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Ca(2+) release activated Ca(2+) (CRAC) channels mediate robust Ca(2+) influx when the endoplasmic reticulum Ca(2+) stores are depleted. This essential process for T-cell activation as well as degranulation of mast cells involves the Ca(2+) sensor STIM1, located in the endoplasmic reticulum and the Ca(2+) selective Orai1 channel in the plasma membrane. Our review describes the CRAC signaling pathway, the activation of which is initiated by a drop in the endoplasmic Ca(2+) level sensed by STIM1. This in term induces multimerisation and puncta-formation of STIM1 proteins is followed by their coupling to and activation of Orai channels. Consequently Ca(2+) entry is triggered through the Orai pore into the cytosol with subsequent closure of the channel by Ca(2+)-dependent inactivation. We will portray a mechanistic view of the events coupling STIM1 to Orai activation based on their structure and biophysics.

Background and purpose Pyrazole derivatives have recently been suggested as selective blockers of transient receptor potential cation (TRPC) channels but their ability to distinguish between the TRPC and Orai pore complexes is ill-defined. This study was designed to characterize a series of pyrazole derivatives in terms of TRPC/Orai selectivity and to delineate consequences of selective suppression of these pathways for mast cell activation. Experimental approach Pyrazoles were generated by microwave-assisted synthesis and tested for effects on Ca2+ entry by Fura-2 imaging and membrane currents by patch-clamp recording. Experiments were performed in HEK293 cells overexpressing TRPC3 and in RBL-2H3 mast cells, which express classical store-operated Ca2+ entry mediated by Orai channels. The consequences of inhibitory effects on Ca2+ signalling in RBL-2H3 cells were investigated at the level of both degranulation and nuclear factor of activated T-cells activation. Key Results Pyr3, a previously suggested selective inhibitor of TRPC3, inhibited Orai1- and TRPC3-mediated Ca2+ entry and currents as well as mast cell activation with similar potency. By contrast, Pyr6 exhibited a 37-fold higher potency to inhibit Orai1-mediated Ca2+ entry as compared with TRPC3-mediated Ca2+ entry and potently suppressed mast cell activation. The novel pyrazole Pyr10 displayed substantial selectivity for TRPC3-mediated responses (18-fold) and the selective block of TRPC3 channels by Pyr10 barely affected mast cell activation. Conclusions and Implications The pyrazole derivatives Pyr6 and Pyr10 are able to distinguish between TRPC and Orai-mediated Ca2+ entry and may serve as useful tools for the analysis of cellular functions of the underlying Ca2+ channels.

The essential molecular components of Ca2+ release-activated Ca2+ (CRAC) channels are represented by the sensor protein STIM1 anchored in the endoplasmatic reticulum (ER) membrane and the pore-forming Orai in the plasma membrane. Channel gating involves direct binding of the cytosolic portion of STIM1 with both N- and C-termini of Orai channel. Recently we have reported (Bergsmann et. al., 2011) that the first half of the N-terminal conserved region of Orai3 is dispensable for its activation either by STIM1 or 2-aminoethoxydiphenyl-borate (2-APB). Here we identified that Orai1 in contrast to Orai3 requires nearly the whole conserved region for store-operated activation. Non-functional Orai1 N-terminal truncation mutants regained partial store-operated function upon substitution of the C-terminus by that of Orai3. Moreover we elucidated that a single point mutation in transmembrane region 1 (TM1) of Orai1 and Orai3 leads to constitutively active channels independent of STIM1. Store-non-responsive Orai1 N-terminal truncation mutants could be revitalized by this single point mutation in TM1. In summary, we identified within the N-terminal conserved region distinct structural requirements for store-operated activation of Orai1 and Orai3. Furthermore, the lack of N-terminal portions essential for Orai1 function can be compensated by domains of Orai3 or single point mutations in TM1. (supported by Austrian Science Fund (FWF) T 466 and P22565)

TRPC3 was repeatedly discussed as a potential component of store-operated signaling pathways and is expressed in mast cells, which display the classical Orai1-mediated CRAC conductance. To elucidate if TRPC3 interferes with this store-operated conductance, we overexpressed TRPC3 in RBL-2H3 mast cells and characterized currents activated by passive store depletion (20mM EGTA). TRPC3 overexpression diminshed the inwardly rectifying CRAC current component significantly when free intracellular Mg2+ was kept at a level of 1.6 mM, and this inhibitory effect was more pronounced when free intracellular Mg2+ was elevated to 8 mM. Moreover, store depletion-induced currents were completely abolished with expression of a pore-dead mutant of TRPC3 (E630K). By contrast, the potential STIM-binding deficient mutant, E697K/E698K lacked effects on CRAC current amplitude. Importantly, at low (1.6 mM) intracellular Mg2+, we observed a reduced Ca2+ selectivity of the store depletion-activated conductance with appearance of a clearly outward rectifying I-V relation. This nonselective conductance was absent at high (8 mM) Mg2+, eliminated by co-expression of the permeation deficient Orai1 E106Q mutation and displayed sensitivity to the TRPC3 blocker Pyr3 (3 μM) as well as to the TRPM7 inhibitor NDGA (10 μM). Biophysical and pharmacological features of the nonselective conductance, which was activated by store depletion at low (physiologic) intracellular Mg2+ levels, favor the concept of generation of a unique membrane conductance by TRPC3 overexpression in RBL cells. Our findings suggest a complex interaction of TRPC3 with Orai1 and probably also TRPM7 in plasma membrane microdomains of mast cells.Supported by FWF, DK-MCD

Ca2+ release activated Ca2+ (CRAC) channels mediate robust Ca2+ influx when the endoplasmic reticulum Ca2+ stores are depleted. This essential process for T-cell activation as well as degranulation of mast cells involves the Ca2+ sensor STIM1, located in the endoplasmic reticulum and the Ca2+ selective Orai1 channel in the plasma membrane. Our review describes the CRAC signaling pathway, the activation of which is initiated by a drop in the endoplasmic Ca2+ level sensed by STIM1. This in term induces multimerisation and puncta-formation of STIM1 proteins is followed by their coupling to and activation of Orai channels. Consequently Ca2+ entry is triggered through the Orai pore into the cytosol with subsequent closure of the channel by Ca2+-dependent inactivation. We will portray a mechanistic view of the events coupling STIM1 to Orai activation based on their structure and biophysics.

