RESUMEN
The mechanisms by which physical forces regulate endothelial cells to determine the complexities of vascular structure and function are enigmatic. Studies of sensory neurons have suggested Piezo proteins as subunits of Ca(2+)-permeable non-selective cationic channels for detection of noxious mechanical impact. Here we show Piezo1 (Fam38a) channels as sensors of frictional force (shear stress) and determinants of vascular structure in both development and adult physiology. Global or endothelial-specific disruption of mouse Piezo1 profoundly disturbed the developing vasculature and was embryonic lethal within days of the heart beating. Haploinsufficiency was not lethal but endothelial abnormality was detected in mature vessels. The importance of Piezo1 channels as sensors of blood flow was shown by Piezo1 dependence of shear-stress-evoked ionic current and calcium influx in endothelial cells and the ability of exogenous Piezo1 to confer sensitivity to shear stress on otherwise resistant cells. Downstream of this calcium influx there was protease activation and spatial reorganization of endothelial cells to the polarity of the applied force. The data suggest that Piezo1 channels function as pivotal integrators in vascular biology.
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Células Endoteliales/citología , Células Endoteliales/fisiología , Fricción , Canales Iónicos/metabolismo , Estrés Mecánico , Animales , Embrión de Mamíferos/irrigación sanguínea , Embrión de Mamíferos/metabolismo , Femenino , Hemorreología , Masculino , RatonesRESUMEN
The concentration of free cytosolic Ca2+ and the voltage across the plasma membrane are major determinants of cell function. Ca2+-permeable non-selective cationic channels are known to regulate these parameters, but understanding of these channels remains inadequate. Here we focus on transient receptor potential canonical 4 and 5 proteins (TRPC4 and TRPC5), which assemble as homomers or heteromerize with TRPC1 to form Ca2+-permeable non-selective cationic channels in many mammalian cell types. Multiple roles have been suggested, including in epilepsy, innate fear, pain, and cardiac remodeling, but limitations in tools to probe these channels have restricted progress. A key question is whether we can overcome these limitations and develop tools that are high-quality, reliable, easy to use, and readily accessible for all investigators. Here, through chemical synthesis and studies of native and overexpressed channels by Ca2+ and patch-clamp assays, we describe compound 31, a remarkable small-molecule inhibitor of TRPC1/4/5 channels. Its potency ranged from 9 to 1300 pm, depending on the TRPC1/4/5 subtype and activation mechanism. Other channel types investigated were unaffected, including TRPC3, TRPC6, TRPV1, TRPV4, TRPA1, TRPM2, TRPM8, and store-operated Ca2+ entry mediated by Orai1. These findings suggest identification of an important experimental tool compound, which has much higher potency for inhibiting TRPC1/4/5 channels than previously reported agents, impressive specificity, and graded subtype selectivity within the TRPC1/4/5 channel family. The compound should greatly facilitate future studies of these ion channels. We suggest naming this TRPC1/4/5-inhibitory compound Pico145.
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Bloqueadores de los Canales de Calcio/química , Bloqueadores de los Canales de Calcio/farmacología , Canales Catiónicos TRPC/antagonistas & inhibidores , Calcio/metabolismo , Células HEK293 , Humanos , Proteína ORAI1/antagonistas & inhibidores , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismoRESUMEN
OBJECTIVE: Vascular endothelial growth factor (VEGF) acts, in part, by triggering calcium ion (Ca(2+)) entry. Here, we sought understanding of a Synta66-resistant Ca(2+) entry pathway activated by VEGF. APPROACH AND RESULTS: Measurement of intracellular Ca(2+) in human umbilical vein endothelial cells detected a Synta66-resistant component of VEGF-activated Ca(2+) entry that occurred within 2 minutes after VEGF exposure. Knockdown of the channel-forming protein Orai3 suppressed this Ca(2+) entry. Similar effects occurred in 3 further types of human endothelial cell. Orai3 knockdown was inhibitory for VEGF-dependent endothelial tube formation in Matrigel in vitro and in vivo in the mouse. Unexpectedly, immunofluorescence and biotinylation experiments showed that Orai3 was not at the surface membrane unless VEGF was applied, after which it accumulated in the membrane within 2 minutes. The signaling pathway coupling VEGF to the effect on Orai3 involved activation of phospholipase Cγ1, Ca(2+) release, cytosolic group IV phospholipase A2α, arachidonic acid production, and, in part, microsomal glutathione S-transferase 2, an enzyme which catalyses the formation of leukotriene C4 from arachidonic acid. Shear stress reduced microsomal glutathione S-transferase 2 expression while inducing expression of leukotriene C4 synthase, suggesting reciprocal regulation of leukotriene C4-synthesizing enzymes and greater role of microsomal glutathione S-transferase 2 in low shear stress. CONCLUSIONS: VEGF signaling via arachidonic acid and arachidonic acid metabolism causes Orai3 to accumulate at the cell surface to mediate Ca(2+) entry and downstream endothelial cell remodeling.
