Figure 4: ORAI1 and ORAI2 form heteromeric channels.

(a) Analysis of SOCE in Orai1-deficient T cells transduced with empty vector (EV), wild type ORAI1 (ORAI1^WT) or a pore-dead ORAI1 mutant (ORAI1^E106Q). Transduced GFP⁺CD4⁺ T cells were FACS sorted and SOCE was analysed following thapsigargin (TG) stimulation and addition of 1 mM extracellular Ca²⁺. (b) Analysis of SOCE in Orai2-deficient T cells transduced with EV, ORAI2^WT or a pore-dead ORAI2 mutant (ORAI2^E80Q). GFP⁺CD4⁺ T cells in a,b were analysed for SOCE (peak F340/380); shown are means±s.e.m. of cells from three mice measured in triplicates. (c) Surface expression of ectopically expressed ORAI1 mutants used in d–g using an anti-ORAI1 (2C1.1) antibody by flow cytometry. (d–g) Analysis of SOCE in Orai1-deficient T cells transduced with (d) EV, ORAI1^WT, ORAI1^E106Q, (e) the STIM1 binding-deficient ORAI1^L273D mutant and (f) the ORAI1^L273D/E106Q double mutant as described in a. (g) Quantification of SOCE (peak F340/380) as shown in d–f; means±s.e.m. of cells from four mice measured in triplicates. (h) Illustration how E106Q and/or L273D mutations affect formation and function of ORAI1:ORAI2 heteromeric channels. (i) Time course of CRAC currents in HEK293 cells overexpressing YFP-ORAI1^WT and CFP-ORAI2^WT together with STIM1 and stimulated with 1 μM TG. Extracellular La³⁺ (150 μM) and 2-APB (50 μM) inhibit the currents in Ringer’s solution containing 20 mM Ca²⁺. Application of DVF solution reveals large Na⁺ currents. (j) Representative I–V relationships of ORAI channels. (k) Summary of peak CRAC current amplitudes measured during step pulses to −100 mV in cells expressing the indicated ORAI isoforms. Eight to nine cells per condition were analysed. (l) Summary of steady-state (SS) CRAC current amplitudes in cells expressing ORAI isoforms at the end of a 100 ms pulse to −100 mV. (m) ORAI1:ORAI2 channels exhibit enhanced fast CDI. Traces depict currents recorded during hyperpolarizing voltage steps to −100 mV. Currents were normalized to their peak values. (n) Summary of fast inactivation during the 100 ms pulse measured as: 1−I_ss/I_peak, where I_peak and I_ss are the peak and steady-state currents during the pulse to −100 mV. k,l,n are means±s.e.m. *P<0.05; **P<0.005; ***P<0.001 in (a,b,g,k,l,n) using unpaired Student’s t-tests.