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 ..
BioActive Compounds: 67
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.
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.
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.
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.
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.
Compounds that demonstrated concentration-dependent efficacy were considered as candidates for further development as GIRK activator probes.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)