Figure 3: Orai2-deficient macrophages have increased SOCE and CRAC currents.

(a,b) Analysis of SOCE following ionomycin stimulation in WT and Orai2-deficient bone marrow-derived macrophages (BMDMs) after readdition of 0.2, 0.5 and 1 mM extracellular Ca²⁺ using a FlexStation plate reader. (b) Quantification of store depletion (AUC_{120 s–400 s}) and SOCE (F340/380 peak) as shown in a; means±s.e.m. of three mice. (c) Mn²⁺ quenching of Fura-2 fluorescence to measure store-operated divalent cation influx via CRAC channels. WT and Orai2-deficient BMDMs were loaded with Fura-2 and stimulated with 0.3 μM ionomycin in Ca²⁺ and Mn²⁺-free Ringer solution, followed by the addition of 1 mM Mn²⁺. Bar graphs show the quantification of Mn²⁺ quench rates in ionomycin-stimulated BMDMs normalized to unstimulated cells; means±s.e.m. of three mice. (d,e) Electrophysiological properties of CRAC currents in WT and Orai2^−/− BMDMs. (d) Leak-corrected CRAC currents measured at −100 mV are plotted over time. The standard extracellular Ringer solution was periodically switched with a Na⁺-based DVF solution, revealing permeation of Na⁺ ions in the absence of extracellular divalents. I_CRAC is completely inhibited by 100 μM LaCl₃. (e) I–V relationship of I_CRAC in 20 mM Ca²⁺ (left) and DVF (middle) solutions in WT and Orai2^−/− BMDMs and inhibition by 50 μM 2-aminoethoxydiphenyl borate (2-APB). Means±s.e.m. of the peak current density in WT and Orai2^−/− BMDMs; 10–12 cells were analysed (right). *P<0.05; **P<0.005; ***P<0.001 in (b,c,e) using unpaired Student’s t-tests.