title: 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.
authors.affiliation: Institute of Biophysics, University of Linz, A-4040 Linz, Austria.
authors.forename: Isabella
authors.initials: I
authors.lastname: Derler
authors.forename: Marc
authors.initials: M
authors.lastname: Fahrner
authors.forename: Martin
authors.initials: M
authors.lastname: Muik
authors.forename: Barbara
authors.initials: B
authors.lastname: Lackner
authors.forename: Rainer
authors.initials: R
authors.lastname: Schindl
authors.forename: Klaus
authors.initials: K
authors.lastname: Groschner
authors.forename: Christoph
authors.initials: C
authors.lastname: Romanin
mesh_terms: D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D020013:Calcium Signaling; D002460:Cell Line; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism*; D000071740:ORAI1 Protein; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D000071737:Stromal Interaction Molecule 1
chemical_list: D015220:Calcium Channels; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; C522867:Orai3 protein, human; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D002118:Calcium