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Aterosclerosis/genética , Canales de Calcio/genética , Calcio/metabolismo , Regulación de la Expresión Génica , ARN/genética , Factor A de Crecimiento Endotelial Vascular/genética , Remodelación Vascular/genética , Animales , Aterosclerosis/metabolismo , Aterosclerosis/patología , Canales de Calcio/biosíntesis , Movimiento Celular , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales de la Vena Umbilical Humana , Humanos , Immunoblotting , Inmunohistoquímica , Masculino , Ratones , Ratones Desnudos , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
CRACR2A protein is described in T cells as an EF-hand-containing modulator of calcium-release-activated calcium (CRAC) channels. Here we sought relevance to calcium entry of endothelial cells. Unexpectedly, short interfering RNA designed to deplete CRACR2A had no effect on CRAC channels in endothelial cells but reduced the abundance of a protein with about twice the mass of CRACR2A. Reference to gene sequence data indicated the potential for a variant transcript encoding a C-terminal Rab GTPase extension of CRACR2A. Full-length cloning demonstrated expression of the long variant in endothelial cells. It was designated CRACR2A-L. Sequence analysis suggested it to be a previously unrecognised member of the Rab GTPase family. It made a positive contribution to endothelial tube formation. The data suggest that endothelial cells contain a long variant of CRACR2A which is an EF-hand-containing Rab protein that lacks impact on CRAC channels.
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Proteínas de Unión al Calcio/metabolismo , Células Endoteliales/citología , Regulación Enzimológica de la Expresión Génica , Proteínas de Unión al GTP rab/metabolismo , Secuencia de Aminoácidos , Calcio/metabolismo , Canales de Calcio/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Células Endoteliales/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Neovascularización Fisiológica , Proteína ORAI1 , Estructura Terciaria de Proteína , ARN Interferente Pequeño/metabolismo , Molécula de Interacción Estromal 1RESUMEN
RATIONALE: Calcium entry through Orai1 channels drives vascular smooth muscle cell migration and neointimal hyperplasia. The channels are activated by the important growth factor platelet-derived growth factor (PDGF). Channel activation is suggested to depend on store depletion, which redistributes and clusters stromal interaction molecule 1 (STIM1), which then coclusters and activates Orai1. OBJECTIVE: To determine the relevance of STIM1 and Orai1 redistribution in PDGF responses. METHODS AND RESULTS: Vascular smooth muscle cells were cultured from human saphenous vein. STIM1 and Orai1 were tagged with green and red fluorescent proteins to track them in live cells. Under basal conditions, the proteins were mobile but mostly independent of each other. Inhibition of sarco-endoplasmic reticulum calcium ATPase led to store depletion and dramatic redistribution of STIM1 and Orai1 into coclusters. PDGF did not evoke redistribution, even though it caused calcium release and Orai1-mediated calcium entry in the same time period. After chemical blockade of Orai1-mediated calcium entry, however, PDGF caused redistribution. Similarly, mutagenic disruption of calcium flux through Orai1 caused PDGF to evoke redistribution, showing that calcium flux through the wild-type channels had been filling the stores. Acidification of the extracellular medium to pH 6.4 caused inhibition of Orai1-mediated calcium entry and conferred capability for PDGF to evoke complete redistribution and coclustering. CONCLUSIONS: The data suggest that PDGF has a nonclustering mechanism by which to activate Orai1 channels and maintain calcium stores replete. Redistribution and clustering become important, however, when the endoplasmic reticulum stress signal of store depletion arises, for example when acidosis inhibits Orai1 channels.
