Whole cell patch clamp

Whole-cell patch clamp was conducted as described (Zhou, et al. 2015). Briefly, NMDG-Cl solutions were used to isolate Cl⁻ currents from UM-SCC-1 cells overexpressing TMEM16A (bath solution was 140mM NMDG-Cl, 10mM HEPES, 1mM MgCl₂, 1.5mM CaCl₂, 5mM glucose; pipette solution was 140mM NMDG-Cl, 10mM HEPES, 1mM MgCl₂, 5mM glucose, 1mM EGTA, 1mM Mg-ATP, and 0.1mM Na-GTP). Membrane current (Im) was measured using a periodic current-voltage (I/V) protocol, which stepped the holding potential from 100 to +100 mV in 20 mV steps. Bath solution ± agonists heated to 37° C was perfused over the cell at 2 ml/min. The percent change in Im was calculated at the 60 mV holding potential. All cells tested exhibited outward rectified I/V curves with a reversal potential equal to the Cl⁻ equilibrium potential, consistent with TMEM 16A activity.