A primary Ca2+ entry pathway in non-excitable cells is determined by the Ca2+ release activated Ca2+ channels. Their two limiting molecular components include the Ca2+-sensor protein STIM1 located in the endoplasmic reticulum and the Orai channel in the plasma membrane. STIM1 senses the luminal Ca2+ content. Store depletion induces STIM1 oligomerization into puncta like structures, thereby triggering coupling to as well as activation of Orai1 channels. A C-terminal STIM1 domain couples to both N- and C-terminal, cytosolic strands of Orai, accomplishing gating of the channel. Here we highlight the inter- and intramolecular choreography of STIM1 and Orai proteins that govern in Ca2+ permeation.

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.

Cardiac transient receptor potential canonical (TRPC) channels are crucial upstream components of Ca 2+ /calcineurin/nuclear factor of activated T cells (NFAT) signaling, thereby controlling cardiac transcriptional programs. The linkage between TRPC-mediated Ca 2+ signals and NFAT activity is still incompletely understood. TRPC conductances may govern calcineurin activity and NFAT translocation by supplying Ca 2+ either directly through the TRPC pore into a regulatory microdomain or indirectly via promotion of voltage-dependent Ca 2+ entry. Here, we show that a point mutation in the TRPC3 selectivity filter (E630Q), which disrupts Ca 2+ permeability but preserves monovalent permeation, abrogates agonist-induced NFAT signaling in HEK293 cells as well as in murine HL-1 atrial myocytes. The E630Q mutation fully retains the ability to convert phospholipase C-linked stimuli into L-type (Ca V 1.2) channel-mediated Ca 2+ entry in HL-1 cells, thereby generating a dihydropyridine-sensitive Ca 2+ signal that is isolated from the NFAT pathway. Prevention of PKC-dependent modulation of TRPC3 by either inhibition of cellular kinase activity or mutation of a critical phosphorylation site in TRPC3 (T573A), which disrupts targeting of calcineurin into the channel complex, converts cardiac TRPC3-mediated Ca 2+ signaling into a transcriptionally silent mode. Thus, we demonstrate a dichotomy of TRPC-mediated Ca 2+ signaling in the heart constituting two distinct pathways that are differentially linked to gene transcription. Coupling of TRPC3 activity to NFAT translocation requires microdomain Ca 2+ signaling by PKC-modified TRPC3 complexes. Our results identify TRPC3 as a pivotal signaling gateway in Ca 2+ -dependent control of cardiac gene expression.

The control of cell adhesion at polymer surfaces is of great interest for applications in medicine and biotechnology research. Chondrocytes, which are the only cells found in cartilage, have proven to rapidly dedifferentiate to a fibroblastic phenotype when cultured under 2D conditions. The dedifferentiated phenotype is characterized by a change in cell morphology which is round in case of chondrocytes and spindle shaped in case of dedifferentiated chondrocytes. Furthermore, the fibroblastic phenotype shows increased expression of collagen I relative to collagen II and Integrin α11β1 relative to Integrin α10β1.The aim of this study was to treat the surface of polystyrene (PS), perfluoro ethylene propylene (FEP) and polyethylene terephthalate (PET) by UV laser irradiation for the improvement of adhesion and differentiation grade of human chondrocytes.Depending on the type of polymer and the micro- or nano-structures at the surfaces induced by the surface pretreatment, the chondrocytes showed round morphology and high cluster formation, low cluster formation, or nearly no cluster formation. In contrast, chondrocytes cultured in well plates without foils showed progressive spreading and spindle shaped morphology. (supported by FFG - NSI3).

We used mechanically embossed polyester films to analyze the dynamics of cell alignment and cell-specific factors modulating the response of Chinese hamster ovary (CHO) cells and of a rat myogenic cell line to the surface topography. The films used had grooves with a periodicity of approximately 750 nm and a depth of 150 nm. Both cell lines responded to the topographical feature. On unpatterned control areas, cells of both lines showed a random distribution with orientation angles close to45∘. Both cell types exhibited an elongated morphology on the patterned surface. CHO cells typically showed bipolar spreading. Their contact area increased almost exclusively along the groove direction. Likewise, freshly seeded rat myoblasts displayed protrusions emerging in parallel with the grooves. However, myoblasts frequently had more than two sites with plasma protrusions pulling the cells along different grooves. They could also develop lamellipodia expanding without a preferred direction and long filopodia.

We present a method for laser lithography of cell-adhesive arrays on a fluoropolymer surface. The method is based on 172 nm excimer-lamp photomodification in ammonia atmosphere followed by microstructuring by laser ablation. The improved wettability of the polymer is caused by new chemical groups on the surface after the UV treatment that we proved by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy analyses. The cell adhesion properties of micropatterned structures were tested by cultivation of mammalian cells. We show that single elongated cells can grow confined to lines with sharply defined boundaries of the cell-covered areas. In preliminary experiments, we also demonstrate that the described technique allows the production of single-cell arrays with variable cell shape.

The control of cell adhesion at polymer surfaces is of great interest for applications in medicine and biotechnology research.We present a method of polystyrene (PS) surface treatment by UV laser irradiation (laser wavelength λ=193 nm) for the improvement of adhesion of Chinese hamster ovary (CHO) cells.We irradiated the PS foils with a total fluence of 200 mJ/cm2 in a circular spot. The irradiation led to formation of specific micro pattern at the surface (assessed by AFM and SEM). Depending on the position at the spot, the pattern structure varies. There is a specific ring area (B in figure), where seeded CHO cells show effective adhesion and pronounced spreading. The cells display here a preferential alignment along the ring. The topography of this area has a height variation of 5 to 10 nm, while the surface region irradiated with higher laser intensity (A in figure) has a higher roughness of hundreds of nm. This area is less favorable for CHO cells adhesion as indicated by the round cell morphology, similar to the flat area outside the ring. (Supported by the Austrian NANO Initiative in the projects NSI_NBPF and NSI_PolyModEUV.View Large Image | View Hi-Res Image | Download PowerPoint Slide

The two proteins STIM1 and Orai1 represent the key components fully reconstituting the Ca2+ release-activated Ca2+ (CRAC) current. While the C-termini of both proteins play an essential role in their store-operated coupling, the role of Orai1 N-terminus in the STIM1/Orai1 signaling machinery remains so far elusive. Orai1 N-terminus contains proline- and arginine-rich regions at the very beginning which are lacking in Orai2 and Orai3. At the end of the N-terminal cytoplasmic stretch close to the first transmembrane segment all Orai proteins include a highly conserved amino acid domain that contains positively charged as well as hydrophobic amino acids. This stretch has also been suggested to interact with STIM1.In attempt to elucidate the role of these conserved amino acids we mutated positively charged to either neutral or negatively charged amino acids. Single neutral mutations had no effect on store-operated currents, however, substantially reduced fast inactivation of Orai1 and Orai3, while double mutations strongly reduced current densities. Single negatively charged amino acids increased store-operated currents in correlation with abolished fast inactivation, while double negative mutations also decreased current density. Furthermore mutation of some hydrophobic amino acids within this conserved region resulted in 1.5-fold increased store-operated currents, while inactivation remained unaffected. In summary, we identified respective amino acids within Orais’conserved N-terminal region that play a modulatory role in Orai function. (supported by the Habilitation Scholarship JKU Linz, Austria and FWF P21118)

