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BioAssay: AID 623909

Activators of the GIRK family of Potassium Channels (GIRK_Confirmatory_CRC)

GIRK potassium channels are a family of inward-rectifying potassium channels also known as the Kir3 family. They are expressed as homo and heterotetramers in with specific subunit combinations expressed in different brain regions and peripheral organ systems notably including the heart. Multiple lines of evidence support important roles for GIRK in a variety of physiological processes including more ..
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 Tested Compounds
 Tested Compounds
All(69)
 
 
Active(67)
 
 
Inactive(2)
 
 
 Tested Substances
 Tested Substances
All(69)
 
 
Active(67)
 
 
Inactive(2)
 
 
AID: 623909
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (rmGlu8_hGIRK 1/2_CRC (GIRK_Confirmatory_CRC))
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-03-28
Hold-until Date: 2013-03-28
Modify Date: 2013-03-29

Data Table ( Complete ):           View Active Data    View All Data
Targets
BioActive Compounds: 67
Related Experiments
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AIDNameTypeProbeComment
488969Assay for HTS of Gi/Go-linked GPCRs using mGluR8: Primary ScreeningScreening depositor-specified cross reference: mGlu8 Primary Screening Assay
504480Assay for HTS of Gi/Go-linked GPCRs using mGluR8: SummarySummary depositor-specified cross reference: mGlu8 Summary Assay
623868Development of Subtype-specific Activators of the GIRK family of Potassium Channels (mGlu8_nonGIRK_Counterscreen)Other depositor-specified cross reference: mGlu8_nonGIRK_Counterscreen
623869Development of Subtype-specific Activators of the GIRK family of Potassium Channels (rmGlu8_Gqi9_Counterscreen)Other depositor-specified cross reference: rmGlu8_Gqi9_Counterscreen
623911Activators of the GIRK family of Potassium Channels (GIRK1/2_Confirmatory)Other depositor-specified cross reference: hGIRK1/2_mGlu8_WaveformData (GIRK1/2_Confirmatory)
623952Activators of the GIRK family of Potassium Channels (minus_mGlu8_GIRK_Confirmatory_CRC)Confirmatory depositor-specified cross reference
623974Activators of the GIRK family of Potassium Channels (GIRK2/3_Confirmatory_EP)Confirmatory depositor-specified cross reference
623975Activators of the GIRK family of Potassium Channels (GIRK1/4_ThalliumFlux_CRC)Confirmatory depositor-specified cross reference
623976Activators of the GIRK family of Potassium Channels (GIRK_1_4_Confirmatory_EP)Confirmatory1 depositor-specified cross reference
623977Activators of the GIRK family of Potassium Channels (GIRK_1_2_Confirmatory_EP)Confirmatory1 depositor-specified cross reference
623983Activators of the GIRK family of Potassium Channels (GIRK2/3_ThalliumFlux_CRC)Confirmatory depositor-specified cross reference
623984Activators of the GIRK family of Potassium Channels (hKir2.1_ThalliumFlux_CRC)Confirmatory depositor-specified cross reference
623986Activators of the GIRK family of Potassium Channels (hKV7.4_ThalliumFlux_CRC)Confirmatory depositor-specified cross reference
623988Activators of the GIRK family of Potassium Channels (hERG_ThalliumFlux_CRC)Confirmatory depositor-specified cross reference
624042Activators of the GIRK family of Potassium Channels (GIRK1/2_PTX_Treated_CRC)Confirmatory depositor-specified cross reference
623932ML297 Competition in Radioligand Binding assays (Ricerca)Other same project related to Summary assay
Description:
BACKGROUND:
GIRK potassium channels are a family of inward-rectifying potassium channels also known as the Kir3 family. They are expressed as homo and heterotetramers in with specific subunit combinations expressed in different brain regions and peripheral organ systems notably including the heart. Multiple lines of evidence support important roles for GIRK in a variety of physiological processes including the control of heart rate and electrical excitability in a variety of neuronal populations. Data concerning GIRK's role in neurons suggest GIRK as a potential target for a variety of therapeutic indications including pain, epilepsy, and reward/addiction. However, a complete lack of selective and effective GIRK activators has prevented any further target validation for the many indications where GIRK activation is speculated to be of potential benefit. And, although GIRK small molecule GIRK inhibitors are known, their lack of selectivity limits their utility as research tools or potential therapeutics for such indications as atrial fibrillation.

