Activators of the GIRK family of Potassium Channels (GIRK_1_4_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 ..
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 closely related GIRK 1/4 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).
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 1/4 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.
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.
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)