Figure 2.

Cytoplasmic Ca²⁺ responses evoked by various STIM1 constructs truncated from the C-terminus. (A) C-terminal truncations on the full-STIM1 (with intact N-terminus) protein up to residue 463 have only small effects on the thapsigargin (Tg, 200 nM) –induced Ca²⁺ elevations. Cells were co-transfected with untagged Orai1 and the indicated mRFP-tagged (in the luminal side) STIM1 constructs driven by the thimidine kinase promoter (TK-STIM1). Means ± S.E.M. are shown (n = 89, 37, 32 and 198 cells for black, green, red and blue traces, respectively obtained in 2–5 separate experiments). (B) The same truncations have significant impact on the abilities of the cytoplasmic STIM1 (STIM1-ct) pieces to activate Orai1 channels after rapamycin-induced clustering. Here the indicated STIM1-ct fragments were fused with the mRFP-FKBP12 module (FK-STIM-ct) and co-expressed with untagged Orai1 and the ER-targeted CFP-FRB. Means ± S.E.M. are shown (n = 356, 254, 259 and 639 cells for black, green, red and blue traces, respectively obtained in 9–10 separate experiments). (C) Truncation of the cytoplasmic STIM1 fragments from the N-terminal direction makes them constitutively active not requiring clustering. Cells were transfected with the indicated constructs as described in panel B. Means ± S.E.M. are shown (n = 208, 82, 124 and 305 cells for black, green, red and blue traces, respectively obtained in 4–6 separate experiments).