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Canales de Calcio/metabolismo , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Estrés Fisiológico , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células Cultivadas , Retículo Endoplásmico/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Microscopía por Video , Músculo Liso Vascular/efectos de los fármacos , Mutación , Miocitos del Músculo Liso/efectos de los fármacos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1 , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Vena Safena/metabolismo , Molécula de Interacción Estromal 1 , Tapsigargina/farmacología , Factores de Tiempo , Transfección , Proteína Fluorescente RojaRESUMEN
Mast cells are infamous for mediating allergic and inflammatory diseases due to their capacity of rapidly releasing a wide range of inflammatory mediators stored in cytoplasmic granules. However, mast cells also have several important physiological roles that involve selective and agonist-specific release of these active mediators. While a filtering mechanism at the plasma membrane could regulate the selective release of some cargo, the plethora of stored cargo and the diversity of mast cell functions suggests the existence of granule subtypes with distinct trafficking pathways. The molecular mechanisms underlying differential trafficking and exocytosis of these granules are not known, neither is it clear how granule trafficking is coupled to the stimulus. In endothelial cells, a Rab GTPase, Rab46, responds to histamine but not thrombin signals, and this regulates the trafficking of a subpopulation of endothelial-specific granules. Here, we sought to explore, for the first time, if Rab46 plays a role in mast cell function. We demonstrate that Rab46 is highly expressed in human and murine mast cells, and Rab46 genetic deletion has an effect on mast cell degranulation that depends on both stimuli and mast cell subtype. This initial insight into the contribution of Rab46 to mast cell function and the understanding of the role of Rab46 in stimuli-dependent trafficking in other cell types necessitates further investigations of Rab46 in mast cell granular trafficking so that novel and specific therapeutic targets for treatment of the diverse pathologies mediated by mast cells can be developed.
RESUMEN
RATIONALE: Orai1 and the associated calcium release-activated calcium (CRAC) channel were discovered in the immune system. Existence also in endothelial cells has been suggested, but the relevance to endothelial biology is mostly unknown. OBJECTIVE: The aim of this study was to investigate the relevance of Orai1 and CRAC channels to vascular endothelial growth factor (VEGF) and endothelial tube formation. METHODS AND RESULTS: In human umbilical vein endothelial cells, Orai1 disruption by short-interfering RNA or dominant-negative mutant Orai1 inhibited calcium release-activated (store-operated) calcium entry, VEGF-evoked calcium entry, cell migration, and in vitro tube formation. Expression of exogenous wild-type Orai1 rescued the tube formation. VEGF receptor-2 and Orai1 partially colocalized. Orai1 disruption also inhibited calcium entry and tube formation in endothelial progenitor cells from human blood. A known blocker of the immune cell CRAC channel (3-fluoropyridine-4-carboxylic acid (2',5'-dimethoxybiphenyl-4-yl)amide) was a strong blocker of store-operated calcium entry in endothelial cells and inhibited calcium entry evoked by VEGF in 3 types of human endothelial cell. The compound lacked effect on VEGF-evoked calcium-release, STIM1 clustering, and 2 types of transient receptor potential channels, TRPC6 and TRPV4. Without effect on cell viability, the compound inhibited human endothelial cell migration and tube formation in vitro and suppressed angiogenesis in vivo in the chick chorioallantoic membrane. The compound showed 100-fold greater potency for endothelial compared with immune cell calcium entry. CONCLUSIONS: The data suggest positive roles for Orai1 and CRAC channels in VEGF-evoked calcium entry and new opportunity for chemical modulation of angiogenesis.
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Canales de Calcio/fisiología , Calcio/metabolismo , Endotelio Vascular/crecimiento & desarrollo , Factores de Crecimiento Endotelial Vascular/fisiología , Animales , Células CHO , Calcio/antagonistas & inhibidores , Canales de Calcio/metabolismo , Células Cultivadas , Embrión de Pollo , Cricetinae , Cricetulus , Endotelio Vascular/citología , Endotelio Vascular/embriología , Células HEK293 , Humanos , Proteína ORAI1RESUMEN
Transient receptor potential canonical 5 (TRPC5) forms cationic channels that are polymodal sensors of factors including oxidized phospholipids, hydrogen peroxide, and reduced thioredoxin. The aim of this study was to expand knowledge of the chemical-sensing capabilities of TRPC5 by investigating dietary antioxidants. Human TRPC5 channels were expressed in HEK 293 cells and studied by patch clamp and intracellular Ca(2+) recording. GFP- and HA-tagged channels were used to quantify plasma membrane localization. Gallic acid and vitamin C suppressed TRPC5 activity if it was evoked by exogenous hydrogen peroxide or lanthanide ions but not by lysophosphatidylcholine or carbachol. Catalase mimicked the effects, suggesting that lanthanide-evoked activity depended on endogenous hydrogen peroxide. Trans-resveratrol, by contrast, inhibited all modes of TRPC5, and its effect was additive with that of vitamin C, suggesting antioxidant-independent action. The IC(50) was â¼10 µM. Diethylstilbestrol, a related hydroxylated stilbene, inhibited TRPC5 with a similar IC(50), but its action contrasted sharply with that of resveratrol in outside-out membrane patches where diethylstilbestrol caused strong and reversible inhibition and resveratrol had no effect, suggesting indirect modulation by resveratrol. Resveratrol did not affect channel surface density, but its effect was calcium-sensitive, indicating an action via a calcium-dependent intermediate. The data suggest previously unrecognized chemical-sensing properties of TRPC5 through multiple mechanisms: (i) inhibition by scavengers of reactive oxygen species because a mode of TRPC5 activity depends on endogenous hydrogen peroxide; (ii) direct channel blockade by diethylstilbestrol; and (iii) indirect, antioxidant-independent inhibition by resveratrol.