Two antiallergic drugs, cromolyn and RU 31156, were examined for their potency to block intermediate-conductance Cl- channels. For this, single Cl- channel activities of mucosal-type mast cells (RBL-2H3) and alternatively, of colonic carcinoma cells (HT29) were monitored employing the patch-clamp technique in the inside-out patch configuration. Here we show that intermediate-conductance Cl- channels of either cell type were blocked by both compounds. Cl- channel inhibition occurred by a slow rather than a flickering block and with a Hill coefficient around 1. RU 31156 was about one order of magnitude more potent (IC50 about 1 microM) than cromolyn. The IC50 values of each compound were not significantly different (p0.01) in the two cell types. Our data provide evidence for two novel Cl- channel blockers, which may be of further use in Cl- channel characterization as well as purification.

TRPC4 is well recognized as a prominent cation channel in the vascular endothelium, but its contribution to agonist-induced endothelial Ca(2+) entry is still a matter of controversy. Here we report that the cellular targeting and Ca(2+) signaling function of TRPC4 is determined by the state of cell-cell adhesions during endothelial phenotype transitions. TRPC4 surface expression in human microvascular endothelial cells (HMEC-1) increased with the formation of cell-cell contacts. Epidermal growth factor recruited TRPC4 into the plasma membrane of proliferating cells but initiated retrieval of TRPC4 from the plasma membrane in quiescent, barrier-forming cells. Epidermal growth factor-induced Ca(2+) entry was strongly promoted by the formation of cell-cell contacts, and both siRNA and dominant negative knockdown experiments revealed that TRPC4 mediates stimulated Ca(2+) entry exclusively in proliferating clusters that form immature cell-cell contacts. TRPC4 co-precipitated with the junctional proteins beta-catenin and VE-cadherin. Analysis of cellular localization of fluorescent fusion proteins provided further evidence for recruitment of TRPC4 into junctional complexes. Analysis of TRPC4 function in the HEK293 expression system identified beta-catenin as a signaling molecule that enables cell-cell contact-dependent promotion of TRPC4 function. Our results place TRPC4 as a Ca(2+) entry channel that is regulated by cell-cell contact formation and interaction with beta-catenin. TRPC4 is suggested to serve stimulated Ca(2+) entry in a specific endothelial state during the transition from a proliferating to a quiescent phenotype. Thus, TRPC4 may adopt divergent, as yet unappreciated functions in endothelial Ca(2+) homeostasis and emerges as a potential key player in endothelial phenotype switching and tuning of cellular growth factor signaling.

Calcium (Ca2+) entry into non-excitable cells is mainly carried by store-operated channels, which serve essential functions ranging from regulation of transcription to cell growth. The best-characterized store-operated current, initially discovered in T lymphocytes and mast cells, is the Ca2+ release-activated Ca2+ (CRAC) current. The search for the molecular components of the CRAC channel has recently identified stromal interaction molecule 1 (STIM1) as the Ca2+ sensor in the endoplasmic reticulum (ER) and Orai1 as the CRAC channel pore. ER store depletion results in formation of STIM1 puncta that trigger Ca2+ influx via Orai1 channels. This review covers the role of domains within STIM1 and Orai and enlightens their function in the STIM1/Orai coupling process. Moreover, a molecular interpretation focuses on interactions between cytosolic portions of STIM1 and Orai together with a mechanistic view on the loss of function of the SCID (severe combined immunodeficiency)-linked Orai1 R91W mutant channel. The architecture of the selectivity filter of Orai channels is finally elucidated based on permeation properties of Orai pore mutants.

Ca(2+) entry in non-excitable cells is mainly carried by store-operated channels among which the CRAC channel is best characterized. Its two limiting molecular components are represented by the Ca(2+) sensor protein STIM1 located in the endoplasmic reticulum and Orai1 in the plasma membrane. STIM1 senses a decrease of the Ca(2+) content in internal stores and triggers its accumulation into puncta like structures resulting in coupling to as well as activation of Orai1 channels. The STIM1-Orai coupling process is determined by an interaction via their C-termini. This review highlights recent developments on domains particularly within the cytosolic part of STIM1 that govern this interaction.

In immune cells generation of sustained Ca2+ levels is mediated by the Ca2+ release activated Ca2+ (CRAC) current. Molecular key players in this process comprise the stromal interaction molecule (STIM1) that functions as a Ca2+ sensor in the endoplasmic reticulum and ORAI1 located in the plasma membrane. Depletion of ER Ca2+ store leads to STIM1 multimerization into discrete punctae that co-cluster with ORAI1 thereby triggering coupling to and activation of ORAI1 channels. The C-terminus of STIM1 is sufficient for the activation of ORAI1 currents independent of store depletion. Here we unmasked an ORAI activating minimal fragment (OAMF) within STIM1 C-terminus that exhibited enhanced interaction with ORAI1 and resulted in three-fold increased Ca2+ currents. STIM1-OAMF still showed the ability of a homomeric interaction similar to longer fragments as well as the full-length form of STIM1 C-terminus. In contrast, further deletion of a thirty amino acid region resulted in a substantial reduction of homomeric interaction concomitant to a loss of coupling to as well as activation of ORAI1. In aggregate, we have identified two key regions within STIM C-terminus that govern ORAI1 activation. (Supported by PhD-Program W1201 from the FWF)

Stim1 in the endoplasmic reticulum and the three Orai (also termed CRACM) channels in the plasma-membrane are main components of native Ca(2+) release-activated Ca(2+) channels. A pharmacological hallmark of these channels is their distinct sensitivity to 2-aminoethoxydiphenyl borate (2-APB). Here we report that Orai3 currents can be robustly stimulated by 75 microm 2-APB independent of Stim1, whereas 2-APB at similar concentrations inhibited store-operated Orai1 currents. 2-APB did not only promote currents through Orai3 channels but also dramatically altered ion selectivity of Orai3 channels. This allowed for permeation of monovalent cations both in the inward as well as outward direction, which is in sharp contrast to the high Ca(2+) selectivity of store-operated Orai3 currents. An Orai3-R66W mutant, which lacked in analogy to the severe combined immune deficiency mutant Orai1-R91W store-operated activation, was also found to be resistant to 2-APB stimulation. The change in selectivity by 2-APB was associated with an increase in Orai3 minimum pore size from about 3.8A to more than 5.34 A. In line with a potential interaction of 2-APB with the Orai3 pore, among three pore mutants tested, the Orai3 E165Q mutant particularly resembled in its permeation properties those of 2-APB stimulated Orai3 and additionally exhibited a reduced response to 2-APB. In aggregate, stimulation of Orai3 currents by 2-APB occurred along with an alteration of the permeation pathway that represents a unique mechanism for regulating ion channel selectivity by chemical compounds.

