Fig. 5: Native CRAC channels are heteromultimers of ORAI isoforms.
From: The native ORAI channel trio underlies the diversity of Ca2+ signaling events
a–d Measurements of ER Ca2+ release (in 0 mM Ca2+) and SOCE (in 2 mM Ca2+) on store depletion with 2 µM thapsigargin (Tg) in HEK293 cells and ORAI1 single-knockout (ORAI1-SKO) cells either untransfected or transfected with ORAI pore mutants: ORAI1-E106Q (a), ORAI1-E106A (b), ORAI2-E80Q (c), and ORAI3-E81Q (d). e Quantification of SOCE magnitude for a–d) (left to right n = 130, 76, 68, 64, 53, 116, 53, 57, 51, and 37; points represent individual cells). f Quantification of oscillations/14 min for a–d; data from ORAI-TKO cells (Fig. 1f) is included as a reference (left to right n = 97, 142, 175, 124, 119, 119, 127, 145, 19, 70, and 84; points represent individual cells). Data are represented as mean ± SEM and were statistically analyzed using the Kruskal–Wallis one-way ANOVA with multiple comparisons (*p < 0.05). Comparisons in e were made to WT HEK293. Comparisons in f were made to the corresponding untransfected control (i.e., WT HEK293 or ORAI1-SKO). g–m CRAC current measurements on store depletion by dialysis of 20 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) through the pipette followed by perfusion of 50 µM 2-APB in the bath. Native CRAC current recordings were performed in HEK293 cells (g), ORAI1-SKO cells (h), ORAI2-SKO cells (i), ORAI3-SKO cells (j), ORAI2,3-DKO cells (k), ORAI1,3-DKO cells (l), and ORAI1,2-DKO cells (m). Top panels: representative current traces elicited by voltage ramps from −140 mV to +100 mV from a holding potential of +30 mV with three pulses of divalent-free solutions to amplify native CRAC currents. The first pulse is performed immediately after whole-cell configuration, the second pulse after CRAC current has been maximally activated (4–5 min after break-in) and the third pulse after 2-APB perfusion (~8 min after break-in). (middle panels) representative I/V curves for maximal CRAC current (black trace) and after 2-APB addition (red trace) taken where indicated in top panels by the color-coded asterisks. (lower panels) statistical analysis of peak background-subtracted Na+ CRAC current densities (represented as mean ± SEM) recorded from several cells in DVF solutions before and after 2-APB perfusion. Data were statistically analyzed using a two-tailed Student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant). Boxplots show the mean, median, and the 75th to 25th percentiles.