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Calcio/metabolismo , Membrana Celular/metabolismo , Depuradores de Radicales Libres/farmacología , Peróxido de Hidrógeno/metabolismo , Estilbenos/farmacología , Canales Catiónicos TRPC/metabolismo , Membrana Celular/genética , Células HEK293 , Humanos , Canales Catiónicos TRPC/antagonistas & inhibidores , Canales Catiónicos TRPC/genéticaRESUMEN
RATIONALE: Transient receptor potential melastatin (TRPM)3 is a calcium-permeable ion channel activated by the neurosteroid pregnenolone sulfate and positively coupled to insulin secretion in beta cells. Although vascular TRPM3 mRNA has been reported, there is no knowledge of TRPM3 protein or its regulation and function in the cardiovascular system. OBJECTIVE: To determine the relevance and regulation of TRPM3 in vascular biology. METHODS AND RESULTS: TRPM3 expression was detected at mRNA and protein levels in contractile and proliferating vascular smooth muscle cells. Calcium entry evoked by pregnenolone sulfate or sphingosine was suppressed by TRPM3 blocking antibody or knock-down of TRPM3 by RNA interference. Low-level constitutive TRPM3 activity was also detected. In proliferating cells, channel activity was coupled negatively to interleukin-6 secretion via a calcium-dependent mechanism. In freshly isolated aorta, TRPM3 positively modulated contractile responses independently of L-type calcium channels. Concentrations of pregnenolone sulfate required to evoke responses were higher than the known plasma concentrations of the steroids, leading to a screen for other stimulators. beta-Cyclodextrin was one of few stimulators of TRPM3, revealing the channels to be partially suppressed by endogenous cholesterol, the precursor of pregnenolone. Elevation of cholesterol further suppressed channel activity and loading with cholesterol to generate foam cells precluded observation of TRPM3 activity. CONCLUSIONS: The data suggest functional relevance of TRPM3 in contractile and proliferating phenotypes of vascular smooth muscle cells, significance of constitutive channel activity, regulation by cholesterol, and potential value of pregnenolone sulfate in therapeutic vascular modulation.
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Colesterol/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Pregnenolona/farmacología , Canales Catiónicos TRPM/metabolismo , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Humanos , Ratones , Contracción Muscular/fisiología , Músculo Liso Vascular/citología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Relación Estructura-Actividad , Canales Catiónicos TRPM/genéticaRESUMEN
Rab46 is a novel Ca2+-sensing Rab GTPase shown to have important functions in endothelial and immune cells. The presence of functional Ca2+-binding, coiled-coil and Rab domains suggest that Rab46 will be important for coupling rapid responses to signalling in many cell types. The molecular mechanisms underlying Rab46 function are currently unknown. Here we provide the first resource for studying Rab46 interacting proteins. Using liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify affinity purified proteins that bind to constitutively active GFP-Rab46 or inactive GFP-Rab46 expressed in endothelial cells, we have revealed 922 peptides that interact with either the GTP-bound Rab46 or GDP-bound Rab46. To identify proteins that could be potential Rab46 effectors we performed further comparative analyses between nucleotide-locked Rab46 proteins and identified 29 candidate effector proteins. Importantly, through biochemical and imaging approaches we have validated two potential effector proteins; dynein and the Na2+/ K+ ATPase subunit alpha 1 (ATP1α1). Hence, our use of affinity purification and LC-MS/MS to identify Rab46 neighbouring proteins provides a valuable resource for detecting Rab46 effector proteins and analysing Rab46 functions.