STIM1 and ORAI1 (also termed CRACM1) are essential components of the classical calcium release-activated calcium current; however, the mechanism of the transmission of information of STIM1 to the calcium release-activated calcium/ORAI1 channel is as yet unknown. Here we demonstrate by Förster resonance energy transfer microscopy a dynamic coupling of STIM1 and ORAI1 that culminates in the activation of Ca(2+) entry. Förster resonance energy transfer imaging of living cells provided insight into the time dependence of crucial events of this signaling pathway comprising Ca(2+) store depletion, STIM1 multimerization, and STIM1-ORAI1 interaction. Accelerated store depletion allowed resolving a significant time lag between STIM1-STIM1 and STIM1-ORAI1 interactions. Store refilling reversed both STIM1 multimerization and STIM1-ORAI1 interaction. The cytosolic STIM1 C terminus itself was able, in vitro as well as in vivo, to associate with ORAI1 and to stimulate channel function, yet without ORAI1-STIM1 cluster formation. The dynamic interaction occurred via the C terminus of ORAI1 that includes a putative coiled-coil domain structure. An ORAI1 C terminus deletion mutant as well as a mutant (L273S) with impeded coiled-coil domain formation lacked both interaction as well as functional communication with STIM1 and failed to generate Ca(2+) inward currents. An N-terminal deletion mutant of ORAI1 as well as the ORAI1 R91W mutant linked to severe combined immune deficiency syndrome was similarly impaired in terms of current activation despite being able to interact with STIM1. Hence, the C-terminal coiled-coil motif of ORAI1 represents a key domain for dynamic coupling to STIM1.

Molecular communication between TRPC and Orai channel proteins has been proposed and is still a matter of debate. We investigated functional crosstalk of TRPC3 and Orai1 in RBL mast cells in terms Orai-mediated NFAT signalng. Overexpression or shRNA knock-down of TRPC3 failed to affect NFAT activation in response to Orai1-mediated Ca2+ entry. Nonetheless, expression of a calcineurin (CaN) binding-deficient TRPC3 mutant (TRPC3P704Q) abolished NFAT translocation initiated by Ca2+ entry into store-depleted RBL cells without significant reduction of store-operated Ca2+ currents. TIRF-FRET experiments revealed a subpopulation of resting Orai1 complexes that are physically coupled to CaN and colocalized with TRPC3 proteins. Overexpressed TRPC3 proteins interfered with CaN recruitment into resting Orai-complexes in that Orai-CaN association was enhanced by TRPC3 and eliminated by TRPC3P704Q expression. Impaired Orai-CaN association in TRPC3P704Q expressing RBL cells was accompanied by reduced targeting of Orai into preformed PM-ER junctional connections. We conclude that TRPC3 governs Orai-mediated activation of NFAT by promoting resting-state Orai-CaN interactions and recruitment of resting-state Orai channels into PM-ER junctions.

Heparin and related polyanions are a new class of compounds interacting with 1,4-dihydropyridine-sensitive L-type Ca2+ channels in a tissue-specific manner. Labeling of membrane-bound Ca2+ channels in rabbit skeletal muscle transverse tubules at the phenylalkylamine, benzothiazepine, and 1,4-dihydropyridine-selective domains was inhibited reversibly by a noncompetitive mechanism as shown by equilibrium saturation analysis and kinetic studies. (+)-cis-diltiazem but not (-)-cis-diltiazem reduced the inhibitory potency of heparin for 1,4-dihydropyridines. Antagonistic but not agonistic 1,4-dihydropyridines reversed heparin inhibition at the benzothiazepine site. Heparin forms a tight complex with the purified Ca2+ channel which is highly sensitive with respect to heparin inhibition (IC50 value: 0.05 microgram/ml) of 1,4-dihydropyridine binding. Reconstituted channel complexes have completely lost 1,4-dihydropyridine binding-inhibition by heparin and are not retained by lectin or heparin affinity columns. In whole cell patch clamp experiments with guinea-pig cardiac myocytes heparin increased the current through L-type Ca2+ channels when applied extracellulary. Synthetic peptides (representing putative heparin binding domains) which were derived from the rabbit skeletal muscle alpha 1-subunit reversed the inhibitory effects of heparin on 1,4-dihydropyridine receptors. Reversal for a peptide representing an extracellular domain occurred by an apparently competitive mechanism. It is suggested that heparin and related polyanions may interact with an evolutionary conserved cluster of basic amino acids in the large putative extracellular domain connecting the fifth and sixth putative transmembrane segment in the first motif of the ionic pore-forming alpha 1-subunit from skeletal muscle.

A novel modified nanocomposite was studied for the adhesion and proliferation of the human umbilical vein endothelial cell (HUVEC) line EA.hy926. The nanocomposite under investigation was poly(carbonate-urea)urethane with silsesquioxane nano-cages, here in the form of a mixture of two polyhedral oligomeric silsesquioxanes. The nanocomposite surfaces were exposed to ultraviolet (UV) light of a Xe(*)(2)-excimer lamp at a wavelength of 172 nm in an ammonia atmosphere. The effects of the irradiation were characterized by atomic force and scanning electron microscopy (AFM, SEM), X-ray photo-electron spectroscopy (XPS), Fourier-transform infrared spectroscopy (FT-IR) using an attenuated total reflection (ATR) device and measurements of advancing water contact angle (CA). The irradiation resulted in the introduction of new hydrophilic N- and O-containing groups into the surface, which was initially amphiphilic, while surface morphology remained mainly unchanged. Slight chemical changes were also observed for the silsesquioxane nano-cages at the surface. Onto the untreated and irradiated samples HUVECs were seeded and grown for various durations in culture. Standard tissue-culture polystyrene (PS) was employed as a positive control to check the efficiency of the cell-culture methods. Viability and proliferation of the cells were then assessed using a non-radioactive assay. Compared to the untreated nanocomposite polymer, irradiation times of at least 5 min resulted in a significantly increased cell proliferation between 3 and 8 days after seeding with the HUVEC line EA.hy926.

