Restoring STIM1 or STIM2 expression rescues IP3-dependent Ca ²⁺ release through a diffuse release mode. (A) Proposed model for STIM regulation of diffuse Ca²⁺ release via IP3R. Under ER Ca²⁺ replete conditions, STIM is at a resting conformation and IP3R-mediated Ca²⁺ release occurs primarily via local Ca²⁺ puffs. Upon a decrease in [Ca²⁺]_ER, STIM adopts an active conformation and promotes a diffuse mode of Ca²⁺ release via IP3R. (B) Average (mean ± SEM) intracellular Ca²⁺ responses of basal and SOCE are shown for control dKO (n = 67) or for dKO cell expressing STIM1 (n = 85), STIM2 (n = 89), or STIM1 D76A (n = 58) cells. (C) Boxplot shows quantification of Tg-induced ER Ca²⁺ release for individual cells shown in B. (D–J) Analysis of global and puff Ca²⁺ release in MDA-MB-231 control dKO cells (n = 13) or in cells expressing EYFP-STIM1 (n = 19), EYFP-STIM2 (n = 23), or EYFP-STIM1 D76A (n = 29). (D) Representative traces of Cal-590 fluorescence ratios (ΔF/F0) recorded from the center of an individual puff site before and after ci-IP3 uncaging by a pulse (1 s) of 405-nm light in the indicated cells. (E and F) Boxplot shows the number of Ca²⁺ puffs (E) or puff sites (F) per cell for the indicated cells. (G) Average global Ca²⁺ responses recorded after UV-induced IP3 uncaging for the indicated cells. (H) Time course of Ca²⁺ release via Ca²⁺ puff in the indicated cells was quantified as in Fig. 5 E. Note that in cells expressing STIM1 or STIM2, a rise in global Ca²⁺ levels (G) is accompanied by a decrease in localized Ca²⁺ release (H). However, in cells expressing the STIM1 D76A mutant, the global Ca²⁺ increase occurs without a corresponding decrease in localized release. (I) Amplitude distributions of local Ca²⁺ puffs in the indicated cells. (J) Curves show the normalized Gaussian fit to the data shown in I for the indicated type of cells. Statistics: one-way ANOVA with Tukey’s post hoc test; *P < 0.05, ***P < 0.001.