Figure 5.
Orai1-GCaMP6f activity differs between puncta. HEK 293A cells were cotransfected with Orai1-GCaMP6f and mCherry-STIM1. (A and B) TIRF micrographs of mCherry-STIM1 (A) and Orai1-GCaMP6f (B) fluorescence at +20 mV holding potential. (C) Difference between Orai1-GCaMP6f fluorescence at −100 and +20 mV. White arrowheads in A–D indicate puncta with little or no change in fluorescence. (D) Map of Orai1-GCaMP6f plateau ΔF/F0 upon stepping from +20 to −100 mV. (E) Map of A-index. Orange arrowheads indicate puncta with high (1) and low (2) A-indices. (F) Whole-cell current for the cell shown in (A–E). (G and H) Orai1-GCaMP6f fluorescence traces corresponding to puncta 1 and 2. (I) Map of relative STIM1/Orai1 ratio; each circle represents an individual punctum. Scale bar in A is 10 µm, applies to B–E and I. Images and trace correspond to cell B (see Fig. S3).

Orai1-GCaMP6f activity differs between puncta. HEK 293A cells were cotransfected with Orai1-GCaMP6f and mCherry-STIM1. (A and B) TIRF micrographs of mCherry-STIM1 (A) and Orai1-GCaMP6f (B) fluorescence at +20 mV holding potential. (C) Difference between Orai1-GCaMP6f fluorescence at −100 and +20 mV. White arrowheads in A–D indicate puncta with little or no change in fluorescence. (D) Map of Orai1-GCaMP6f plateau ΔF/F0 upon stepping from +20 to −100 mV. (E) Map of A-index. Orange arrowheads indicate puncta with high (1) and low (2) A-indices. (F) Whole-cell current for the cell shown in (A–E). (G and H) Orai1-GCaMP6f fluorescence traces corresponding to puncta 1 and 2. (I) Map of relative STIM1/Orai1 ratio; each circle represents an individual punctum. Scale bar in A is 10 µm, applies to B–E and I. Images and trace correspond to cell B (see Fig. S3).

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