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Células Endoteliales/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Western Blotting , Cromatografía de Gases y Espectrometría de Masas , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Inmunoprecipitación , Espectrometría de Masas , ProteómicaRESUMEN
CRAC channel regulator 2 A (CRACR2A) is a large Rab GTPase that is expressed abundantly in T cells and acts as a signal transmitter between T cell receptor stimulation and activation of the Ca2+-NFAT and JNK-AP1 pathways. CRACR2A has been linked to human diseases in numerous genome-wide association studies, however, to date no patient with damaging variants in CRACR2A has been identified. In this study, we describe a patient harboring biallelic variants in CRACR2A [paternal allele c.834 gaG> gaT (p.E278D) and maternal alelle c.430 Aga > Gga (p.R144G) c.898 Gag> Tag (p.E300*)], the gene encoding CRACR2A. The 33-year-old patient of East-Asian origin exhibited late onset combined immunodeficiency characterised by recurrent chest infections, panhypogammaglobulinemia and CD4+ T cell lymphopenia. In vitro exposure of patient B cells to a T-dependent stimulus resulted in normal generation of antibody-secreting cells, however the patient's T cells showed pronounced reduction in CRACR2A protein levels and reduced proximal TCR signaling, including dampened SOCE and reduced JNK phosphorylation, that contributed to a defect in proliferation and cytokine production. Expression of individual allelic mutants in CRACR2A-deleted T cells showed that the CRACR2AE278D mutant did not affect JNK phosphorylation, but impaired SOCE which resulted in reduced cytokine production. The truncated double mutant CRACR2AR144G/E300* showed a pronounced defect in JNK phosphorylation as well as SOCE and strong impairment in cytokine production. Thus, we have identified variants in CRACR2A that led to late-stage combined immunodeficiency characterized by loss of function in T cells.
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Canales de Calcio Activados por la Liberación de Calcio/genética , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Citocinas/biosíntesis , Mutación , Enfermedades de Inmunodeficiencia Primaria/genética , Enfermedades de Inmunodeficiencia Primaria/fisiopatología , Receptores de Antígenos de Linfocitos T/metabolismo , Adulto , Pueblo Asiatico , Canales de Calcio Activados por la Liberación de Calcio/inmunología , Citocinas/genética , Variación Genética , Humanos , Enfermedades de Inmunodeficiencia Primaria/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Endothelial cells selectively release cargo stored in Weibel-Palade bodies (WPBs) to regulate vascular function, but the underlying mechanisms are poorly understood. Here we show that histamine evokes the release of the proinflammatory ligand, P-selectin, while diverting WPBs carrying non-inflammatory cargo away from the plasma membrane to the microtubule organizing center. This differential trafficking is dependent on Rab46 (CRACR2A), a newly identified Ca2+-sensing GTPase, which localizes to a subset of P-selectin-negative WPBs. After acute stimulation of the H1 receptor, GTP-bound Rab46 evokes dynein-dependent retrograde transport of a subset of WPBs along microtubules. Upon continued histamine stimulation, Rab46 senses localized elevations of intracellular calcium and evokes dispersal of microtubule organizing center-clustered WPBs. These data demonstrate for the first time that a Rab GTPase, Rab46, integrates G protein and Ca2+ signals to couple on-demand histamine signals to selective WPB trafficking.
