| Assay Method Information | |
| | Automated Whole-Cell Patch Clamp Assay |
| Description: | Cell Culture and PreparationFisher rat thyroid (FRT) cells stably expressing human TMEM16A (TMEM16Aabc variant; Dr Luis Galietta, Insituto Giannina, Italy) were cultured in T-75 flasks in Hams F-12 media with Coon's modification (Sigma) supplemented with 10% (v/v) foetal bovine serum, penicillin-streptomycin (10,000 U/mL/10000 pg/mL), G-418 (750 pg/mL), L-glutamine (2 mM) and sodium bicarbonate solution (7.5% v/v). At −90% confluence cells were harvested for experiments by detachment with a 2:1 (v/v) mixture of Detachin (BMS Biotechnology) and 0.25% (w/v) trypsin-EDTA. Cells were diluted to a density of 3.5-4.5×106 cells/mL with media consisting of CHO-S-SFM (Sigma), 25 mM HEPES (Sigma) and Soy bean trypsin inhibitor (Sigma).Whole-Cell Patch Clamp RecordingFRT-TMEM16A cells were whole-cell patch clamped using an automated planar patch clamp system (Qpatch, Sophion). Briefly, once high resistance (GOhm) seals were established between the cells and the planar recording array the patch was ruptured using suction pulses to establish the whole-cell recording configuration of the patch clamp technique. The assay employed the following solutions (all reagents Sigma): Intracellular solution (mM): N-methyl-D-glucamine 130, CaCh 18.2, MgCh 1. HEPES 10, EGTA 10, BAPTA 20, Mg-ATP 2, pH 7.25, 325 mOsm with sucrose.Extracellular solution (mM): N-methyl-D-glucamine 130, CaCl2 2, MgCh 1, HEPES 10, pH 7.3, 320 mOsm with sucrose.The intracellular solution buffers intracellular calcium at levels required to give −20% activation of the maximal TMEM16A mediated current (EC20 for calcium ions). Cells were voltage clamped at a holding potential of −70 mV and a combined voltage step (to +70 mV)/ramp (−90 my to +90 mV) was applied at 0.05 Hz. After a period of current stabilisation test compounds, solubilised in 100% (v/v) DMSO and subsequently diluted into extracellular solution, were applied to generate a cumulative concentration response curve. Each concentration of test compound was incubated for 5 minutes before addition of the next concentration. After the final concentration was tested a supramaximal concentration of either a known active positive modulator or the TMEM16A inhibitor. CaCCinhAOI (Del La Fuente et al, 2008) was added to define the upper and lower limits of the assay.Compound activity was quantified by measuring the increase in current upon compound addition and expressing this as a percentage increase of baseline TMEM16A current level. Percentage increases in current were determined for each concentration and the data plotted as a function of concentration using either the Qpatch software or Graphpad Prism v6.05 providing the concentration which gave 50% of its maximal effect (EC50) and maximum efficacy (percentage of baseline increase). |
| Affinity data for this assay | |
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