[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. · 4.73 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.
AJP Heart and Circulatory Physiology 09/2013; · 4.01 Impact Factor
[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. · 5.37 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.
Journal of Biological Chemistry 08/2013; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We recently showed in primary vascular smooth muscle cells (VSMC), 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. 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 leukotrieneC4 (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 leukotrieneC4 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 co-localize with Orai1 either under basal conditions or in response to thrombin. However, STIM1 is pre-coupled to Orai3 and Orai3/Orai1 channels under basal conditions as shown using 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 07/2013; · 6.06 Impact Factor
[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.
Biochimica et Biophysica Acta 02/2013; · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rationale: Through largely unknown mechanisms, Ca(2+) signaling plays important roles in vascular smooth muscle cell (VSMC) remodeling. Orai1-encoded store-operated Ca(2+) entry (SOCE) has recently emerged as an important player in VSMC remodeling. However, the role of the exclusively mammalian Orai3 protein in native VSMC Ca(2+) entry pathways, its upregulation during VSMC remodeling and its contribution to neointima formation remain unknown. Objective: The goal of this study was to determine the agonist-evoked Ca(2+) entry pathway contributed by Orai3; Orai3 potential upregulation and role during neointima formation after balloon-injury of rat carotid arteries. Methods and Results: Ca(2+) imaging and patch clamp recordings showed that while the platelet-derived growth factor (PDGF) activates the canonical Ca(2+) release-activated Ca(2+) (CRAC) channels via store depletion in VSMC, the pathophysiological agonist thrombin activates a distinct Ca(2+)-selective channel contributed by Orai1, Orai3 and STIM1 in the same cells. Unexpectedly, Ca(2+) store depletion is not required for activation of Orai1/3 channel by thrombin. Rather, the signal for Orai1/3 channel activation is cytosolic leukotrieneC4 produced downstream thrombin receptor stimulation through the catalytic activity of leukotrieneC4 synthase. Importantly, Orai3 is upregulated in an animal model of VSMC neointimal remodeling and in vivo Orai3 knockdown inhibits neointima formation. Conclusions: These results demonstrate that distinct native Ca(2+)-selective Orai channels are activated by different agonists/pathways and uncover a mechanism whereby leukotrieneC4 acts through hitherto unknown intracrine mode to elicit store-independent Ca(2+) signaling that promotes vascular occlusive disease. Orai3 and Orai3-containing channels provide novel targets for control of VSMC remodeling during vascular injury or disease.
Circulation Research 01/2013; · 11.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: As the molecular composition of calcium-release activated calcium (CRAC) channels has been unknown for two decades, elucidation of selective inhibitors has been considerably hampered. By the identification of the two key components of CRAC channels, STIM1 and Orai1 have emerged as promising targets for CRAC blockers. The aim of this study was to thoroughly characterize the effects of two selective CRAC channel blockers on currents derived from STIM1/Orai heterologoulsy expressed in HEK293 cells. The novel compounds GSK-7975A and GSK-5503A were tested for effects on STIM1 mediated Orai1 or Orai3 currents by whole-cell patch-clamp recordings and for the effects on STIM1 oligomerisation or STIM1/Orai coupling by FRET microscopy. To investigate their site of action, inhibitory effects of these molecules were explored using Orai pore mutants. The GSK blockers inhibited Orai1 and Orai3 currents with an IC(50) of approximately 4μM and exhibited a substantially slower rate of onset than the typical pore blocker La(3+), together with almost no current recovery upon wash-out over 4min. For the less Ca(2+)-selective Orai1 E106D pore mutant, I(CRAC) inhibition was significantly reduced. FRET experiments indicated that neither STIM1-STIM1 oligomerization nor STIM1-Orai1 coupling was affected by these compounds. These CRAC channel blockers are acting downstream of STIM1 oligomerization and STIM1/Orai1 interaction, potentially via an allosteric effect on the selectivity filter of Orai. The elucidation of these CRAC current blockers represents a significant step toward the identification of CRAC channel-selective drug compounds.
