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BioAssay: AID 623974

Activators of the GIRK family of Potassium Channels (GIRK2/3_Confirmatory_EP)

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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AID: 623974
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (human_GIRK 2/3_Voltage_clamp (GIRK2/3_Confirmatory_EP))
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 ):           View All Data
Targets
Sequence: GIRK 2
Description ..   
Protein Family: Inward rectifier potassium channel

Gene:KCNJ6     Related Protein 3D Structures     More BioActivity Data..


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Tested Compound:
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
623909Activators of the GIRK family of Potassium Channels (GIRK_Confirmatory_CRC)Confirmatory depositor-specified cross reference: rmGlu8_hGIRK1/2_CRC (GIRK_Confirmatory_CRC) - Dry powder reorder
623911Activators of the GIRK family of Potassium Channels (GIRK1/2_Confirmatory)Other depositor-specified cross reference: hGIRK1/2_mGlu8_WaveformData (GIRK1/2_Confirmatory)
623932ML297 Competition in Radioligand Binding assays (Ricerca)Other depositor-specified cross reference: ML297 Competition in Radioligand Binding assays (Ricerca)
623952Activators of the GIRK family of Potassium Channels (minus_mGlu8_GIRK_Confirmatory_CRC)Confirmatory depositor-specified cross reference: hGIRK_1/2_Thallium_Flux_minus_mGlu8 - Dry powder reorder
623975Activators of the GIRK family of Potassium Channels (GIRK1/4_ThalliumFlux_CRC)Confirmatory depositor-specified cross reference: human_GIRK1/4_ThalliumFlux (GIRK1/4_ThalliumFlux_CRC)
623976Activators of the GIRK family of Potassium Channels (GIRK_1_4_Confirmatory_EP)Confirmatory1 depositor-specified cross reference: human_GIRK1/4_Voltage_clamp (GIRK1_4_Confirmatory_EP)
623977Activators of the GIRK family of Potassium Channels (GIRK_1_2_Confirmatory_EP)Confirmatory1 depositor-specified cross reference: human_GIRK1/2_Voltage_clamp (GIRK1_2_Confirmatory_EP)
623983Activators of the GIRK family of Potassium Channels (GIRK2/3_ThalliumFlux_CRC)Confirmatory depositor-specified cross reference: human_GIRK2/3_ThalliumFlux (GIRK2_3_ThalliumFlux_CRC)
623984Activators of the GIRK family of Potassium Channels (hKir2.1_ThalliumFlux_CRC)Confirmatory depositor-specified cross reference: human_Kir2.1_ThalliumFlux (hKir2.1_ThalliumFlux_CRC)
623986Activators of the GIRK family of Potassium Channels (hKV7.4_ThalliumFlux_CRC)Confirmatory depositor-specified cross reference: human_KV7.4_ThalliumFlux (hKV7.4_ThalliumFlux_CRC)
623988Activators of the GIRK family of Potassium Channels (hERG_ThalliumFlux_CRC)Confirmatory depositor-specified cross reference: human_hERG_kV11.1_ThalliumFlux (hERG_ThalliumFlux_CRC)
624042Activators of the GIRK family of Potassium Channels (GIRK1/2_PTX_Treated_CRC)Confirmatory depositor-specified cross reference: rmGlu8_hGIRK1/2_PTX (GIRK1/2_PTX_Treated_CRC)
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 2/3 subunit combination using voltage-clamp in order to confirm the general activity profile of GIRK activator probe, SID 135363148, established using the thallium flux assays (AID 623952, AID 623975, and AID 623983).
Protocol
Standard Whole-Cell Voltage-Clamp Screening:
The GIRK activator probe, SID 135363148, was serially diluted in DMSO manually from a 100 mM DMSO stock further diluted to make test samples in 20 mM KCl, 140 mM NaCl, 0.5 mM CaCl2, 2 mM MgCl2 and 10 mM HEPES (pH 7.4), referred to hereafter as 20K Extracellular Solution. A single cell suspension of HEK-293 cells stably transfected with hGIRK 2/3 were plated into TC-treated 35 mm polystyrene dishes at ~15% confluence and incubate in a 5% CO2 incubator at 37 degrees C. Whole-cell patch-clamp recordings were performed 24-72 h after plating. Borosilicate glass electrodes (5-7 mOhm resistance) were filled with Intracellular Solution (130 mM KCl, 20 mM NaCl, 5 mM EGTA, 5.46 mM MgCl2, and 10 mM HEPES, pH 7.4).

Whole cell currents were recorded in 20K Extracellular Solution at -70 mV using a Pico1 amplifier (Tecella) with WINWCP 4.X (University of Strathclyde) software interface. Signals were compensated for electrode capacitance, cellular capacitance and series resistance. Signals were filtered at 1 kHz, and digitized at 5 kHz. Solutions were applied to the cell with an Automate Scientific Valvelink 8.2 rapid, valve-controlled perfusion system with in-house-made local superfusion manifold. Ba2+-sensitive currents measurements were obtained by applying the non-selective Kir inhibitor, 2 mM BaCl2, in Extracellular 20K Solution in the absence and presence of test compounds and measuring the amplitude of the Ba2+-sensitive current.

Data Analysis:
Raw current amplitudes were converted to current density by dividing by each cell's membrane capacitance estimated from capacitance compensation. Barium-sensitive, steady-state currents density values measured at different compound concentrations were evaluated using a four-parameter logistic equation. Data were normalized using the current evoked by an 80 mM concentration of the non-selective GIRK activator methyl pentanediol as a standard.

REFERENCES
1. Aryal P, Dvir H, Choe S, Slesinger PA. (2009) A discrete alcohol pocket involved in GIRK channel activation. Nat Neurosci.12(8):988-95.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
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 maximally effective concentration of SID 135363148 in each cell testedFloat
11SLOPE_UCISlope Upper Confidence limitFloat
12SLOPE_LCISlope Lower Confidence limit Float
13R1_0.0017_uM (0.0017μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
14R1_0.015_uM (0.015μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
15R1_0.046_uM (0.046μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
16R1_0.137_uM (0.137μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
17R1_0.412_uM (0.412μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
18R1_1.2_uM (1.2μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
19R1_3.7_uM (3.7μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
20R1_11.0_uM (11μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
21R2_0.0017_uM (0.0017μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
22R2_0.015_uM (0.015μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
23R2_0.046_uM (0.046μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
24R2_0.137_uM (0.137μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
25R2_0.412_uM (0.412μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
26R2_1.2_uM (1.2μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
27R2_3.7_uM (3.7μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
28R2_11.0_uM (11μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
29R3_0.0017_uM (0.0017μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
30R3_0.015_uM (0.015μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
31R3_0.046_uM (0.046μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
32R3_0.137_uM (0.137μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
33R3_0.412_uM (0.412μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
34R3_1.2_uM (1.2μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
35R3_3.7_uM (3.7μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float
36R3_11.0_uM (11μM**)Barium-sensitive, steady-state current density normalized to current at 80 mM concentration of the non-selective GIRK activator methyl pentanediol (see Protocol)Float

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

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