Figure 2.

IP₃- and BAPTA-mediated whole-cell currents in isolated HEK293 cells co-expressing Stim1 and Orai1. (A) Average time course of SOC current development after activation with 100 μM inositol 1,4,5-trisphosphate (IP₃) and 10 mM BAPTA in the pipette solution in HEK293 cells expressing YFP alone (Control: black dashed trace), Orai1 (Orai1 alone: grey dashed trace), Orai1 and Stim1 (Orai1 + Stim1: black trace), or Orai1 and Stim1 under the presence of 1 μM Gd³⁺ (Orai1 + Stim1 + 1 μM Gd³⁺: grey trace). External solution contained 10 mM Ca²⁺ in these experiments to keep experimental conditions consistent between YFP alone and overexpressing cells. (B) Same as (A), except the internal stores were passively depleted with only 10 mM BAPTA in the intracellular pipette solution. (C) Current-voltage relationship recorded from HEK293 cells expressing only YFP (Control: black trace), Orai1 and Stim1 depleted of their internal Ca²⁺ stores by the addition of 100 μM IP₃ and 10 mM BAPTA (Orai1 + Stim1-IP₃: black trace), or 10 mM BAPTA alone (Orai1 + Stim1-BAPTA: grey trace). (D) Representative time course of the transient divalent-free potentiation of Orai1 + Stim1 currents, followed by the inhibitory effect of nominally Ca²⁺ free conditions (NCF) on the current recorded at −100 mV (n=7). (E) Typical time course showing both the potentiation at low (5 μM) and inhibition at high (30 μM) concentration of 2APB (n=3). Error bars indicate mean ± SEM (n=7 for Orai1 + Stim1 in A, n=5 for Orai1 + Stim1 in B, all others n=5).