[Show abstract][Hide abstract] ABSTRACT: TRP proteins mostly assemble to homomeric channels but can also heteromerize, preferentially within their subfamilies. The TRPC1 protein is the most versatile member and forms various TRPC channel combinations but also unique channels with the distantly related TRPP2 and TRPV4. We show here a novel cross-family interaction between TRPC1 and TRPV6, a Ca(2+) selective member of the vanilloid TRP subfamily. TRPV6 exhibited substantial co-localization and in vivo interaction with TRPC1 in HEK293 cells, however, no interaction was observed with TRPC3, TRPC4, or TRPC5. Ca(2+) and Na(+) currents of TRPV6-overexpressing HEK293 cells are significantly reduced by co-expression of TRPC1, correlating with a dramatically suppressed plasma membrane targeting of TRPV6. In line with their intracellular retention, remaining currents of TRPC1 and TRPV6 co-expression resemble in current-voltage relationship that of TRPV6. Studying the N-terminal ankyrin like repeat domain, structurally similar in the two proteins, we have found that these cytosolic segments were sufficient to mediate a direct heteromeric interaction. Moreover, the inhibitory role of TRPC1 on TRPV6 influx was also maintained by expression of only its N-terminal ankyrin-like repeat domain. Our experiments provide evidence for a functional interaction of TRPC1 with TRPV6 that negatively regulates Ca(2+) influx in HEK293 cells.
Journal of Biological Chemistry 08/2012; 287(42):35612-20. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Store-operated Ca(2+) entry describes the phenomenon that connects a depletion of internal Ca(2+) stores to an activation of plasma membrane-located Ca(2+) selective ion channels. Tremendous progress towards the underlying molecular mechanism came with the discovery of the two respective limiting components, STIM and Orai. STIM1 represents the ER-located Ca(2+) sensor and transmits the signal of store depletion to the plasma membrane. Here it couples to and activates Orai, the highly Ca(2+)-selective pore-forming subunit of Ca(2+) release-activated Ca(2+) channels. In this review, we focus on the molecular steps that these two proteins undergo from store-depletion to their coupling, the activation, and regulation of Ca(2+) currents.
Cellular and Molecular Life Sciences CMLS 07/2012; · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Control of endothelial phenotype involves a variety of signaling pathways and transcriptional regulators, including the junctional protein β-catenin. This multifunctional signaling molecule is part of adhesion contacts in the endothelium and is able to translocate into the nucleus to activate genetic programs and control proliferation and the fate of the cells. We investigated the influence of laser-generated nanopatterns on polymeric cell culture substrates on endothelial tissue architecture, proliferation and β-catenin signaling. For our experiments human microvascular endothelial cells or CD34(+) endothelial progenitor cells, isolated from human adipose tissue, were cultured on polyethylene terephthalate (PET) substrates with oriented nanostructures with lateral periodicities of 1.5 μm and 300 nm, respectively. The surface topography and chemistry of the PET substrates were characterized by electron microscopy, atomic force microscopy, water contact angle measurement and X-ray photoelectron spectroscopy. Analysis of cell phenotype markers as well as β-catenin signaling revealed that short-term culture of endothelial cells on nanostructured substrates generates a proliferative cell phenotype associated with nuclear accumulation of β-catenin and activation of specific β-catenin target genes. The effects of the nanostructures were not directly correlated with nanostructure-induced alignment of cells and were also clearly distinguishable from the effects of altered PET surface chemistry due to photomodification. In summary, we present a novel mechanism of surface topology-dependent control of transcriptional programs in mature endothelium and endothelial progenitor cells.
[Show abstract][Hide abstract] ABSTRACT: Frequently observed structures in laser-surface processing are ripples, also denoted as laser-induced periodic surface structures (LIPSS). Ripples originate from the interference of the incident/refracted laser light with the scattered or diffracted light near the surface. For many polymer surfaces, organized nano-ripple structures surfaces can be induced by irradiation with pulsed UV lasers with pulse lengths in the order of some nanoseconds at fluences well below the ablation threshold and with a large number of laser pulses N. After exposure to linearly polarized radiation at normal incidence, the lateral period of the observed LIPSS is close to the wavelength λ. This type of structures is usually called low spatial frequency LIPSS (LSFL). For femto-second laser light, ripples at polymer surfaces are also observed at a laser fluence above the ablation threshold, even with low numbers of laser pulses N. Under special conditions, another type of ripples with periods as small as λ/3 has been reported. This type of ripples is called high spatial frequency LIPSS (HSFL). We summarize here our work on LIPSS generation at polymers and describe potential applications in the field of self-organized formation of gold nano-wires and nano-structure induced alignment of biological cells cultivated on polymer substrates.
Transparent Optical Networks (ICTON), 2012 14th International Conference on; 01/2012