PURPOSE:
The purpose of this assay was to determine whether confirmed hits showing activity consistent with GIRK 1/2 activators chosen using AIDs 623869, 623911, 623868 demonstrated concentration dependent efficacy.

REFERENCES
1. Niswender CM, Myers KA, Lou Q, Ayala J, Kim C, Conn PJ, Weaver CD. (2008) Development of a Novel and Direct Assay for High-Throughput Screening of Gi/o-linked GPCRs using Thallium Flux Through GIRK Channels. J. Mol Pharm, 73(4):1213-24.
Protocol
High-Throughput Screening:
Compounds selected as putative GIRK activators were purchased as dry samples from commercial vendors and dissolved to a concentration of 30 mM in DMSO. These samples were transferred to 384-well polypropylene plates and serially diluted in DMSO using and Agilent Bravo (11-points, 3-fold dilutions). Serially diluted plates were dispensed to daughter plates using a Labcyte Echo555 and diluted to 2-fold over their target assay concentration with 20 mM HEPES-buffered (pH 7.4) Hanks Balanced Salt Solution (HBSS), hereafter referred to as Assay Buffer, using a Thermo Fisher Combi. Twenty-thousand HEK-293 cells/well stably transfected with rmGlu8 and hGIRK 1/2 were plated into 384-well, black-walled, clear-bottom, poly-D-lysine coated plates in 20 uL/well Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% (v/v) dialyzed fetal bovine serum and incubated overnight in a 5% CO2 incubator at 37 degrees C. Cell culture medium was removed from cell plates using a BioTek ELx405 cell washer and replaced with 20 uL/well Assay Buffer. Twenty uL/well of 0.5 mM FluoZin-2 AM in Assay Buffer was added to cell plates using a Thermo Fisher Combi. Cell plates were incubated for ~60 min at room temperature and then the FluoZin-2 solution was removed from cell plates using the BioTek ELx405 cell washer and replaced with 20 uL/well Assay Buffer. Dye loaded and washed cell plates were transferred to a Hamamatsu FDSS 6000 and a double-addition protocol was initiated. After 10 seconds, 20 uL/well of 20 microM test compound in 0.2% DMSO and Assay Buffer was added. After 4 minutes 10 uL/well of a 5x sodium bicarbonate-based thallium stimulus buffer (20 mM HEPES pH 7.4, 135 mM NaHCO3, 2 mM CaSO4, 1 mM MgSO4, 5 mM glucose, 12 mM Tl2SO4) containing and EC20 concentration of glutamate was added and 2 more minutes of data collection followed. Fluorescence data were collected at 1 Hz.
Data Analysis:
Waveform signals (fluorescence intensity versus time normalized by dividing each fluorescence value (F) by the initial fluorescence value for each trace (F0)) were reduced to single values by subtracting the average normalized waveform from vehicle control wells from each wave on the plate followed by obtaining the slope of the change in fluorescence immediately after the addition of the thallium stimulus. Slope values were normalized as a percentage of the slope obtained by the addition of a maximally effective concentration of glutamate. Curve fits were attempted for normalized slope values using a four-parameter logistic equation.
Comment
Compounds that demonstrated concentration-dependent efficacy were considered as candidates for further development as GIRK activator probes.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1POTENCY_uM*Potency value in micromolarFloatμM
2POTENCY_UCI_uMPotency Upper Confidence Limit in micromolarFloatμM
3POTENCY_LCI_uMPotency Lower Confidence Limit in micromolarFloatμM
4TOPCalculated value for the Top curve fit parameterFloat
5TOP_UCIUpper Confidence limit for Top curve fit parameterFloat
6TOP_LCILower Confidence limit for Top curve fit parameterFloat
7BOTTOMCalculated value for the Bottom curve fit parameterFloat
8BOTTOM_UCIUpper Confidence limit for Bottom curve fit parameterFloat
9BOTTOM_LCILower Confidence limit for Bottom curve fit parameterFloat
10SLOPESlope values normalized as percentage of the slope obtained by addition of maximally effective concentration of glutamateFloat
11SLOPE_UCISlope Upper Confidence limitFloat
12SLOPE_LCISlope Lower Confidence limit Float
13R1_Value_0.00152_uM (0.00152μM**)Fluorescence intensity normalized to baseline at concentration for R1 replicate (see Protocol)Float
14R1_Value_0.00457_uM (0.00457μM**)Fluorescence intensity normalized to baseline at concentration for R1 replicate (see Protocol)Float