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Señalización del Calcio/genética , Proteínas de Unión al Calcio/genética , Receptores Histamínicos H1/genética , Cuerpos de Weibel-Palade/genética , Membrana Celular/genética , Dineínas/genética , Exocitosis/genética , Proteínas de Unión al GTP/genética , Histamina/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Microtúbulos/genética , Selectina-P/genética , Transporte de Proteínas/genética , Transducción de Señal/genética , Cuerpos de Weibel-Palade/metabolismoRESUMEN
BACKGROUND: The neuronal endoplasmic reticulum (ER) is an extensive, complex endomembrane system, containing Ca2+ pumps, and Ca2+ channels that permit it to act as a dynamic calcium store. Currently, there is controversy over the continuity of the ER in neurones, how this intersects with calcium signalling and the possibility of physical compartmentalisation. Unfortunately, available probes of ER structure such as vital dyes are limited by their membrane specificity. The introduction of ER-targeted GFP plasmids has been a considerable step forward, but these are difficult to express in neurones through conventional transfection approaches. To circumvent such problems we have engineered a novel ER-targeted GFP construct, termed pIN-KDEL, into a 3rd generation replication-defective, self-inactivating lentiviral vector system capable of mediating gene transduction in diverse dividing and post-mitotic mammalian cells, including neurones. RESULTS: Following its expression in HEK293 (or COS-7) cells, LV-pIN-KDEL yielded a pattern of fluorescence that co-localised exclusively with the ER marker sec61beta but with no other major organelle. We found no evidence for cytotoxicity and only rarely inclusion body formation. To explore the utility of the probe in resolving the ER in live cells, HEK293 or COS-7 cells were transduced with LV-pIN-KDEL and, after 48 h, imaged directly at intervals from 1 min to several hours. LV-pIN-KDEL fluorescence revealed the endoplasmic reticulum as a tubular lattice structure whose morphology can change markedly within seconds. Although GFP can be phototoxic, the integrity of the cells and ER was retained for several weeks and even after light exposure for periods up to 24 h. Using LV-pIN-KDEL we have imaged the ER in diverse fixed neuronal cultures and, using real-time imaging, found evidence for extensive, dynamic remodelling of the neuronal ER in live hippocampal cultures, brain slices, explants and glia. Finally, through a Fluorescence Loss in Photobleaching (FLIP) approach, continuous irradiation at a single region of interest removed all the fluorescence of LV-pIN-KDEL-transduced nerve cells in explant cultures, thus, providing compelling evidence that in neurons the endoplasmic reticulum is not only dynamic but also continuous. CONCLUSION: The lentiviral-based ER-targeted reporter, LV-pIN-KDEL, offers considerable advantages over present systems for defining the architecture of the ER, especially in primary cells such as neurones that are notoriously difficult to transfect. Images and continuous photobleaching experiments of LV-pIN-KDEL-transduced neurones demonstrate that the endoplasmic reticulum is a dynamic structure with a single continuous lumen. The introduction of LV-pIN-KDEL is anticipated to greatly facilitate a real-time visualisation of the structural plasticity and continuous nature of the neuronal ER in healthy and diseased brain tissue.
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Retículo Endoplásmico/fisiología , Vectores Genéticos/fisiología , Oligopéptidos/genética , Animales , Línea Celular Transformada , Chlorocebus aethiops , Cricetinae , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Técnicas In Vitro , Oligopéptidos/metabolismo , Procesamiento Proteico-Postraduccional , Señales de Clasificación de Proteína/genética , Transfección/métodos , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Endothelial barrier formation is maintained by intercellular communication through junctional proteins. The mechanisms involved in maintaining endothelial communication subsequent to barrier disruption remain unclear. It is known that low numbers of endothelial cells can be interconnected by homotypic actin-driven tunneling nanotubes (TNTs) which could be important for intercellular transfer of information in vascular physiology. Here we sought insight into the triggers for TNT formation. Wheat germ agglutinin, a C-type lectin and known label for TNTs, unexpectedly caused striking induction of TNTs. A succinylated derivative was by contrast inactive, suggesting mediation by a sialylated protein. Through siRNA-mediated knockdown we identified that this protein was likely to be CD31, an important sialylated membrane protein normally at endothelial cell junctions. We subsequently considered thrombin as a physiological inducer of endothelial TNTs because it reduces junctional contact. Thrombin reduced junctional contact, redistributed CD31 and induced TNTs, but its effect on TNTs was CD31-independent. Thrombin-induced TNTs nevertheless required PKCα, a known mediator of thrombin-dependent junctional remodelling, suggesting a necessity for junctional proteins in TNT formation. Indeed, TNT-inducing effects of wheat germ agglutinin and thrombin were both correlated with cortical actin rearrangement and similarly Ca2+-dependent, suggesting common underlying mechanisms. Once formed, Ca2+ signalling along TNTs was observed.
