Abstract
Store-operated Ca(2+) entry (SOCE) due to activation of Ca(2+) release-activated Ca(2+) (CRAC) channels leads to sustained elevation of cytoplasmic Ca(2+) and activation of lymphocytes. CRAC channels consisting of four pore-forming Orai1 subunits are activated by STIM1, an endoplasmic reticulum Ca(2+) sensor that senses intracellular store depletion and migrates to plasma membrane proximal regions to mediate SOCE. One of the fundamental properties of CRAC channels is their Ca(2+)-dependent fast inactivation. To identify the domains of Orai1 involved in fast inactivation, we have mutated residues in the Orai1 intracellular loop linking transmembrane segment II to III. Mutation of four residues, V(151)SNV(154), at the center of the loop (MutA) abrogated fast inactivation, leading to increased SOCE as well as higher CRAC currents. Point mutation analysis identified five key amino acids, N(153)VHNL(157), that increased SOCE in Orai1 null murine embryonic fibroblasts. Expression or direct application of a peptide comprising the entire intracellular loop or the sequence N(153)VHNL(157) blocked CRAC currents from both wild type (WT) and MutA Orai1. A peptide incorporating the MutA mutations had no blocking effect. Concatenated Orai1 constructs with four MutA monomers exhibited high CRAC currents lacking fast inactivation. Reintroduction of a single WT monomer (MutA-MutA-MutA-WT) was sufficient to fully restore fast inactivation, suggesting that only a single intracellular loop can block the channel. These data suggest that the intracellular loop of Orai1 acts as an inactivation particle, which is stabilized in the ion permeation pathway by the N(153)VHNL(157) residues. These results along with recent reports support a model in which the N terminus and the selectivity filter of Orai1 as well as STIM1 act in concert to regulate the movement of the intracellular loop and evoke fast inactivation.
Highlights
The Intracellular Loop of Orai1 between TM II and III Inhibits Ca2؉ release-activated Ca2؉ (CRAC) Currents—To determine the functional role of the intracellular loop of Orai1 located between TM II and TM III, which contains hydrophobic residues and is well conserved throughout evolution (Fig. 1a and supplemental Fig. 1), we systematically mutated the loop residues and studied their role in store-operated Ca2ϩ entry
In Naϩ channels, the role of an inactivation particle has been demonstrated with channel mutants lacking inactivation and where inhibition was restored by a short peptide with a sequence similar to that of the inactivation particle [39, 40]
mutant in the intracellular loop (MutA) monomers are depicted in red. b, measurement of store-operated Ca2ϩ entry (SOCE) in Orai1-null Murine Embryonic Fibroblast (MEF) expressing concatenated tetramers with different stoichiometry of wild type (WT) and MutA subunits
Summary
Reagents—Thapsigargin, puromycin, and 2-aminoethoxydiphenyl borate were purchased from EMD Biochemicals. HeLa OϩS cells were generated by transducing HeLa cells with retroviruses encoding STIM1 and Orai proteins. STIM1 cDNA was encoded from a plasmid co-expressing hCD25 [35, 36], and positively transduced cells were selected by using hCD25coated magnetic beads (Invitrogen). Orai cDNA was encoded from a pMSCV-CITE-PGK-Puro vector, and transduced cells were selected by puromycin (1 g/ml). Phoenix cells stably expressing gag-pol and ecotropic env (ATCC) were transfected with plasmids encoding Orai proteins to produce ecotropic, replication-incompetent retrovirus using the calcium phosphate transfection method. 2, 4, and 5, HEK293T cells were co-transfected with plasmids encoding STIM1 and WT or MutA Orai at a molar ratio of 1:1 using Lipofectamine 2000 (Invitrogen). The extent of inactivation may have been underestimated as a result of removing the first 2 ms of recording
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