Figure 3. His¹³⁴ mutations switch Orai1 between generating constitutively-active, store-dependent, or suppressed currents.

(A) Time course of whole cell patch-clamp recordings of HEK cells overexpressing Orai1 His¹³⁴ (Ala, Cys, Glu, Gly, Met, Val) mutants. These mutants show constitutive activity (n = 7 – 14 cells, from at least 2 individual transfections). (B) Analogous experiments to those conducted on Orai1 His¹³⁴ mutants as in (A) or wild-type Orai1 co-expressed with STIM1 (n = 7 – 14 cells, from at least 2 individual transfections). (C,D) Time courses for store-dependent activation for Orai1 His¹³⁴ (Gln, Asn) mutants or wild-type Orai1 coexpressed with STIM1 (D) or reduced currents generated by Orai1 His¹³⁴ (Phe, Trp, Tyr, Leu) coexpressed with STIM1 (n = 6 – 14 cells, from at least 2 individual transfections). All currents were recorded at -86 mV in a 10 mM Ca²⁺-containing bath solution and store-depletion was induced by 20 mM EGTA in the pipette. (E) Representative images of CFP-OASF and YFP-Orai1 wild-type (upper panel) and Orai1-H134W mutant (lower panel). Scale bar, 10 µm. (F) Intensity plots for regions close to the plasma membrane measured at the dashed lines for individual cells(E) (n= 4 – 16 cells, from at least 2 individual transfections). (G) Representative current-voltage relationships for maximum currents of Orai1 His¹³⁴ mutants of (A) are shown.