Assay Method Information | |
| TREK1 Assay |
Description: | The hTREK1 stable cell lines were seeded at a density of 10 000 cells/well in a 12-well plate. Whole-cell membrane currents were amplified using the Axopatch 200A patch-clamp system. Currents were elicited by 1-second voltage ramps descending from +50 mV to -150 mV (from a holding potential of -70 mV). Data acquisition was controlled by pclamp 10.2 software (Molecular Devices, Sunnyvale,CA, USA). A Digidata 1322A interface was used to convert digital-analog signals between amplifier and computer. Data were sampled at 5 kHz and filtered at 2 kHz. Cell membrane capacitance was measured using the 'membrane test' protocolbuilt into pClampex. The bath solution contained (in mM) 150 NaCl, 10 HEPES, 3 KCl, 2 CaCl2, 2 MgCl2, and 5.5 glucose adjusted to pH 7.3 with NaOH. The pipette solution contained (in mM) 145 KCl, 0.5 CaCl2, 10 HEPES, 4 Mg-ATP, 0.3 Na3-GTPadjusted to pH 7.2 with KOH. All experiments were performed at a room temperature of 20-22 °C. TREK1 currents were recorded in the presence of each 10 μm hit compounds. The block percentage of inhibitors was obtained by measuring the percentage of remaining current. |
Affinity data for this assay | |
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