Fig. 1

AC8- and cav-binding site on ORAI1 are required for ORAI1 CDI. a Schematic of consensus domains unique to ORAI1. The first N-terminal 63 amino acids are exclusive to ORAI1 (black). ORAI1β (red) starts at Methionine-64. The putative calmodulin (CaM)-binding domain is found in both ORAI1 and ORAI1β. Recordings are from ORAI-KO cells co-expressing eYFP-STIM1 with either (b, e, h) ORAI1-CFP or (c, f, i) ORAI1β-CFP. Representative currents using either 10 mM EGTA (b, c), or 20 mM BAPTA (e, f) in patch pipette with 20 mM Ca²⁺ bath solution, or 10 mM EGTA in patch pipette with 20 mM Ba²⁺ bath solution (h, i). CDI was revealed by applying a voltage-step protocol from a holding potential of + 30 mV as depicted in (Supplementary Fig. 1f; see Methods). d, g, j The extent of CRAC channel inactivation for ORAI1 and ORAI1β represented as current remaining at the end of the pulse (at 146 ms; see Methods). Each data point represents mean ± SEM. k–o Representative currents from ORAI-KO cells co-expressing eYFP-STIM1 with either k WT ORAI1-CFP, l ORAI1-CFP mutant deficient in AC8-binding (R31–33A), or m ORAI1-CFP mutant deficient in caveolin binding (Y52A, W55A). The extent of CDI for ORAI1 R31–33A (n) and ORAI1 Y52A, W55A (o) compared with the respective side-by-side recordings from WT ORAI1 are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, two-tailed Student’s t test was used for (d, g, j, n, o)