The closely related TRPC4 and TRPC5 proteins, members of the canonical transient receptor potential (TRPC) family, assemble into either homo- or heterotetrameric, non-selective cation-channels. To elucidate domains that mediate channel complex formation, we evaluated dominant negative effects of N- or C-terminal TRPC4/5 fragments on respective currents of full-length proteins overexpressed in HEK293 cells with whole-cell electrophysiology. Confocal Forster Resonance Energy Transfer (FRET) measurements enabled to probe the interaction potential of these CFP/YFP-labelled fragments in vivo. Only N-terminal fragments that included the first ankyrin-like repeat potently down-regulated TRPC4/TRPC5 currents, while fragments including either the second ankyrin-like repeat and the coiled-coil domain or the C-terminus remained ineffective. Total internal reflection fluorescence (TIRF) microscopy data suggested that the dominant negative N-terminal fragments led to a predominantly intracellular localisation of coexpressed TRPC5 proteins. FRET measurements clearly revealed that only fragments including the first ankyrin-like repeat were able to multimerise. Moreover a TRPC5 mutant that lacked the first ankyrin-like repeat was unable to homo-multimerise, failed to interact with wild-type TRPC5 and resulted in non-functional channels.

Introduction The activity of many types of calcium channels decreases rapidly when contact between membrane and cytosol is disrupted. This phenomenon, called runnlown, is wide spread among voltage gated calcium channels and appears to be related to regulatory factors present in the cytosol, which are rapidly lost upon patch excision. Even in whole cell experiments currents exhibit a slow run-down, suggesting that endogenous cytoplasmic factors are subject to wash-out. ' It is now generally recognized that run-down of ion channel activity is not due to an artificial artefact.^ Run-down is time-dependent and generally fast, so that the complete closure of the channel occurs within a few minutes. Run-down has never been reported so far to show any voltage dependence. The loss of channel activity does not result from a lack of Ca^* channel integrity, ' because of the following reasons: a. The single channel current amplitude remains stable during run-down b. The run-down in excised patches corresponds to the spontaneous drops in either the channel open probability (PQ) or the number of diannels (N) present in multi-channel patches or both Afiier run-down, calcium channels can be re-activated excluding a proteolytic process interfering with channel activity.

The large family of mammalian TRP (transient receptor potential) ion channels encompasses diverse sensory functions. TRP proteins consist of six transmembrane domains, with a pore–loop motif between the fifth and sixth domains and cytosolic N- and C-termini. The intracellular strands not only interact with various proteins and lipids, but also include essential multimerization regions. This review summarizes the current knowledge of the intrinsic assembly domains that assure tetrameric TRP channel formation.

We describe here the modification of various polymers (polytetrafluoroethylene, polyethyleneterephthalate, and polyvinyl alcohol) by UV-irradiation with wavelengths below 200 nm in an inert or reactive atmosphere. The light sources employed are F2- or excimer lasers and excimer lamps. The reactive gases include ammonia (NH3), acetylene (C2H2), and oxygen (O2). Photo-dissociated fragments of these gases can react with the polymers or be deposited thereon, resulting in new chemical groups at the surface. Special emphasis is put to improved adhesion of biological cells at these modified surfaces. Potentials applications include cell coated medical implants and prostheses as well as cell micro-arrays for high throughput screening.© (2006) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Oriented stable binding of functional proteins on surfaces is of fundamental interest for receptor/ligand studies in atomic force microscopy (AFM) and surface plasmon resonance (SPR) experiments. Here we have chosen the His 6 -tagged carboxyl-tail (C-tail) of the α 1 C -subunit of the L-type Ca 2 + channel and calmodulin (CaM) as its cognitive partner as a model system to develop a new functional surface. Covalently attached self-assembled monolayers on ultraflat gold containing NTA-thiols to which the Hiss-tagged C-tail was bound and thiols with triethylene-glycol groups as matrix-thiols represented the system of choice. The topography of this surface was characterized using AFM; its ability to bind C-tail proteins oriented and stable was confirmed by SPR measurements and by complementary force spectroscopy experiments with a CaM 4 -construct covalently attached to the tip. The developed anchoring strategy can now be used to study receptor/ligand interactions in general applying force spectroscopy and SPR on His6-tagged proteins oriented immobilized onto this new NTA-functionalized self-assembled monolayer.

UV-irradiation in a reactive atmosphere can improve the biocompatibility of poly-tetrafluoroethylene (PTFE, [C 2 F 4 ] n ) in a single step with high efficiency and single parameter control. Now we extend our investigations to well defined micro-patterns on PTFE. Potential applications are cell arrays for in-situ gene product analysis of living cells.

The article describes the modification of fluoropolymers by UV-irradiation with wavelengths below 200 nm in a reactive or inert atmosphere. Light sources employed are excimer lamps, excimer-lasers or F 2 -lasers. Special emphasis is put on adhesion phenomena of fluids, biological cells and metal coatings at the modified surfaces.

Complexes formed between beta2GPI (beta2-glycoprotein I), a human plasma protein, and biological membranes are considered to be targets of macrophages and antiphospholipid autoantibodies involved in autoimmune diseases, such as antiphospholipid syndrome or systemic lupus erythematosus. The positively charged lysine-rich fifth domain of beta2GPI facilitates its interaction with phospholipid membranes containing acidic phospholipids, which normally become exposed by apoptotic processes. In the present study, atomic force microscopy was applied to visualize the binding of beta2GPI to a mixed phospholipid model membrane at physiological ionic strength. On supported lipid bilayers the formation of supramolecular assemblies of the protein with a height of approx. 3.3 nm was observed, suggesting a lateral agglomeration of beta2GPI. Detailed analysis of kinetic constants using surface plasmon resonance revealed that the binding can be described by a two-state reaction model, i.e. a very fast interaction step, depending on the content of acidic phospholipids in the bilayer, and a second step with significantly lower k(on) and k(off) values. Taken together, our results suggest a biphasic interaction mechanism: a fast step of beta2GPI binding to negatively charged lipids, mainly based on electrostatic interactions, and a slower phase of agglomeration of the protein on the bilayer surface accompanied by a protein-induced rigidification of the membrane, as revealed by electron paramagnetic resonance.

