CaTAr1 inhibits Ca ²⁺ tunneling. (A) HeLa cells expressing GFP-MAPPER and loaded with the Ca²⁺ indicator Calbryte 590. (B) Changes in intracellular Ca²⁺ during a tunneling experiment from an untransfected cell (Ctr) and a cell expressing GFP-MAPPER (MAP). (C) Violin plots of the amplitude of the tunneling signal in Ctr and MAPPER expressing cells (MAP) (n = 439/118; 12 dishes; unpaired t test, P = 0.65). (D) Violin plot of the slope of the initial phase of tunneling in MAPPER expressing cells n = 427/110, unpaired t test, P = 0.0008). (E) Example averaged traces of Ca²⁺ release and SOCE in control (Ctr; no detectable CaTAr1 expression) and CatAr1 expressing cells from the same dish. Histamine-induced (His, 100 μM) Ca²⁺ release from stores was followed by CPA to deplete Ca²⁺ store, a wash period in Ca²⁺-free media to allow for SERCA to be active, and then the addition of Ca²⁺ to activate SOCE (n = 9/5). (F) Violin plots summarizing the levels of Ca²⁺ release in response to histamine in a Ca²⁺-free solution (n = 192/77; 5 dishes; unpaired t test, P = 0.085). (G and H) Enlarged traces from the red rectangle in E and violin plots summarizing the levels of SOCE in cells that did not express CatAr1 (Ctr) and CaTAr1 expressing cells (n = 192/77, unpaired t test, P = 0.10). (I) SOCE amplitude after thapsigargin application (1 µM) on control cells (Ctr) and cells expressing CaTAr1 (n = 125/51, 3 dishes, unpaired t test, P = 0.0175). (J) Quantification of NFAT nuclear translocation in Ctr and CaTAr1 expressing cells (n = 269/109, 5 dishes, unpaired t test, P = 0.58). (K) HeLa cells expressing CaTAr1 and loaded with Calbryte 590. The numbers correspond to the cells in L. (L) Traces showing the Ca²⁺ tunneling transient in response to His+Ca²⁺ following the standard store depletion with CPA and wash (not shown for clarity). A second histamine application after a delay in Ca²⁺-containing media confirms that all cells including those expressing CatAr1 refill their stores. (M and N) Violin plots summarizing the levels of Ca²⁺ tunneling (M, n = 262/83, six dishes, unpaired t test, P < 0.0001) and Ca²⁺ release in response to His following the second application in Ca²⁺-containing media (N, n = 182/56, five dishes, unpaired t test, P = 0.0250).