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Células Endoteliales/citología , Trombina/metabolismo , Triticum/metabolismo , Aglutininas del Germen de Trigo/genética , Aglutininas del Germen de Trigo/metabolismo , Señalización del Calcio , Comunicación Celular , Células Endoteliales/metabolismo , Técnicas de Silenciamiento del Gen , Células Endoteliales de la Vena Umbilical Humana , Humanos , Uniones Intercelulares/metabolismo , Proteína Quinasa C-alfa/metabolismo , Imagen de Lapso de Tiempo , Triticum/genéticaRESUMEN
BACKGROUND: The identification of protein trafficking signals, and their interacting mechanisms, is a fundamental objective of modern biology. Unfortunately, the analysis of trafficking signals is complicated by their topography, hierarchical nature and regulation. Powerful strategies to test candidate motifs include their ability to direct simpler reporter proteins, to which they are fused, to the appropriate cellular compartment. However, present reporters are limited by their endogenous expression, paucity of cloning sites, and difficult detection in live cells. RESULTS: Consequently, we have engineered a mammalian expression vector encoding a novel trafficking reporter--pIN-G--consisting of a simple, type I integral protein bearing permissive intra/extracellular cloning sites, green fluorescent protein (GFP), cMyc and HA epitope tags. Fluorescence imaging, flow cytometry and biochemical assays of transfected HEK293 cells, confirm the size, topology and surface expression of PIN-G. Moreover, a pIN-G fusion construct, containing a Trans-Golgi Network (TGN) targeting determinant, internalises rapidly from the cell surface and localises to the TGN. Additionally, another PIN-G fusion protein and its mutants reveal trafficking determinants in the cytoplasmic carboxy terminus of Kv1.4 voltage-gated potassium channels. CONCLUSION: Together, these data indicate that pIN-G is a versatile, powerful, new reporter for analysing signals controlling membrane protein trafficking, surface expression and dynamics.
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Riñón/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/ultraestructura , Microscopía Fluorescente/métodos , Mapeo de Interacción de Proteínas/métodos , Señales de Clasificación de Proteína/fisiología , Transporte de Proteínas/fisiología , Línea Celular , HumanosRESUMEN
BACKGROUND AND PURPOSE: The Sigma-1 receptor (Sig1R) impacts on calcium ion signalling and has a plethora of ligands. This study investigated Sig1R and its ligands in relation to endogenous calcium events of endothelial cells and transient receptor potential (TRP) channels. EXPERIMENTAL APPROACH: Intracellular calcium and patch clamp measurements were made from human saphenous vein endothelial cells and HEK 293 cells expressing exogenous human TRPC5, TRPM2 or TRPM3. Sig1R ligands were applied and short interfering RNA was used to deplete Sig1R. TRP channels tagged with fluorescent proteins were used for subcellular localization studies. KEY RESULTS: In endothelial cells, 10-100 µM of the Sig1R antagonist BD1063 inhibited sustained but not transient calcium responses evoked by histamine. The Sig1R agonist 4-IBP and related antagonist BD1047 were also inhibitory. The Sig1R agonist SKF10047 had no effect. Sustained calcium entry evoked by VEGF or hydrogen peroxide was also inhibited by BD1063, BD1047 or 4-IBP, but not SKF10047. 4-IBP, BD1047 and BD1063 inhibited TRPC5 or TRPM3, but not TRPM2. Inhibitory effects of BD1047 were rapid in onset and readily reversed on washout. SKF10047 inhibited TRPC5 but not TRPM3 or TRPM2. Depletion of Sig1R did not prevent the inhibitory actions of BD1063 or BD1047 and Sig1R did not co-localize with TRPC5 or TRPM3. CONCLUSIONS AND IMPLICATIONS: The data suggest that two types of Sig1R ligand (BD1047/BD1063 and 4-IBP) are inhibitors of receptor- or chemically activated calcium entry channels, acting relatively directly and independently of the Sig1R. Chemical foundations for TRP channel inhibitors are suggested.
