tested Compounds
 Tested Compounds
All(54)
 
 
Active(50)
 
 
Inactive(4)
 
 
 Tested Substances
 Tested Substances
All(56)
 
 
Active(52)
 
 
Inactive(4)
 
 
AID: 623898
Data Source: Johns Hopkins Ion Channel Center (JHICC_KCNK9_inh_QPatch)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-03-28
Hold-until Date: 2013-03-27
Modify Date: 2013-03-27

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: potassium channel subfamily K member 9 [Homo sapiens]
Description ..   

     More BioActivity Data..
BioActive Compounds: 50
Related Experiments
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AIDNameTypeComment
488964Summary of probe development for inhibitors of the two-pore domain potassium channel KCNK9Summarydepositor-specified cross reference
488922Primary cell-based screen for identification of compounds that inhibit the two-pore domain potassium channel KCNK9Screeningsame project related to Summary assay
492992Confirmatory screen for identification of compounds that inhibit the two-pore domain potassium channel (KCNK9)Screeningsame project related to Summary assay
492993Specificity screen against Kir2.1 for compounds that modulate the two-pore domain potassium channel (KCNK9)Screeningsame project related to Summary assay
492997Second counter screen for compounds that modulate the two-pore domain potassium channel (KCNK9)Screeningsame project related to Summary assay
504846SAR analysis for compounds that inhibit the two-pore domain potassium channel KCNK9Confirmatorysame project related to Summary assay
504902SAR Analysis for the identification of selective inhibitors of KCNK9 against parental cells: FluxOR Assay CRCConfirmatorysame project related to Summary assay
504920SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in hERG expressing cells: FluxOR Assay CRC for hERG SpecificityConfirmatorysame project related to Summary assay
504922SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in Kir2.1 expressing cells: FluxOR Assay CRCConfirmatorysame project related to Summary assay
540321SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in Kir2.1 expressing cells: FluxOR Assay CRC 2Confirmatorysame project related to Summary assay
540322SAR analysis for compounds that inhibit the two-pore domain potassium channel KCNK9 IIConfirmatorysame project related to Summary assay
540323SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in hERG expressing cells: FluxOR Assay CRC 2Confirmatorysame project related to Summary assay
540324SAR Analysis for the identification of selective inhibitors of KCNK9 against parental cells: FluxOR Assay CRC 2Confirmatorysame project related to Summary assay
588724SAR analysis for compounds that inhibit the two-pore domain potassium channel KCNK9 IIIConfirmatorysame project related to Summary assay
588741SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in hERG expressing cells: FluxOR Assay CRC 3Confirmatorysame project related to Summary assay
588759SAR Analysis for the identification of selective inhibitors of KCNK9 against parental cells: FluxOR Assay CRC 3Confirmatorysame project related to Summary assay
588760SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in Kir2.1 expressing cells: FluxOR Assay CRC 3Confirmatorysame project related to Summary assay
588761SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in KCNQ2 expressing cells: FluxOR Assay CRCConfirmatorysame project related to Summary assay
588776SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in KCNK3 expressing cells: FluxOR Assay CRC 1Confirmatorysame project related to Summary assay
588798SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in KCNQ2 expressing cells: FluxOR Assay CRC 3Confirmatorysame project related to Summary assay
588800SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in KCNQ2 expressing cells: FluxOR Assay CRC 2Confirmatorysame project related to Summary assay
623881SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in Kir2.1 expressing cells: FluxOR Assay CRC 4Confirmatorysame project related to Summary assay
623883SAR Analysis for the identification of inhibitors of the two-pore domain potassium channel KCNK9 - Selectivity assay against hERG: FluxOR Assay CRC 4Confirmatorysame project related to Summary assay
623884SAR Analysis for the identification of selective inhibitors of KCNK9 against parental cells: FluxOR Assay CRC 4Confirmatorysame project related to Summary assay
623886SAR Analysis for the identification of inhibitors of the two-pore domain potassium channel KCNK9 - Selectivity assay against KCNQ2: FluxOR Assay CRCConfirmatorysame project related to Summary assay
623887SAR analysis for compounds that inhibit the two-pore domain potassium channel KCNK9 4Confirmatorysame project related to Summary assay
623894SAR Analysis for the identification of inhibitors of the two-pore domain potassium channel KCNK9 - Selectivity assay against KCNK3: FluxOR Assay CRC 3Confirmatorysame project related to Summary assay
623912SAR Analysis for the identification of inhibitors of the two-pore domain potassium channel KCNK9 - Selectivity assay against KCNK3: FluxOR Assay CRC 2Confirmatorysame project related to Summary assay
