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Fig. 3. ML-9 dose-dependently inhibits SOCE and I_crac. (A) Relative intracellular Ca²⁺ concentrations were monitored in wild-type HEK293 cells treated with 100 µM ML-9 (red trace) or left untreated (black trace). Ca²⁺ stores were depleted with 2 µM thapsigargin in nominally Ca²⁺-free extracellular medium, and extracellular Ca²⁺ was restored to 1.8 mM 15 minutes later to reveal SOCE. ML-9 was removed at the end of the experiment to demonstrate the reversibility of ML-9 inhibition. Each trace represents the averaged response of all cells measured on a single coverslip. (B) The average peak SOCE responses above baseline from experiments performed as described in panel A were averaged for untreated control cells (n=150; five coverslips) or cells treated with 1 µM (n=78; three coverslips), 10 µM (n=76; three coverslips), 50 µM (n=84; three coverslips) or 100 µM (n=64; three coverslips) ML-9. Data are reported as the percentage of untreated control±s.e.m.; ^*, significant difference compared with control (P<0.001) based on one-way ANOVA. (C) Experiments were performed as described in panel A, but ML-9 was added 5 minutes following restoration of extracellular Ca²⁺ (red trace). (D) For experiments performed as described in panel C, the baseline-subtracted 340/380 ratio 5 minutes following ML-9 addition was divided by that just before addition. Data are reported as the percentage of untreated control±s.e.m. for untreated controls (n=151; five coverslips), and for 1 µM (n=90; four coverslips), 10 µM (n=80, three coverslips), 50 µM (n=61, three coverslips) and 100 µM (n=74, three coverslips) ML-9; ^*, significant difference compared with control (P<0.001) based on one-way ANOVA. (E) Whole-cell patch clamp analysis was performed with a pipette solution containing 20 mM BAPTA and 25 µM Ins(1,4,5)P₃ to deplete intracellular Ca²⁺ stores. The cell was initially perfused with an extracellular solution containing 10 mM Ca²⁺. At the time indicated, perfusion was switched to a divalent-free (DVF) solution, which resulted in the development of a Na⁺ current. Perfusion was then returned to 10 mM Ca²⁺, and 100 µM ML-9 was added at the time indicated. Note the decrease in the Ca²⁺ current upon ML-9 addition. In the continued presence of ML-9, perfusion was again switched to DVF solution. At the end of the experiment, 10 mM extracellular Ca²⁺ was restored and ML-9 was removed to demonstrate reversal of inhibition of the Ca²⁺ current. (F) For experiments performed as described in panel E, the peak Na⁺ currents at the initial switch to DVF solution in the absence of ML-9 (control) and that at the second switch to DVF in the presence of 100 µM ML-9 were averaged and are expressed as mean±s.e.m. (n=6); ^*, significant difference compared with control (P<0.005) based on Student's t test.

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