Activators of the GIRK family of Potassium Channels (hERG_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 ..
BioActive Compound: 1
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 1/2 activators in a cell line that expressed the voltage-gated potassium channel, hERG in order to determine the selectivity of the GIRK activator probe, SID 135363148, for a more distantly related potassium channel family member.
The GIRK activator probe, SID 135363148, was serially diluted in DMSO manually from a 100 mM DMSO stock and transferred to Echo compatible source plates by hand. Compounds were then transferred to daughter plates as described in AID 623909 with a high-potassium Assay Buffer containing : 88 mM NaCl, 60 mM KCl, 1.3 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 20 mM HEPES pH 7.4, referred to hereafter as 60K Assay Buffer.
Twenty-thousand T-REx-293 cells/well stably transfected with hKv7.4 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. The cell plates were 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 uM 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 uM test compound in 0.2% DMSO and Assay Buffer was added. After 20 minutes, 10 uL/well of a 5x sodium gluconate-based thallium stimulus buffer (125 mM sodium gluconate, 2.4 mM Tl2SO4, 1 mM MgSO4, 1.8 mM calcium gluconate, 5 mM glucose, 10 mM HEPES, pH 7.3) and 2 more minutes of data collection followed. Fluorescence data were collected at 1/5 Hz for all but the 10 seconds immediately preceding an all time points following the thallium addition which were obtained 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 wells treated with a maximally effective concentration of the hERG inhibitor, dofetilide, 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 from vehicle-treated control wells. Curve fits were attempted for normalized slope values from triplicate wells using a four-parameter logistic equation.
1. Schmalhofer WA, Swensen AM, Thomas BS, Felix JP, Haedo RJ, Solly K, Kiss L, Kaczorowski GJ, Garcia ML.(2010) A pharmacologically validated, high-capacity, functional thallium flux assay for the human Ether-a-go-go related gene potassium channel. Assay Drug Dev Technol. 8(6):714-26.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)