Figure 1. Store depletion results in FRET between Orai1–CFP and STIM1–YFP.

A, HEK293 cells expressing Orai1–CFP and STIM1–YFP were imaged in 2 mm Ca²⁺ (pre-TG) and 300 s following the addition of 1 μm TG in Ca²⁺-free Ringer solution (post-TG). TG-induced store depletion alters the distribution of STIM1–YFP and Orai1–CFP from diffuse expression to overlapping puncta at the cell periphery. This is accompanied by increase in FRET. The graph on the right plots the time course of E_app between Orai1–CFP and STIM1–YFP in a different cell following depletion of Ca²⁺ stores. B, the increase in FRET between Orai1–CFP and STIM1–YFP triggered by store depletion is reversed by washout of the reversible SERCA-pump inhibitor, CPA. Trace shows E_app in a single HEK293 cell and is representative of 5/5 experiments. A Ringer solution containing a high concentration of extracellular Ca²⁺ (20 mm) was employed to facilitate store refilling following washout of CPA. C, an EF-hand STIM1 mutation, D76ASTIM1, abolishes the FRET change with store depletion. The trace shows a E_app measurement in a single cell expressing D76ASTIM1–YFP + Orai1–CFP. D, summary of 3-cube E_app values in cells expressing WT STIM1–YFP and Orai1–CFP (n = 64 cells) at rest and 300 s after store depletion with 1 μm TG, and in cells expressing D76ASTIM1–YFP (n = 9 cells) at rest.