15R1_Value_0.0137_uM (0.0137μM**)Fluorescence intensity normalized to baseline at concentration for R1 replicate (see Protocol)Float
16R1_Value_0.04115_uM (0.04115μM**)Fluorescence intensity normalized to baseline at concentration for R1 replicate (see Protocol)Float
17R1_Value_0.1235_uM (0.1235μM**)Fluorescence intensity normalized to baseline at concentration for R1 replicate (see Protocol)Float
18R1_Value_0.3704_uM (0.3704μM**)Fluorescence intensity normalized to baseline at concentration for R1 replicate (see Protocol)Float
19R1_Value_1.111_uM (1.111μM**)Fluorescence intensity normalized to baseline at concentration for R1 replicate (see Protocol)Float
20R1_Value_3.333_uM (3.333μM**)Fluorescence intensity normalized to baseline at concentration for R1 replicate (see Protocol)Float
21R1_Value_10.0_uM (10μM**)Fluorescence intensity normalized to baseline at concentration for R1 replicate (see Protocol)Float
22R1_Value_30.0_uM (30μM**)Fluorescence intensity normalized to baseline at concentration for R1 replicate (see Protocol)Float
23R1_Value_90.0_uM (90μM**)Fluorescence intensity normalized to baseline at concentration for R1 replicate (see Protocol)Float
24R2_Value_0.00152_uM (0.00152μM**)Fluorescence intensity normalized to baseline at concentration for R2 replicate (see Protocol)Float
25R2_Value_0.00457_uM (0.00457μM**)Fluorescence intensity normalized to baseline at concentration for R2 replicate (see Protocol)Float
26R2_Value_0.0137_uM (0.0137μM**)Fluorescence intensity normalized to baseline at concentration for R2 replicate (see Protocol)Float
27R2_Value_0.04115_uM (0.04115μM**)Fluorescence intensity normalized to baseline at concentration for R2 replicate (see Protocol)Float
28R2_Value_0.1235_uM (0.1235μM**)Fluorescence intensity normalized to baseline at concentration for R2 replicate (see Protocol)Float
29R2_Value_0.3704_uM (0.3704μM**)Fluorescence intensity normalized to baseline at concentration for R2 replicate (see Protocol)Float
30R2_Value_1.111_uM (1.111μM**)Fluorescence intensity normalized to baseline at concentration for R2 replicate (see Protocol)Float
31R2_Value_3.333_uM (3.333μM**)Fluorescence intensity normalized to baseline at concentration for R2 replicate (see Protocol)Float
32R2_Value_10.0_uM (10μM**)Fluorescence intensity normalized to baseline at concentration for R2 replicate (see Protocol)Float
33R2_Value_30.0_uM (30μM**)Fluorescence intensity normalized to baseline at concentration for R2 replicate (see Protocol)Float
34R2_Value_90.0_uM (90μM**)Fluorescence intensity normalized to baseline at concentration for R2 replicate (see Protocol)Float
35R3_Value_0.00152_uM (0.00152μM**)Fluorescence intensity normalized to baseline at concentration for R3 replicate (see Protocol)Float
36R3_Value_0.00457_uM (0.00457μM**)Fluorescence intensity normalized to baseline at concentration for R3 replicate (see Protocol)Float
37R3_Value_0.0137_uM (0.0137μM**)Fluorescence intensity normalized to baseline at concentration for R3 replicate (see Protocol)Float
38R3_Value_0.04115_uM (0.04115μM**)Fluorescence intensity normalized to baseline at concentration for R3 replicate (see Protocol)Float
39R3_Value_0.1235_uM (0.1235μM**)Fluorescence intensity normalized to baseline at concentration for R3 replicate (see Protocol)Float
40R3_Value_0.3704_uM (0.3704μM**)Fluorescence intensity normalized to baseline at concentration for R3 replicate (see Protocol)Float
41R3_Value_1.111_uM (1.111μM**)Fluorescence intensity normalized to baseline at concentration for R3 replicate (see Protocol)Float
42R3_Value_3.333_uM (3.333μM**)Fluorescence intensity normalized to baseline at concentration for R3 replicate (see Protocol)Float
43R3_Value_10.0_uM (10μM**)Fluorescence intensity normalized to baseline at concentration for R3 replicate (see Protocol)Float
44R3_Value_30.0_uM (30μM**)Fluorescence intensity normalized to baseline at concentration for R3 replicate (see Protocol)Float
45R3_Value_90.0_uM (90μM**)Fluorescence intensity normalized to baseline at concentration for R3 replicate (see Protocol)Float

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH076398

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