Auxiliary beta-subunits bound to the cytoplasmic alpha(1)-interaction domain of the pore-forming alpha(1C)-subunit are important modulators of voltage-gated Ca(2+) channels. The underlying mechanisms are not yet well understood. We investigated correlations between differential modulation of inactivation by beta(1a)- and beta(2)- subunits and structural responses of the channel to transition into distinct functional states. The NH(2)-termini of the alpha(1C)- and beta-subunits were fused with cyan or yellow fluorescent proteins, and functionally coexpressed in COS1 cells. Fluorescence resonance energy transfer (FRET) between them or with membrane-trapped probes was measured in live cells under voltage clamp. It was found that in the resting state, the tagged NH(2)-termini of the alpha(1C)- and beta-subunit fluorophores are separated. Voltage-dependent inactivation generates strong FRET between alpha(1C) and beta(1a) suggesting mutual reorientation of the NH(2)-termini, but their distance vis-à-vis the plasma membrane is not appreciably changed. These voltage-gated rearrangements were substantially reduced when the beta(1a)-subunit was replaced by beta(2). Differential beta-subunit modulation of inactivation and of FRET between alpha(1C) and beta were eliminated by inhibition of the slow inactivation. Thus, differential beta-subunit modulation of inactivation correlates with the voltage-gated motion between the NH(2)-termini of alpha(1C)- and beta-subunits and targets the mechanism of slow voltage-dependent inactivation.

CaT1, the calcium transport protein 1 encoded by TRPV6, is able to generate a Ca(2+) conductance similar but not identical to the classical CRAC current in mucosal-type mast cells. Here we show that CaT1-derived Ca(2+) entry into HEK293 cells is effectively inhibited either by expression of various dominant negative N-terminal fragments of CaT1 (N(334)-CaT1, N(198)-CaT1 and N(154)-CaT1) or by antisense suppression. By contrast, the endogenous CRAC current of the mast cells was unaffected by CaT1 antisense and siRNA knockdown but markedly suppressed by two (N(334)-CaT1, N(198)-CaT1) of the dominant negative N-CaT1 fragments. Inhibition of CRAC current was not an unspecific, toxic effect, as inward rectifier K(+) and MagNuM currents of the mast cells were not significantly affected by these N-CaT1 fragments. The shortest N(154)-CaT1 fragment inhibited CaT1-derived currents in mast cells, but failed to inhibit CRAC currents. Thus, the structural requirements of rCaT N-terminal fragments for inhibition of rCaT1 and CRAC channels are different. These results together with the lack of CaT1 antisense and siRNA effects on currents render it unlikely that CaT1 is a component of native CRAC channels in mast cells. The data further demonstrate a novel strategy for CRAC current inhibition by an N-terminal structure of CaT1.

While long-term effects of temperature treatment in respect of, e.g., gene-expression and cellular function have already been studied in some detail, nothing is known on the physiological responses of lymphocytes during short-term hypothermal shifts. In this report, we characterized the effects of such a stimulation using the human lymphocyte cell line Jurkat E6.1 and present evidence that warming from 4 to 37 degrees C for only 2 min is sufficient to cause co-localization of CD3, prion protein and the lipid-raft ganglioside GM1 paralleling lymphocyte activation as observed by Ca(2+) mobilization and mitogen-activated protein kinase-phosphorylation.

TRPC3 has been suggested as a key component of phospholipase C-dependent Ca(2+) signaling. Here we investigated the role of TRPC3-mediated Na(+) entry as a determinant of plasmalemmal Na(+)/Ca(2+) exchange. Ca(2+) signals generated by TRPC3 overexpression in HEK293 cells were found to be dependent on extracellular Na(+), in that carbachol-stimulated Ca(2+) entry into TRPC3 expressing cells was significantly suppressed when extracellular Na(+) was reduced to 5 mm. Moreover, KB-R9743 (5 microm) an inhibitor of the Na(+)/Ca(2+) exchanger (NCX) strongly suppressed TRPC3-mediated Ca(2+) entry but not TRPC3-mediated Na(+) currents. NCX1 immunoreactivity was detectable in HEK293 as well as in TRPC3-overexpressing HEK293 cells, and reduction of extracellular Na(+) after Na(+) loading with monensin resulted in significant rises in intracellular free Ca(2+) (Ca(2+)(i)) of HEK293 cells. Similar rises in Ca(2+)(i) were recorded in TRPC3-overexpressing cells upon the reduction of extracellular Na(+) subsequent to stimulation with carbachol. These increases in Ca(2+)(i) were associated with outward membrane currents at positive potentials and inhibited by KB-R7943 (5 microm), chelation of extracellular Ca(2+), or dominant negative suppression of TRPC3 channel function. This suggests that Ca(2+) entry into TRPC3-expressing cells involves reversed mode Na(+)/Ca(2+) exchange. Cell fractionation experiments demonstrated co-localization of TRPC3 and NCX1 in low density membrane fractions, and co-immunoprecipitation experiments provided evidence for association of TRPC3 and NCX1. Glutathione S-transferase pull-down experiments revealed that NCX1 interacts with the cytosolic C terminus of TRPC3. We suggest functional and physical interaction of nonselective TRPC cation channels with NCX proteins as a novel principle of TRPC-mediated Ca(2+) signaling.

We studied the adhesion and proliferation of human endothelial cells on photochemically modified polytetrafluoroethylene samples. The polymer surfaces were modified by exposure to the ultraviolet light of a Xe(2)(*)-excimer lamp at a wavelength of 172 nm in an ammonia atmosphere. Treatment times were between 10 and 20 min. The endothelial cell density was determined 1, 3 and 8 days after seeding by image analysis. Surface modification of the samples resulted in a significant increase in the number of adhering cells and in the formation of a confluent cell layer after 3-8 days. The results were comparable than those obtained on polystyrene Petri dishes, which are used as standard substrates in cell cultivation. Thus modified PTFE appears to be a promising material for the fabrication of artificial vascular prostheses coated with endothelial cells.

We studied the adhesion and proliferation of human umbilical-vein endothelial cells (HUVEC) and human aortic smooth-muscle cells (HASMC) on modified polymer samples. The polymers under investigation were either PTFE (polytetrafluoroethylene) or PET [poly(ethylene terephthalate)], which are among the most frequently employed biomaterials in reconstructive medicine. The polymer surfaces were modified by exposure to the UV light of an excited-xenon-dimer (Xe2*) Excimer lamp (Heraeus-Noblelight, Hanau, Germany) at a wavelength of 172 nm in an NH3 atmosphere. On modified PTFE, this resulted in a significant increase in the number of adhering HUVEC or HASMC 1 day after seeding and in the formation of a confluent cell layer after 3-4 days. On PET, HUVEC adhesion and proliferation rates were already high on untreated samples and could not be significantly increased by the UV-light treatment, but the cells were distributed more homogenously on the treated samples. Cell proliferation was in all cases comparable with, or even better than, that obtained on standard polystyrene (PS) Petri dishes used in cell cultivation. As on PS Petri dishes, the proliferation of HASMC on modified PTFE was faster than that of HUVEC under similar culture conditions.

