Activators of the GIRK family of Potassium Channels (minus_mGlu8_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: 4
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 test the concentration-dependent efficacy of putative GIRK activators in a cell line that did not contain a heterologously over-expressed GPCR (e.g. mGlu8)
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.
The four, structurally distinct compounds that showed concentration-dependent efficacy when tested in AID 624042 were selected for further testing. These four compounds (PubChem SIDs: 849688, 4244565, 4258786, 22410373) were serially diluted and transferred to daughter plates as described in AID 623909. In addition, all novel compounds synthesized as part of the development of the GIRK activator probe, SID 135363148, were tested in this assay. These samples were diluted from 10 mM DMSO stocks using the protocol described in AID 623909.
Twenty-thousand HEK-293 cells/well stably transfected with 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 mM 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.
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 compound, SID 4258786. When testing the GIRK subunit combination-selectivity of the probe compound, SID 135363148, an 80 mM concentration of the non-selective GIRK activator, methyl pentanediol (MPD) was used for normailzation. 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)