A Ca2(+ )release-activated Ca2(+) (CRAC) modulatory domain (CMD) within STIM1 mediates fast Ca2(+)-dependent inactivation of ORAI1 channels

Abstract

STIM1 and ORAI1, the two limiting components in the Ca(2+) release-activated Ca(2+) (CRAC) signaling cascade, have been reported to interact upon store depletion, culminating in CRAC current activation. We have recently identified a modulatory domain between amino acids 474 and 485 in the cytosolic part of STIM1 that comprises 7 negatively charged residues. A STIM1 C-terminal fragment lacking this domain exhibits enhanced interaction with ORAI1 and 2-3-fold higher ORAI1/CRAC current densities. Here we focused on the role of this CRAC modulatory domain (CMD) in the fast inactivation of ORAI1/CRAC channels, utilizing the whole-cell patch clamp technique. STIM1 mutants either with C-terminal deletions including CMD or with 7 alanines replacing the negative amino acids within CMD gave rise to ORAI1 currents that displayed significantly reduced or even abolished inactivation when compared with STIM1 mutants with preserved CMD. Consistent results were obtained with cytosolic C-terminal fragments of STIM1, both in ORAI1-expressing HEK 293 cells and in RBL-2H3 mast cells containing endogenous CRAC channels. Inactivation of the latter, however, was much more pronounced than that of ORAI1. The extent of inactivation of ORAI3 channels, which is also considerably more prominent than that of ORAI1, was also substantially reduced by co-expression of STIM1 constructs missing CMD. Regarding the dependence of inactivation on Ca(2+), a decrease in intracellular Ca(2+) chelator concentrations promoted ORAI1 current fast inactivation, whereas Ba(2+) substitution for extracellular Ca(2+) completely abrogated it. In summary, CMD within the STIM1 cytosolic part provides a negative feedback signal to Ca(2+) entry by triggering fast Ca(2+)-dependent inactivation of ORAI/CRAC channels.

FIGURE 1.

Fast inactivation of ORAI1 currents is preserved as long as CMD is present in STIM1. a, b, d, and e, fast inactivation of STIM1- (a), STIM1-(1–474)- (b), STIM1-(1–485)- (d), and STIM1 7xA-mediated (e) ORAI1 Ca²⁺ currents upon voltage steps from a holding potential of 0 mV to −30, −50, −70, and −90 mV. c and f, mean fast inactivation of normalized current traces of STIM1, STIM1-(1–535), STIM1-(1–485), STIM1-(1–474), and STIM1 7xA upon voltage steps to −90 mV. g, scheme of various STIM1 deletion mutants (STIM1, STIM1-(1–535), STIM1-(1–485), STIM1-(1–474), STIM1-(1–450)) as well as STIM1 7xA and EF-hand mutant STIM1 D76A in comparison with the extent of inactivation of ORAI1 currents at t = 90 ms (p < 0.05 for STIM1 when compared with STIM1-(1–474), STIM1-(1–450), and STIM1 7xA) and maximum current densities. wt, wild type; SAM, sterile α motif; TM, transmembrane; pA, picoampere; pF, picofarad.

FIGURE 2.

Fast inactivation of ORAI1 currents is diminished after deletion of CMD in cytosolic STIM1 C-terminal fragments HEK 293 as well as RBL mast cells. a and b, fast inactivation of STIM1-(233–685)- (a) and STIM1-(233–685) 7xA-mediated (b) ORAI1 Ca²⁺ currents upon voltage steps from a holding potential of 0 mV to −30, −50, −70, and −90 mV. c, mean fast inactivation of normalized current traces of STIM1-(233–685), -(233–685) 7xA (D/E → A, aa 475, 476, 478, 479, 481–483), -(233–685) 5xA (D/E → A, aa 478, 479, 481–483), and -(233–685) 3xA (D/E → A, aa 481–483) upon voltage steps to −90 mV. d, scheme of various STIM1 C terminus deletion mutants (233–685, 233–535, 233–485, 233–474), STIM1-(233–685) 7xA, 5xA, 3xA, and STIM1-(233–485) 7xA in comparison with the extent of inactivation of ORAI1 currents t = 90 ms (p < 0.05 for STIM1-(233–685) when compared with STIM1-(233–474), STIM1-(233–450), STIM1-(233–685) 7xA, STIM1-(233–485) 7xA), and maximum current densities). ERM, ezrin-radixin-moesin. pA, picoampere; pF, picofarad. e and f, fast inactivation of STIM1- (e) and STIM1 7xA-mediated (f) RBL CRAC currents upon voltage steps from a holding potential of 0 mV to −30, −50, −70, or −90 mV. g, mean fast inactivation of normalized current traces of e and f upon voltage steps to −90 mV (t = 90 ms, p < 0.05).

FIGURE 3.

Fast inactivation of ORAI1 channels occurs in a Ca²⁺-dependent manner. a, fast inactivation profiles as well as block diagrams for t = 90 ms of STIM1 D76A-mediated constitutive ORAI1 currents in the presence of 20 mm EGTA in comparison with 100 μm EGTA intracellular solution (t = 30 ms, p < 0.05). The scheme on the right side represents STIM1 D76A in the presence of the two distinct Ca²⁺ buffering conditions. SAM, sterile α motif; TM, transmembrane. b and c, activation of STIM1- (b) and STIM1-(1–474)-mediated (c) ORAI1 Ba²⁺ currents upon voltage steps from a holding potential of 0 mV to −30, −50, −70, and −90 mV. d, mean activation of STIM1- and STIM1-(1–474)-mediated ORAI1 Ba²⁺ current in comparison with fast inactivation of STIM1- and STIM1-(1–474)-mediated ORAI1 Ca²⁺ currents. e, scheme of STIM1 deletion mutants in the presence of Ca²⁺ or Ba²⁺ in comparison with the extent of inactivation of ORAI1 currents t = 90 ms (p < 0.05 for STIM1 (Ca²⁺), when compared with STIM1-(1–474) (Ca²⁺), STIM1 (Ba²⁺), STIM1-(1–474) (Ba²⁺), and maximum current densities. pA, picoamperes; pF, picofarads.