Figure 1. Ca²⁺ store refilling reverses the rearrangement of EYFP-Stim1.

TIRFM fluorescence intensity and relative intracellular Ca²⁺ concentrations were measured simultaneously in the same HEK293 cells overexpressing EYFP-Stim1 and the m5 muscarinic receptor. As indicated, cells were treated with 300 μM carbachol in nominally Ca²⁺-free extracellular medium to deplete intracellular Ca²⁺ stores. Carbachol signaling was then terminated by the addition of 50 μM atropine, after which extracellular Ca²⁺ was restored to 1.8 mM. Fifteen minutes later the cells were treated with 2 μM thapsigargin to demonstrate that store refilling had occurred. This protocol was performed with cells treated in the absence (A) or presence (B) of 5 μM Gd³⁺ throughout. Note in (B) that SOCE did not occur upon restoration of extracellular Ca²⁺, and EYFP-Stim1 was not reversed. The upper traces show the TIRFM intensity profiles, and the bottom traces show the 360/380 fluorescence intensities representative of relative Ca²⁺ responses; each trace represents the average response of four cells measured in a single experiment. The bottom panels show TIRFM images taken at the times indicated (i-iv) in the intensity profiles. Scalebars = 10 μm.