CTID controls SCDI by SARAF. (a) Effect of SARAF on store-independent Ca²⁺ influx was measured in HEK293 cells transfected with STIM1, SOAR, and the indicated deletion mutants and SARAF. The traces are the means of 30–40 cells and shown as mean ± SEM. SARAF inhibited Ca²⁺ influx by SOAR, STIM1(Δ447–460), and STIM1(Δ448–490) but not by STIM1(Δ490–521), STIM1(Δ448–521), and STIM1(Δ448–530). (b) siSARAF reduces SCDI. HEK cells were treated with siSARAF for 48 h that reduced SARAF mRNA by 70% (right). The cells were then transfected with Orai1 and either STIM1 or STIM1(Δ447–460). The Orai1 current was measured 24 h after transfection. The results are plotted as normalized mean ± SEM current at −100mV of the number of experiments indicated in parentheses. Note that STIM1(Δ447–460) increased the rate of SCDI whereas siSARAF markedly reduced SCDI. (c–e) The mean ± SEM of the normalized current at −100 mV measured in HEK cells transfected with Orai1 and the indicated STIM1 C-terminal deletion mutants in the presence and absence of SARAF. Current measurement started by perfusing a solution containing 10 mM Ca²⁺ after 5-min incubation in Ca²⁺-free solution in whole cell configuration with pipette solution containing 1.2 mM EGTA. Note that SCDI is markedly reduced when Orai1 is activated by SOAR, STIM1(Δ490–521), and STIM1(Δ448–530) (c and d), whereas SCDI is accelerated with STIM1(Δ447–460) and STIM1(Δ448–490) (e). In addition, SARAF increased SCDI recorded with STIM1 and SOAR (c), had no effect with STIM1(Δ490–521) and STIM1(Δ448–530) (d), and potently inhibited Orai1 current activated by STIM1(Δ447–460) and STIM1(Δ447–460) (e). In panel e, after normalization, the currents were adjusted to the maximal currents to illustrate the inhibition of the current by SARAF. The dashed traces in d and e mark the SCDI recorded with STIM1 (control).