Bookmark and Share
BioAssay: AID 623975

Activators of the GIRK family of Potassium Channels (GIRK1/4_ThalliumFlux_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 ..
_
   
 Tested Compounds
 Tested Compounds
All(1)
 
 
Active(1)
 
 
 Tested Substances
 Tested Substances
All(1)
 
 
Active(1)
 
 
AID: 623975
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (human_GIRK 1/4_ThalliumFlux (GIRK1/4_ThalliumFlux_CRC))
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-04-03
Hold-until Date: 2013-04-02
Modify Date: 2013-04-02

Data Table ( Complete ):           Active    All
Targets
Sequence: GIRK 1
Description ..   
Protein Family: Inward rectifier potassium channel

Gene:KCNJ3     Related Protein 3D Structures     More BioActivity Data..


more...  
BioActive Compound: 1
Depositor Specified Assays
Show more
AIDNameTypeProbeComment
504480Assay for HTS of Gi/Go-linked GPCRs using mGluR8: Summarysummary mGlu8 Summary Assay
488969Assay for HTS of Gi/Go-linked GPCRs using mGluR8: Primary Screeningscreening mGlu8 Primary Screening Assay
623911Activators of the GIRK family of Potassium Channels (GIRK1/2_Confirmatory)other hGIRK1/2_mGlu8_WaveformData (GIRK1/2_Confirmatory)
623869Development of Subtype-specific Activators of the GIRK family of Potassium Channels (rmGlu8_Gqi9_Counterscreen)other rmGlu8_Gqi9_Counterscreen
623868Development of Subtype-specific Activators of the GIRK family of Potassium Channels (mGlu8_nonGIRK_Counterscreen)other mGlu8_nonGIRK_Counterscreen
623909Activators of the GIRK family of Potassium Channels (GIRK_Confirmatory_CRC)confirmatory rmGlu8_hGIRK1/2_CRC (GIRK_Confirmatory_CRC) - Dry powder reorder
623952Activators of the GIRK family of Potassium Channels (minus_mGlu8_GIRK_Confirmatory_CRC)confirmatory hGIRK_1/2_Thallium_Flux_minus_mGlu8 - Dry powder reorder
624042Activators of the GIRK family of Potassium Channels (GIRK1/2_PTX_Treated_CRC)confirmatory rmGlu8_hGIRK1/2_PTX (GIRK1/2_PTX_Treated_CRC)
623983Activators of the GIRK family of Potassium Channels (GIRK2/3_ThalliumFlux_CRC)confirmatory human_GIRK2/3_ThalliumFlux (GIRK2_3_ThalliumFlux_CRC)
623984Activators of the GIRK family of Potassium Channels (hKir2.1_ThalliumFlux_CRC)confirmatory human_Kir2.1_ThalliumFlux (hKir2.1_ThalliumFlux_CRC)
623986Activators of the GIRK family of Potassium Channels (hKV7.4_ThalliumFlux_CRC)confirmatory human_KV7.4_ThalliumFlux (hKV7.4_ThalliumFlux_CRC)
623988Activators of the GIRK family of Potassium Channels (hERG_ThalliumFlux_CRC)confirmatory human_hERG_kV11.1_ThalliumFlux (hERG_ThalliumFlux_CRC)
623977Activators of the GIRK family of Potassium Channels (GIRK_1_2_Confirmatory_EP)confirmatory1 human_GIRK1/2_Voltage_clamp (GIRK1_2_Confirmatory_EP)
623976Activators of the GIRK family of Potassium Channels (GIRK_1_4_Confirmatory_EP)confirmatory1 human_GIRK1/4_Voltage_clamp (GIRK1_4_Confirmatory_EP)
623974Activators of the GIRK family of Potassium Channels (GIRK2/3_Confirmatory_EP)confirmatory human_GIRK2/3_Voltage_clamp (GIRK2/3_Confirmatory_EP)
623932ML297 Competition in Radioligand Binding assays (Ricerca)other ML297 Competition in Radioligand Binding assays (Ricerca)
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 test the concentration-dependent efficacy of putative GIRK 1/2 activators in a cell line that expressed the closely related GIRK 1/4 subunit combination in order to determine intra-GIRK subunit combination selectivity of the GIRK activator probe, SID 135363148.
Protocol
High-throughput Screening:
The GIRK activator probe, SID 135363148, was serially diluted and transferred to daughter plates as described in AID 623909. Twenty-thousand HEK-293 cells/well stably transfected with hGIRK 1/4 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 microL/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, 2.4 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 an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD). Curve fits were attempted for normalized slope values from triplicate wells using a four-parameter logistic equation.

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.
Result Definitions
Show more
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_UCLUpper Confidence limit for Top curve fit parameterFloat
6TOP_LCLLower 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 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD). Float
11SLOPE_UCISlope Upper Confidence limitFloat
12SLOPE_LCISlope Lower Confidence limit Float
13R1_Value_0.00051_uM (0.00051μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
14R1_Value_0.00152_uM (0.00152μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
15R1_Value_0.00457_uM (0.00457μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
16R1_Value_0.0137_uM (0.0137μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
17R1_Value_0.04115_uM (0.04115μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
18R1_Value_0.1235_uM (0.1235μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
19R1_Value_0.3704_uM (0.3704μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
20R1_Value_1.111_uM (1.111μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
21R1_Value_3.333_uM (3.333μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
22R1_Value_10.0_uM (10μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
23R1_Value_30.0_uM (30μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
24R2_Value_0.00051_uM (0.00051μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
25R2_Value_0.00152_uM (0.00152μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
26R2_Value_0.00457_uM (0.00457μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
27R2_Value_0.0137_uM (0.0137μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
28R2_Value_0.04115_uM (0.04115μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
29R2_Value_0.1235_uM (0.1235μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
30R2_Value_0.3704_uM (0.3704μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
31R2_Value_1.111_uM (1.111μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
32R2_Value_3.333_uM (3.333μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
33R2_Value_10.0_uM (10μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
34R2_Value_30.0_uM (30μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
35R3_Value_0.00051_uM (0.00051μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
36R3_Value_0.00152_uM (0.00152μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
37R3_Value_0.00457_uM (0.00457μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
38R3_Value_0.0137_uM (0.0137μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
39R3_Value_0.04115_uM (0.04115μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
40R3_Value_0.1235_uM (0.1235μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
41R3_Value_0.3704_uM (0.3704μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
42R3_Value_1.111_uM (1.111μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
43R3_Value_3.333_uM (3.333μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
44R3_Value_10.0_uM (10μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float
45R3_Value_30.0_uM (30μM**)Fluorescence intensity values (F) corrected to baseline Fluorescence (F0) (see Protocol) and normalized to cells treated with an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD).Float

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

Data Table (Concise)
Classification
PageFrom: