[Show abstract][Hide abstract] ABSTRACT: Store-operated Ca(2+) entry (SOCE) is a universal Ca(2+) influx pathway that is important for the function of many cell types. SOCE occurs upon depletion of endoplasmic reticulum (ER) Ca(2+) stores and relies on a complex molecular interplay between the plasma membrane (PM) Ca(2+) channel ORAI1 and the ER Ca(2+) sensor stromal interaction molecule (STIM) 1. Patients with null mutations in ORAI1 or STIM1 genes present with severe combined immunodeficiency (SCID)-like disease. Here, we describe the molecular mechanisms by which a loss-of-function STIM1 mutation (R429C) in human patients abolishes SOCE. R429 is located in the third coiled-coil (CC3) domain of the cytoplasmic C terminus of STIM1. Mutation of R429 destabilizes the CC3 structure and alters the conformation of the STIM1 C terminus, thereby releasing a polybasic domain that promotes STIM1 recruitment to ER-PM junctions. However, the mutation also impairs cytoplasmic STIM1 oligomerization and abolishes STIM1-ORAI1 interactions. Thus, despite its constitutive localization at ER-PM junctions, mutant STIM1 fails to activate SOCE. Our results demonstrate multifunctional roles of the CC3 domain in regulating intra- and intermolecular STIM1 interactions that control (i) transition of STIM1 from a quiescent to an active conformational state, (ii) cytoplasmic STIM1 oligomerization, and (iii) STIM1-ORAI1 binding required for ORAI1 activation.
Proceedings of the National Academy of Sciences 04/2015; 112(19). DOI:10.1073/pnas.1418852112 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Store-operated Ca2+ entry, essential for the adaptive immunity, is initiated by the endoplasmic reticulum (ER) Ca2+ sensor STIM1. Ca2+ entry occurs through the plasma membrane resident Ca2+ channel Orai1 that directly interacts with the C-terminal STIM1 domain, named SOAR/CAD. Depletion of the ER Ca2+ store controls this STIM1/Orai1 interaction via transition to an extended STIM1 C-terminal conformation, exposure of the
SOAR/CAD domain, and STIM1/Orai1 co-clustering. Here we developed a novel approach termed FRET-derived Interaction in a Restricted
Environment (FIRE) in an attempt to dissect the interplay of coiled-coil (CC) interactions in controlling STIM1 quiescent
as well as active conformation and cluster formation. We present evidence of a sequential activation mechanism in the STIM1
cytosolic domains where the interaction between CC1 and CC3 segment regulates both SOAR/CAD exposure and CC3-mediated higher-order
oligomerization as well as cluster formation. These dual levels of STIM1 auto-inhibition provide efficient control over the
coupling to and activation of Orai1 channels.
[Show abstract][Hide abstract] ABSTRACT: Transient receptor potential vanilloid subfamily member 6 (TRPV6) is a highly selective calcium channel that has been considered as a part of store-operated calcium entry (SOCE). Despite its first discovery in the early 2000s, the role of this channel in prostate cancer (PCa) remained, until now, obscure. Here we show that TRPV6 mediates calcium entry, which is highly increased in PCa due to the remodeling mechanism involving the translocation of the TRPV6 channel to the plasma membrane via the Orai1/TRPC1-mediated Ca(2+)/Annexin I/S100A11 pathway, partially contributing to SOCE. The TRPV6 calcium channel is expressed de novo by the PCa cell to increase its survival by enhancing proliferation and conferring apoptosis resistance. Xenografts in nude mice and bone metastasis models confirmed the remarkable aggressiveness of TRPV6-overexpressing tumors. Immunohistochemical analysis of these demonstrated the increased expression of clinical markers such as Ki-67, prostate specific antigen, synaptophysin, CD31, and CD56, which are strongly associated with a poor prognosis. Thus, the TRPV6 channel acquires its oncogenic potential in PCa due to the remodeling mechanism via the Orai1-mediated Ca(2+)/Annexin I/S100A11 pathway.
Proceedings of the National Academy of Sciences 08/2014; 111(37). DOI:10.1073/pnas.1413409111 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Orai proteins contribute to Ca(2+) entry into cells through both store-dependent, Ca(2+) release-activated Ca(2+) (CRAC) channels (Orai1) and store-independent, arachidonic acid (AA)-regulated Ca(2+) (ARC) and leukotriene C4 (LTC4)-regulated Ca(2+) (LRC) channels (Orai1/3 heteromultimers). Although activated by fundamentally different mechanisms, CRAC channels, like ARC and LRC channels, require stromal interacting molecule 1 (STIM1). The role of endoplasmic reticulum-resident STIM1 (ER-STIM1) in CRAC channel activation is widely accepted. Although ER-STIM1 is necessary and sufficient for LRC channel activation in vascular smooth muscle cells (VSMCs), the minor pool of STIM1 located at the plasma membrane (PM-STIM1) is necessary for ARC channel activation in HEK293 cells. To determine whether ARC and LRC conductances are mediated by the same or different populations of STIM1, Orai1, and Orai3 proteins, we used whole-cell and perforated patch-clamp recording to compare AA- and LTC4-activated currents in VSMCs and HEK293 cells. We found that both cell types show indistinguishable nonadditive LTC4- and AA-activated currents that require both Orai1 and Orai3, suggesting that both conductances are mediated by the same channel. Experiments using a nonmetabolizable form of AA or an inhibitor of 5-lipooxygenase suggested that ARC and LRC currents in both cell types could be activated by either LTC4 or AA, with LTC4 being more potent. Although PM-STIM1 was required for current activation by LTC4 and AA under whole-cell patch-clamp recordings in both cell types, ER-STIM1 was sufficient with perforated patch recordings. These results demonstrate that ARC and LRC currents are mediated by the same cellular populations of STIM1, Orai1, and Orai3, and suggest a complex role for both ER-STIM1 and PM-STIM1 in regulating these store-independent Orai1/3 channels.
The Journal of General Physiology 03/2014; 143(3):345-59. DOI:10.1085/jgp.201311084 · 4.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Orai1 calcium channels in the plasma membrane are activated by stromal interaction molecule-1 (STIM1), an endoplasmic reticulum calcium sensor, to mediate store-operated calcium entry (SOCE). The cytosolic region of STIM1 contains a long putative coiled-coil (CC)1 segment and shorter CC2 and CC3 domains. Here we present solution nuclear magnetic resonance structures of a trypsin-resistant CC1-CC2 fragment in the apo and Orai1-bound states. Each CC1-CC2 subunit forms a U-shaped structure that homodimerizes through antiparallel interactions between equivalent α-helices. The CC2:CC2' helix pair clamps two identical acidic Orai1 C-terminal helices at opposite ends of a hydrophobic/basic STIM-Orai association pocket. STIM1 mutants disrupting CC1:CC1' interactions attenuate, while variants promoting CC1 stability spontaneously activate Orai1 currents. CC2 mutations cause remarkable variability in Orai1 activation because of a dual function in binding Orai1 and autoinhibiting STIM1 oligomerization via interactions with CC3. We conclude that SOCE is activated through dynamic interplay between STIM1 and Orai1 helices.
[Show abstract][Hide abstract] ABSTRACT: Ca (2+) influx via store-operated Ca (2+) release activated Ca (2+) (CRAC) channels represents a main signaling pathway for T-cell activation as well as mast-cell degranulation. The ER-located Ca (2+)-sensor, STIM1 and the Ca (2+)-selective ion pore, Orai1 in the membrane are sufficient to fully reconstitute CRAC currents. Their identification, but even more the recent structural resolution of both proteins by X-ray crystallography has substantially advanced the understanding of the activation mechanism of CRAC channels. In this review, we provide a detailed description of the STIM1/Orai1 signaling pathway thereby focusing on the critical domains mediating both, intra- as well as intermolecular interactions and on the ion permeation pathway. Based on the results of functional studies as well as the recently published crystal structures, we portray a mechanistic view of the steps in the CRAC channel signaling cascade ranging from STIM1 oligomerization over STIM1-Orai1 coupling to the ultimate Orai1 channel activation and permeation.
