Fig. 4.

Cl⁻ flux analysis of BEST1_TripleA and Ca²⁺ clasp mutants. (A–D) For each assay, proteoliposomes were diluted 100-fold into flux assay buffer containing either 1 mM EGTA (- Ca²⁺) or 1 mM EGTA-CaSO₄ (+ Ca²⁺, [Ca²⁺]_free ∼ 2 μM). The arrow indicates when the assay was initiated by the addition of the proton ionophore CCCP. (A) Cl⁻ flux of BEST1_WT is Ca²⁺ dependent. (B) Cl⁻ flux through BEST1_TripleA is observed regardless of the presence or absence of Ca²⁺. (C) Mutation of aspartate amino acids that coordinate Ca²⁺ in the Ca²⁺ clasp to alanine (D301A/D304A) abolishes Cl⁻ flux and makes the channel unresponsive to 2 µM Ca²⁺. (D) Combining the D301A/D304A and the BEST1_TripleA mutations produces a constitutively active channel that is not responsive to the Ca²⁺ concentration. The increased flux observed for BEST1_TripleA relative to BEST1_WT is partially due to the nature of the assay, where Ca²⁺ is only added to the outside of the vesicles. For BEST1_WT, only those channels with their Ca²⁺ sensors facing outside are reactivated, whereas Cl⁻ flux through BEST1_TripleA channels in both orientation is detected because these channels conduct Cl⁻ in a Ca²⁺-independent manner.