Figure 3. Ion flux through H206A Orai_cryst in liposomes.

(a) Schematic of the fluorescence-based flux assay. Vesicles containing WT or H206A Orai_cryst or those prepared without protein (empty vesicles) were loaded with 150 mM KCl and were diluted 50-fold into flux buffer containing a fluorescent pH indicator (ACMA) and 150 mM N-methyl-D-glucamine (NMDG) to establish a K⁺ gradient (Materials and methods). After stabilization of the fluorescence signal (150 s), a proton ionophore (CCCP) was added. An electric potential arising from K⁺ efflux was used to drive the uptake of protons, which quenches the fluorescence of ACMA. A red ‘X’ indicates that ACMA is not membrane-permeable in the protonated form. (b) K⁺ flux measurements for WT and H206A Orai_cryst. The time-dependent decrease in fluorescence observed for H206A Orai_cryst after the addition of CCCP is indicative of K⁺ flux. Valinomycin (val) was added after 990 s to render all vesicles permeable to K⁺ and establish a baseline fluorescence. Traces were normalized by dividing by the initial fluorescence value, which was within ±10% for each experiment. (c) K⁺ flux through H206A Orai_cryst is inhibited by Ca²⁺, Mg²⁺ and Gd³⁺.