[Show abstract][Hide abstract] ABSTRACT: Sirolimus (rapamycin) is used in drug-eluting stent strategies and proved clearly superior in this application as compared to other immunomodulators such as pimecrolimus. The molecular basis of this action of sirolimus in the vascular system is still incompletely understood. Measurements of cell proliferation in human coronary smooth muscle cells (hCASM) demonstrated a higher antiproliferative activity of sirolimus as compared to pimecrolimus. Although sirolimus lacks inhibitory effects on calcineurin, NFAT activation in hCASM was suppressed to a similar extent by both drugs at 10 µM. Sirolimus, but not pimecrolimus, inhibited agonist-induced and store-operated Ca(2+) entry as well as CREB phosphorylation in human arterial smooth muscle suggesting the existence of an as yet unrecognized inhibitory effect of sirolimus on Ca(2+) signaling and Ca(2+)-dependent gene transcription. Electrophysiological experiments revealed that only sirolimus but not pimecrolimus significantly blocked the classical STIM/Orai-mediated, store-operated Ca(2+) current reconstituted in HEK293 cells. A link between Orai function and proliferation was confirmed by dominant negative knockout of Orai in hCASM. Analysis of the effects of sirolimus on cell proliferation and CREB activation in an in vitro model of arterial intervention using human aorta corroborated the ability of sirolimus to suppress stent implantation-induced CREB activation in human arteries. We suggest inhibition of store-operated Ca(2+) entry based on Orai channels and the resulting suppression of Ca(2+) transcription coupling as a key mechanism underlying the antiproliferative activity of sirolimus in human arteries. This mechanism of action is specific for sirolimus and not a general feature of drugs interacting with FKBPs.
[Show abstract][Hide abstract] ABSTRACT: We recently showed, in primary vascular smooth muscle cells (VSMCs), that the platelet-derived growth factor activates canonical store-operated Ca(2+) entry and Ca(2+) release-activated Ca(2+) currents encoded by Orai1 and STIM1 genes. However, thrombin activates store-independent Ca(2+) selective channels contributed by both Orai3 and Orai1. These store-independent Orai3/Orai1 channels are gated by cytosolic leukotriene C4 (LTC4) and require STIM1 downstream LTC4 action. However, the source of LTC4 and the signaling mechanisms of STIM1 in the activation of this LTC4-regulated Ca(2+) (LRC) channel are unknown. Here, we show that upon thrombin stimulation, LTC4 is produced through the sequential activities of phospholipase C, diacylglycerol lipase, 5-lipo-oxygenease, and leukotriene C4 synthase. We show that the endoplasmic reticulum-resident STIM1 is necessary and sufficient for LRC channel activation by thrombin. STIM1 does not form sustained puncta and does not colocalize with Orai1 either under basal conditions or in response to thrombin. However, STIM1 is precoupled to Orai3 and Orai3/Orai1 channels under basal conditions as shown using Forster resonance energy transfer (FRET) imaging. The second coiled-coil domain of STIM1 is required for coupling to either Orai3 or Orai3/Orai1 channels and for LRC channel activation. We conclude that STIM1 employs distinct mechanisms in the activation of store-dependent and store-independent Ca(2+) entry pathways
Molecular and Cellular Biology 09/2013; 33(18):3715-3723. · 4.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: STIM1 and Orai1 represent the two molecular key components of the Ca2+ release-activated Ca2+-channels. Their activation involves STIM1 C-terminus coupling to both Orai-N- and -C-terminus. Here we focused on the extended transmembrane Orai1 N-terminal (ETON, aa73-90) region, conserved among the Orai family forming an elongated helix of TM1 as recently shown by X-ray crystallography. In order to identify hot spot-residues in the ETON binding interface for STIM1-interaction, numerous Orai1-constructs with N-terminal truncations or point mutations within the ETON region were generated. N-terminal truncations of the first four residues of the ETON region or beyond completely abolished STIM1-dependent Orai1-function. Loss of Orai1-function resulted from neither an impairment of plasma membrane targeting nor pore damage, but from a disruption of STIM1-interaction. Complementary we monitored STIM1-Orai-interaction via Orai1-V102A by determining restored Ca2+-selectivity as a consequence of STIM1-coupling. Orai1 N-terminal truncations that led to a loss of function consistently failed to restore Ca2+-selectivity of Orai1-V102A in the presence of STIM1 demonstrating impairment of STIM1-binding. Hence, the major portion of the ETON region (aa76-90) is essential for STIM1-binding and Orai1-activation. Mutagenesis within the ETON region revealed several hydrophobic and basic hot spot-residues that appear to control STIM1-coupling to Orai1 in a concerted manner. Moreover, we identified two basic residues, which protrude into the elongated pore to redound to Orai1-gating. In aggregate, we suggest that several hot spot-residues in the ETON region contribute to the binding of STIM1, which in turn is coupled to a conformational reorientation of the gate.
