| Assay Method Information | |
| | Electrophysiology Assay |
| Description: | HEK293 cells were cultured in DMEM with 4.5 g L-1 glucose, L-glutamine, and sodium pyruvate (Mediatech) containing 10% (v/v) FBS (Axenia BioLogix) and 1% (v/v) penicillin-streptomycin, at 37° C. and with 5% CO2. Cells were lifted with trypsin-EDTA (Life Technologies) and passaged to 6-well plates (Warner Instruments) 3-4 d before recording. Transient transfection was performed with Lipofectamine 2000 (Thermo Fisher Scientific) 2 days before recording. The plasmids of human Kir6.2 and SUR1 were the gift from Dr. Show-Ling Shyng (Oregon Health and Science University), and we fused mCherry fluorescent protein to the C-terminus of Kir6.2. The vector ratio for co-transfection of Kir6.2 to SUR1 was 1:10. Before recording, cells were lifted with trypsin-EDTA, kept in modified Tyrode's saline (140 mM NaCl, 5 mM KCl, 10 mM HEPES, 2 mM CaCl2), 1 mM MgCl2, 10 mM glucose, pH 7.2 ˜ 7.3 with HCl), and were used within 8 hours. For recording, an aliquot of cells was transferred to a recording chamber on a Nikon-TE2000 Inverted Scope (Nikon Instruments), and transfection was confirmed with fluorescent microscopy. The pipette solution contained: 145 mM KCl, 1 mM MgCl2, 5 mM EGTA, 2 mM CaCl2), 20 mM HEPES, 0.3 mM K2-ATP and 0.3 mM K2-ADP. Patch borosilicate pipettes (Sutter Instrument) were pulled from a Sutter P-97 puller with resistances of 2-3 MΩ. Data were acquired using a Axopatch 200B amplifier controlled by Clampex 10.2 via Digidata 1550 Å (Axon Instruments), sampled at 10 kHz, filtered at 2 kHz. Membrane capacitance was around 15 pF. Rs was around 5 MΩ. The membrane potential was held at −80 mV and a ramp to +80 mV (1 mV/ms) was applied every second. Bath was switched to 150 mM KCl, 10 mM HEPES, 2 mM CaCl2), and the chemical to be tested was dissolved in it and puffed with VC3-8xP pressurized perfusion system (ALA Science). |
| Affinity data for this assay | |
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