623913SAR Analysis for the identification of inhibitors of the two-pore domain potassium channel KCNK9 - Selectivity assay against KCNQ2: FluxOR Assay CRC 4Confirmatorysame project related to Summary assay
623914SAR Analysis for the identification of inhibitors of the two-pore domain potassium channel KCNK9 - Selectivity assay against KCNQ2: FluxOR Assay CRC 2Confirmatorysame project related to Summary assay
623915SAR analysis for the identification of selective inhibitors of the two-pore domain potassium channel (KCNK9) -selectivity assay against KCNK3: Automated ElectrophysiologyConfirmatorysame project related to Summary assay
624121SAR analysis for the identification of selective inhibitors of the two-pore domain potassium channel (KCNK9): Manual ElectrophysiologyOthersame project related to Summary assay
Description:
Principle of the assay
The purpose of this assay is to evaluate dose dependent compound inhibition of KCNK9 two pore domain potassium channels using an automated electrophysiology voltage clamp technique. The inhibitory effects of test compounds on KCNK9 channels were evaluated in electrophysiologcal assays using a QPatch16 automated electrophysiology instrument. Whole cell recordings were made from HEK293 cells stably expressing KCNK9 channels. For each experiment, channel function was tested first in 4mM potassium solution. After stabilization, external buffer was switched to one containing 140mM potassium. Following external buffer switch, the effects of test compounds were measured in the presence of 140mM potassium. Application of 2 mM Ba2+ in at pH5.8 solution was used as reference for 100% inhibition for each cell tested.
Protocol
Stable HEK 293 cells with inducible KCNK9 expression were seeded and induced (0.5ug/ml Tetracycline) in a 15 cm dish two days before testing on a QPatch16 automated patch clamp system.
Cells were detached into single cell suspension and allowed to recover for at least 1hr before running in single-hole mode on QPatch16 instrument. Whole cell currents were filtered at 1 kHz (4-pole Bessel) and sampled at 5 kHz. The series resistance was compensated by 20% with a cut-off frequency of 0.2 kHz. KCNK9 currents were elicited by voltage ramp protocols consisting of 50ms at -80 mV, 50ms at -100 mV, followed by a 600 ms ramp to +20 mV, 5 ms at +20 mV, and then back to -80 mV for 40 ms. The voltage protocol was applied every 5 sec from a holding potential of -80 mV. After a 3 minute stabilization period in external bath solution containing 4 mM potassium, the bath solution was exchanged to the 140 mM potassium solution. After a 3 minute baseline period in 140 mM potassium external solution (Imax), cumulative dose response experiments were performed by applying compounds in a dual addition mode in which external solution was fully exchanged twice at each compound concentration. Compound effects were determined after a 5 minute incubation period at each concentration (Itest). A reference 140 mM potassium solution containing 2 mM barium with pH 5.8 was then applied to fully block KCNK9 channels and determine maximal possible block (Ibaseline).
Current amplitudes were measured at -30 mV. Fractional channel block at each compound concentration was calculated as ((Imax-Itest)/(Imax-Ibaseline)). IC50 values were determined for each cell by fitting fractional block values versus compound concentration with a Hill equation with the maximal and minimal values fixed to one and zero, respectively, and the slope fixed to one.
Solutions:
Internal solution (in mM): 140 KCl, 2 MgCl2, 10 EGTA, 5 MgATP, and 10 HEPES, pH 7.30 with KOH.
4K External solution (in mM): 150 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, and 10 HEPES, pH 7.40, with NaOH.
140K external solution (in mM): 14 NaCl, 140 KCl, 1.8 CaCl2, 1 MgCl2, and 10 HEPES, pH 7.40, with NaOH.
1. Outcome assignment:
If the test compound causes greater than 50% inhibition of KCNK9 at 10 microM, the compound is considered to be active (Outcome=2). Otherwise, the compound is designated as inactive (Outcome=1).
2. Score assignment:
An inactive test compound is assigned the score of 0. An active test compound is assigned a score between 1 and 100 according to the following criteria: 25 for compounds with 10 microM3. Result Definitions:
A. IC50
B. Number of cells included in each determination.
List of reagents:
1. Stable HEK 293 cell line with inducible KCNK9 expression
2. PBS: pH7.4 (Invitrogen Cat#10010023)
3. Medium: (Dulbecco's Modification of Eagle's Medium (DMEM)-L-Glutamine (Mediatech, Cat#10-013-CV)
4. Fetal Bovine Serum (Gibco, Cat#26140)
5. 100x Penicillin-Streptomycin (Mediatech, Cat#30-001-CI)
6. Detachin (Genlantis , Cat#T100110 )
7. CHO-S-SFMII (Invitrogen Cat#12052)
8. Tetracycline Hydrochloride (Sigma,Cat#T7660)
9. QPlate16 (Sophion Bioscience, Ballerup, Denmark)
10. Blasticidin S (Research Products Internationl Corp., Cat#B12150)
11. Hygromycin (Mediatech, Cat#30-240-CR)
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: HEK293
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50*IC50FloatμM
2NNumber of cellsInteger

* Activity Concentration.
Additional Information
Grant Number: R03 MH090849-01

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