[Show abstract][Hide abstract] ABSTRACT: We recently showed, in primary vascular smooth muscle cells (VSMCs), that the platelet-derived growth factor activates canonical
store-operated Ca2+ entry and Ca2+ release-activated Ca2+ currents encoded by Orai1 and STIM1 genes. However, thrombin activates store-independent Ca2+ selective channels contributed by both Orai3 and Orai1. These store-independent Orai3/Orai1 channels are gated by cytosolic
leukotriene C4 (LTC4) and require STIM1 downstream LTC4 action. However, the source of LTC4 and the signaling mechanisms of STIM1 in the activation of this LTC4-regulated Ca2+ (LRC) channel are unknown. Here, we show that upon thrombin stimulation, LTC4 is produced through the sequential activities of phospholipase C, diacylglycerol lipase, 5-lipo-oxygenease, and leukotriene
C4 synthase. We show that the endoplasmic reticulum-resident STIM1 is necessary and sufficient for LRC channel activation by
thrombin. STIM1 does not form sustained puncta and does not colocalize with Orai1 either under basal conditions or in response
to thrombin. However, STIM1 is precoupled to Orai3 and Orai3/Orai1 channels under basal conditions as shown using Forster
resonance energy transfer (FRET) imaging. The second coiled-coil domain of STIM1 is required for coupling to either Orai3
or Orai3/Orai1 channels and for LRC channel activation. We conclude that STIM1 employs distinct mechanisms in the activation
of store-dependent and store-independent Ca2+ entry pathways.
[Show abstract][Hide abstract] ABSTRACT: Utilizing a novel molecular model of TRPC3, based on the voltage-gated sodium channel from Arcobacter butzleri (NaVAB) as template, we performed structure-guided mutagenesis experiments to identify amino acid residues involved in divalent permeation and gating. Substituted cysteine accessibility screening within the predicted selectivity filter uncovered amino acids 629-631 as the narrowest part of the permeation pathway with an estimated pore diameter of <5.8Å. E630 was found to govern not only divalent permeability but also sensitivity of the channel to block by ruthenium red. Mutations in a hydrophobic cluster at the cytosolic termini of transmembrane segment 6, corresponding to the S6 bundle crossing structure in NaVAB, distorted channel gating. Removal of a large hydrophobic residue (I667A or I667E) generated channels with approximately 60% constitutive activity, suggesting I667 as part of the dynamic structure occluding the permeation path. Destabilization of the gate was associated with reduced Ca(2+) permeability, altered cysteine cross-linking in the selectivity filter and promoted channel block by ruthenium red. Collectively, we present a structural model of the TRPC3 permeation pathway and localize the channel's selectivity filter and the occluding gate. Moreover, we provide evidence for allosteric coupling between the gate and the selectivity filter in TRPC3.
[Show abstract][Hide abstract] ABSTRACT: RTN1A is a reticulon protein with predominant localization in the endoplasmic reticulum (ER). It was previously shown that RTN1A is expressed in neurons of the mammalian central nervous system but functional information remains sparse. To elucidate the neuronal function of RTN1A, we chose to focus our investigation on identifying possible novel binding partners specifically interacting with the unique N-terminus of RTN1A. Using a nonbiased approach involving GST pull-downs and MS analysis, we identified the intracellular calcium release channel ryanodine receptor 2 (RyR2) as a direct binding partner of RTN1A. The RyR2 binding site was localized to a highly conserved 150-amino acid residue region. RTN1A displays high preference for RyR2 binding in vitro and in vivo and both proteins colocalize in hippocampal neurons and Purkinje cells. Moreover, we demonstrate the precise subcellular localization of RTN1A in Purkinje cells and show that RTN1A inhibits RyR channels in [3H]ryanodine binding studies on brain synaptosomes. In a functional assay, RTN1A significantly reduced RyR2-mediated Ca2+ oscillations. Thus, RTN1A and RyR2 might act as functional partners in the regulation of cytosolic Ca2+ dynamics in neurons.