TMEM16A Cl- Current Activation Protocols

Table 1 lists the composition of external solutions (ES) and internal solutions (IS) used to record the Cl⁻ current activated by intracellular Ca²⁺ or V_m (I_Cl). IS with buffered Ca²⁺ was prepared using EGTA and Ca²⁺; the free [Ca²⁺]_i was estimated using MAXCHELATOR (maxchelator.stanford.edu). ES-140Cl and IS-40Cl/0.2Ca are control solutions. An IS with 5 µM [Ca²⁺]_i (IS-40Cl/5Ca) was used to activate fully TMEM16A and record I_Cl with long depolarizations in cells bathed with ES-140Cl. To analyse the activation of TMEM16A in zero [Ca²⁺]_i we used IS-40Cl/0Ca containing 25.2 mM EGTA and 50 mM HEPES. The Cl⁻ dependence of activation was studied using ES containing 140, 109, 70.5, 30, 10 or 1.5 mM [Cl⁻]_o. The pH of each solution was adjusted to 7.3 with TEA-OH or NaOH. ES was made hypertonic relative to the internal solutions to avoid activation of volume-sensitive Cl⁻ channels present in HEK293 cells [23 (link)]. Osmolarity was adjusted by adding D-mannitol and measured using the vapour pressure point method (VAPRO, Wescor Inc., South Logan, UT, USA). All chemicals were purchased from Sigma-Aldrich (Co. St. Louis, MO, USA.).