This study was designed to investigate the role of voltage-independent and voltage-dependent Ca2+ channels in the Ca2+ signaling associated with intracellular alkalinization in A7r5 vascular smooth muscle cells. Extracellular administration of ammonium chloride (20 mmol/L) resulted in elevation of intracellular pH and activation of a sustained Ca2+ entry that was inhibited by 2-amino-ethoxydiphenyl borate (2-APB, 200 micromol/L) but not by verapamil (10 micro;mol/L). Alkalosis-induced Ca2+ entry was mediated by a voltage-independent cation conductance that allowed permeation of Ca2+ (PCa/PNa approximately 6), and was associated with inhibition of L-type Ca2+ currents. Alkalosis-induced inhibition of L-type Ca2+ currents was dependent on the presence of extracellular Ca2+ and was prevented by expression of a dominant-negative mutant of calmodulin. In the absence of extracellular Ca2+, with Ba2+ or Na+ as charge carrier, intracellular alkalosis failed to inhibit but potentiated L-type Ca2+ channel currents. Inhibition of Ca2+ currents through voltage-independent cation channels by 2-APB prevented alkalosis-induced inhibition of L-type Ca2+ currents. Similarly, 2-APB prevented vasopressin-induced activation of nonselective cation channels and inhibition of L-type Ca2+ currents. We suggest the existence of a pH-controlled Ca2+ entry pathway that governs the activity of smooth muscle L-type Ca2+ channels due to control of Ca2+/calmodulin-dependent negative feedback regulation. This Ca2+ entry pathway exhibits striking similarity with the pathway activated by stimulation of phospholipase-C-coupled receptors, and may involve a similar type of cation channel. We demonstrate for the first time the tight functional coupling between these voltage-independent Ca2+ channels and classical voltage-gated L-type Ca2+ channels.

The intestinal Ca(2+) transport protein CaT1 encoded by TRPV6 has been reported (Yue, L., Peng, J. B., Hediger, M. A., and Clapham, D. E. (2001) Nature 410, 705-709) to be all or a part of the Ca(2+) release-activated Ca(2+) channel (CRAC). The major characteristic of CRAC is its activation following store depletion. We expressed CaT1 in HEK293 cells and rat basophilic leukemia (RBL) mast cells and measured whole-cell currents by the patch clamp technique. In HEK293 cells, the expression of CaT1 consistently yielded a constitutively active current, the size of which was strongly dependent on the holding potential and duration of voltage ramps. In CaT1-expressing RBL cells, the current was either activated by store depletion or was constitutively active at a higher current density. CaT1 currents could be clearly distinguished from endogenous CRAC by their typical current-voltage relationship in divalent free solution. 2-aminoethoxydiphenyl borate (2-APB), which is considered a blocker of CRAC, was tested for its inhibitory effect on both cell types expressing CaT1. Endogenous CRAC as well as store-dependent CaT1-derived currents of RBL cells were largely blocked by 75 microm 2-APB, whereas constitutively active CaT1 currents in both RBL and HEK293 cells were slightly potentiated. These results indicate that despite the difference in the permeation properties of CRAC and CaT1 channels, the latter are similarly able to form store depletion-activated conductances in RBL mast cells that are inhibited by 2-APB.

Among store-operated Ca2+ entry (SOCE) pathways the Ca2+ release-activated Ca2+ (CRAC) channel is best characterized. STIM1 and Orai1 have been identified as the molecular key components of the CRAC channel. STIM1 acts as a Ca2+ sensor in the ER. Upon store-depletion STIM1 proteins oligomerize and translocate close to the plasma membrane. Thereby Orai proteins, representing pore-forming subunits in the plasma membrane, are activated by coupling to STIM1 multimers.Here we focused especially on the role of Orai N-termini. It is noticeable that this cytosolic strand includes an among all Orai proteins conserved region close to the first TM domain, which contains positively charged as well as hydrophobic amino acids.Up to now the role of these residues has not been clarified. Moreover this N-terminal conserved region functions not only as a binding partner for STIM1, but also for CaM and CRACR2A. The series of positively charged amino acids further resembles a PIP2 binding domain. With respect to these overlapping regions, we examined the role of positively charged and hydrophobic amino acids by generating point mutants. Single point mutations of positively charged amino acids reduced fast inactivation, possibly due to impairment of CaM binding. Replacement of hydrophobic amino acids did not affect fast inactivation excluding the disturbance of CaM binding. However, store-operated currents of these hydrophobic amino acid mutants were significantly increased, suggesting a possible connection with lipid-mediated processes.Mag. Lackner Barbara is a Recipient of a DOC/DOC-fFORTE-fellowship of the Austrian Academy of Sciences at the Institute of Biophysics at the Johannes-Kepler University of Linz.

Ca2+ influx in non-excitable cells is mainly carried by store-operated channels (SOCs), where Orai1 (CRACM1) and STIM1 represent the two molecular key players in this process. STIM1 functions as an endoplasmic reticulum located Ca2+ sensor and transmits the signal of store depletion to the plasma membrane by coupling to Orai1, which in turn causes channel activation. STIM1 C-terminus itself can act as a surrogate of full length STIM1 and is sufficient for the activation of Orai1 currents. 2-aminoethoxydiphenylborate (2-APB) has been shown to induce enhanced association of STIM1 C-terminus with Orai1 suggesting a conformational change within the former that drives this association. Indeed we were able to monitor 2-APB induced STIM1 C-terminal conformational rearrangement by fluorescence microscopy. In the absence of 2-APB the change in conformation was only seen for STIM1 C-terminus coupled to Orai1 but not for that part remaining in the cytosol suggesting this conformational change crucial for Orai1 binding. Moreover, we were able to mimic this conformational rearrangement by introducing selective point mutations into STIM1 C-terminus, which in line substantially increased binding to Orai1. In aggregate, our data support the theory of flexible regions within STIM C-terminus that undergo conformational rearrangement upon coupling to Orai1. (Supported by FWF-P21118)

The role of the 80-amino acid motif 1572-1651 in the C-terminal tail of alpha(1C) Ca(2+) channel subunits was studied by comparing properties of the conventional alpha(1C,77) channel expressed in HEK-tsA201 cells to three isoforms carrying alterations in this motif. Replacement of amino acids 1572-1651 in alpha(1C,77) with 81 non-identical residues leading to alpha(1C,86) impaired membrane targeting and cluster formation of the channel. Similar to alpha(1C, 86), substitution of its 1572-1598 (alpha(1C,77L)) or 1595-1652 (alpha(1C,77K)) segments into the alpha(1C,77) channel yielded single-channel Ba(2+) currents with increased inactivation, reduced open probability and unitary conductance, when compared to the alpha(1C,77) channel. Thus, the C-terminal sequence 1572-1651 of the alpha(1C) subunit is important for membrane targeting, permeation and open probability of L-type Ca(2+) channels.

