Figure 4.

Chloride-selective, Ca²⁺-dependent channels are formed by BEST1₃₄₅ and BEST1_S358E. (A–C) Example I-V relationships are shown for voltages stepped from −100 to +100 mV for BEST1_WT (A; adapted with permission from Vaisey et al., 2016), BEST1₃₄₅ (B), and BEST1_S358E (C) for indicated conditions (cis/trans KCl concentrations in millimolar and 300 nM Ca²⁺ [+Ca²⁺] or 10 mM EGTA [−Ca²⁺]). (D–F) Reactivation of BEST1 currents by increasing the [Ca²⁺]_free. After observing currents in symmetrical solutions, 10 mM EGTA was added to the cis side to deactivate channels with their cytosolic side facing that direction. Then, using perfusion, the [Ca²⁺]_free in the trans solution was varied from ∼0 to 350 nM with a Ca–EGTA buffer system (Materials and methods). The Ca²⁺ response curves for BEST1_WT (D; adapted from Vaisey et al., 2016), BEST1₃₄₅ (E), and BEST1_S358E (F) are plotted as average currents observed at −100 mV for different [Ca²⁺]_free as a fraction of current recorded from the [Ca²⁺]_free = 350 nM condition (I_max). Three separate experiments were used to calculate SD, and the data were fit to a one-site binding model.