STIM1 and ORAI1, the two limiting components in the CRAC signalling cascade, have been reported to couple tightly upon store-depletion culminating in CRAC current activation. Based on the homology within the ORAI protein family, an analogous scenario might be assumed for ORAI2 as well as ORAI3 channels as both are activated in a similar store- and STIM1-dependent manner. A combined approach of electrophysiology and confocal Forster Resonance Energy Transfer (FRET) microscopy revealed a general mechanism in the communication of STIM1 with ORAI proteins that involved the predicted second coiled-coil motif in STIM1 C-terminus and the conserved putative coiled-coil domain in the respective ORAI C-terminus. Of the latter, a much higher coiled-coil probability is predicted for ORAI2 as well as ORAI3 than for ORAI1, compatible with our observation that a single point coiled-coil mutation in ORAI1 C-terminus abrogated communication with STIM1 C-terminus, while an analogous mutation in ORAI2 and ORAI3 still allowed for their moderate activation. Conversely, destabilizing the second coiled-domain of STIM1 C-terminus by a single point mutation still enabled partial stimulation of ORAI2 and ORAI3 channels but not of ORAI1. A double mutation within the second coiled-coil motif of STIM1 C-terminus fully disrupted communication with all three ORAI channels. In aggregate, the impairment in the overall communication between STIM1 and ORAI channels upon mutual destabilization of putative coiled-coil domains in either C-terminus would be compatible with their heteromeric interaction.Supported by FWF P18169.

The endoplasmic reticulum Ca2+ sensor STIM1 forms together with the Ca2+ channel Orai1 the molecular basis for Ca2+ release activated Ca2+ (CRAC) channels. The recent crystal structure of Orai from Drosophila melanogaster shows a unique Ca2+ channel composed of a hexameric subunit complex. The pore structure is formed by transmembrane (TM) 1 helices, surrounded by two ring-like structures, formed by TM2 and TM3 as well as TM4. Employing a combined approach of patch-clamp, molecular biology, biochemical techniques, molecular modeling and structure guided mutagenesis; we discovered a novel key mutation in the second trans-membrane helix of Orai1 that results in a Ca2+ selective, STIM1 independent, constitutively active current. Substitution of this essential residue to a hydrophobic amino-acid retained store-operated activation, yet with largely reduced Orai1 currents. In addition, we took advantage of the constitutively active Orai1 mutant, to evaluate reorientation of the gate located within the cytosolic region of TM1 helices. Cysteine scanning mutagenesis within the TM1 helix enabled identification of gating residues, the dimerization of which was altered in the constitutively opened and closed Orai1 channel conformation. In molecular dynamic simulations of an all-atom model of human Orai1 we will show the interaction network of these identified residues and predict implication of mutations on conformational changes. Our experiments will be summarized in a unique gating model, and we will moreover discuss how STIM1 binding might trigger the open channel conformation. This work was supported by the Austrian Science Foundation (FWF): P26067 to R.S. and P25172 to C.R. Irene Frischauf is an Elise Richter Scholarship holder: V286.

Let me first take the opportunity to thank all the authors for their excellent contributions to this special issue dedicated to the relatively young field of “STIM/Orai” research. I would also like to express my sincere thanks to the Channels Editor-in-Chief, Gerald Zamponi, who approached me more than a year ago with this project and the extraordinary dedicated Channels staff, in particular, Harmony Zambrano, who successfully helped to get this issue wrapped up. The concept of store-operated channels (SOCs) was first introduced by James Putney, almost 30 years ago, suggesting that the depletion of an intracellular Ca2+ store leads to the activation of Ca2+ entry, the extent of which is regulated by the filling state of the endoplasmic reticulum. The prototypic SOC is represented by the Ca2+ release-activated Ca2+ (CRAC) current which has been initially characterized by electrophysiological experiments in mast cells and T-lyphocytes. In 2005 and 2006, the molecular key components of CRAC channels have been identified as STIM1 and Orai, respectively. The latest breakthrough, providing structural resolution, has been obtained in 2012 by the crystallization of active entities of both STIM1 and Orai. In the time period between the identification of these key components and their structural resolution, substantial progress has been achieved in understanding the mechanism of STIM/Orai coupling culminating in CRAC channel activation. Moreover, additional proteins have emerged that modulate STIM-Orai communication. Even store-independent activation has been identified by arachidonate- (ARC) or leukotrieneC4- regulated Ca2+-channels involving STIM1/Orai. All of these findings have meanwhile paved the way for elucidation of the (patho)physiological role of STIM and Orai proteins in native Ca2+ signaling pathways, for instance in regulating gene expression or immunity and their involvement in pathogenesis of various diseases. Particularly with respect to the latter, molecular pharmacology on the STIM/Orai system has just started in an attempt to provide tissue- and disease-specific drugs for therapeutic interventions. The purpose of this special issue of Channels is to cover the above aspects in a manner that provides students or scientists interested in the field with up-to-date information on the current concepts and developments in the STIM/Orai research, which we expect to gain increasing importance as molecular structures and mechanisms will be linked to (patho)physiological processes.

1. The novel cation channel blocker, LOE 908, was tested for its effects on Ca2+ entry and membrane currents activated by depletion of intracellular Ca2+ stores in human endothelial cells. 2. LOE 908 inhibited store-operated Ca2+ entry induced by direct depletion of Ca2+ stores with 100 nM thapsigargin or 100 nM ionomycin with an EC50 of 2 microM and 4 microM, respectively. 3. LOE 908 did not affect thapsigargin- or ionomycin-induced Ca2+ release from intracellular stores up to concentrations of 3 microM. 4. LOE 908 reversibly suppressed thapsigargin- as well as ionomycin-induced whole-cell membrane currents. 5. The LOE 908-sensitive membrane conductance corresponded to a cation permeability of 5.5 and 6.9 fold selectivity for Ca2+ over K+ in the presence of thapsigargin and ionomycin, respectively. 6. Our results suggest that the isoquinoline, LOE 908 is a novel, potent inhibitor of the store-operated (capacitive) Ca2+ entry pathway in endothelial cells.