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Señalización del Calcio , Endotelio Vascular/metabolismo , Receptores sigma/metabolismo , Canales Catiónicos TRPC/metabolismo , Canales Catiónicos TRPM/metabolismo , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Células HEK293 , Histamina/metabolismo , Humanos , Cinética , Ligandos , Potenciales de la Membrana/efectos de los fármacos , Moduladores del Transporte de Membrana/farmacología , Técnicas de Placa-Clamp , Transporte de Proteínas/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño , Receptores sigma/agonistas , Receptores sigma/antagonistas & inhibidores , Receptores sigma/genética , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Canales Catiónicos TRPC/antagonistas & inhibidores , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPM/antagonistas & inhibidores , Canales Catiónicos TRPM/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor Sigma-1RESUMEN
BACKGROUND AND PURPOSE: The aim was to advance the understanding of Orai proteins and identify a specific inhibitor of the associated calcium entry mechanism in vascular smooth muscle cells (VSMCs). EXPERIMENTAL APPROACH: Proliferating VSMCs were cultured from human saphenous veins. Intracellular calcium was measured using fura-2, whole-cell current was recorded using patch-clamp and cell migration quantified in modified Boyden chambers. Subcellular protein localization was determined by microscopy. Isometric tension was recorded from mouse aortic rings. KEY RESULTS: Molecular disruption and rescue experiments indicated the importance of Orai1 in calcium entry caused by store depletion evoked passively or by platelet-derived growth factor (PDGF), suggesting the presence of Ca(2+) release-activated Ca(2+) (CRAC) channels like those of the immune system. The CRAC channel blocker, S66, was a potent inhibitor of the VSMC signals, IC(50) 26 nM, which was almost two orders of magnitude greater than with leucocytes. S66 had no effect on PDGF- and ATP-evoked calcium release, overexpressed transient receptor potential canonical (TRPC)5 channels, native TRPC1/5-containing channels, stromal interaction molecule 1 clustering, non-selective cationic current evoked by store depletion and phenylephrine-evoked aortic contraction. S66 reduced PDGF-evoked VSMC migration while having only modest effects on cell proliferation and no effect on cell viability. CONCLUSIONS AND IMPLICATIONS: The data suggest that Orai1 has a role in human VSMC migration, and that a CRAC channel inhibitor has high potency and selectivity for the associated calcium entry, suggesting a distinct characteristic of vascular CRAC channels and the potential for selective chemical suppression of vascular remodelling.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Movimiento Celular/fisiología , Miocitos del Músculo Liso/efectos de los fármacos , Animales , Canales de Calcio/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/fisiología , Proteína ORAI1 , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismoRESUMEN
The identification of motifs that control the intracellular trafficking of proteins is a fundamental objective of cell biology. Once identified, such regions should, in principle, be both necessary and sufficient to direct any randomly distributed protein, acting as a reporter, to the subcellular compartment in question. However, most reporter proteins have limited versatility owing to their endogenous expression and limited modes of detection--especially in live cells. To surmount such limitations, we engineered a plasmid--pIN-G--encoding an entirely artificial, type I transmembrane reporter protein (PIN-G), containing HA, cMyc and GFP epitope, and fluorescence tags. Although originally designed for trafficking studies, pIN technology is a powerful tool applicable to almost every area of biology. Here we describe the methodologies used routinely in analyzing pIN constructs and some of their derivatives.
Asunto(s)
Genes Reporteros , Proteínas de la Membrana/metabolismo , Transducción de Señal , Línea Celular , Citometría de Flujo , Fluorescencia , Técnica del Anticuerpo Fluorescente , Humanos , Proteínas de la Membrana/genéticaRESUMEN
The dynamic expression of voltage-gated potassium channels (Kvs) at the cell surface is a fundamental factor controlling membrane excitability. In exploring possible mechanisms controlling Kv surface expression, we identified a region in the extracellular linker between the first and second of the six (S1-S6) transmembrane-spanning domains of the Kv1.4 channel, which we hypothesized to be critical for its biogenesis. Using immunofluorescence microscopy, flow cytometry, patch clamp electrophysiology, and mutagenesis, we identified a single threonine residue at position 330 within the Kv1.4 S1-S2 linker that is absolutely required for cell surface expression. Mutation of Thr-330 to an alanine, aspartate, or lysine prevented surface expression. However, surface expression occurred upon co-expression of mutant and wild type Kv1.4 subunits or mutation of Thr-330 to a serine. Mutation of the corresponding residue (Thr-211) in Kv3.1 to alanine also caused intracellular retention, suggesting that the conserved threonine plays a generalized role in surface expression. In support of this idea, sequence comparisons showed conservation of the critical threonine in all Kv families and in organisms across the evolutionary spectrum. Based upon the Kv1.2 crystal structure, further mutagenesis, and the partial restoration of surface expression in an electrostatic T330K bridging mutant, we suggest that Thr-330 hydrogen bonds to equally conserved outer pore residues, which may include a glutamate at position 502 that is also critical for surface expression. We propose that Thr-330 serves to interlock the voltage-sensing and gating domains of adjacent monomers, thereby yielding a structure competent for the surface expression of functional tetramers.
Asunto(s)
Membrana Celular/metabolismo , Canal de Potasio Kv1.4/química , Canales de Potasio con Entrada de Voltaje/metabolismo , Treonina/química , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Electrofisiología , Evolución Molecular , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Homología de Secuencia de AminoácidoRESUMEN
The last decade has witnessed an exponential increase in interest in one of the great mysteries of nerve cell biology: Specifically, how do neurons know where to place the ion channels that control their excitability? Many of the most important insights have been gleaned from studies on the voltage-gated potassium channels (Kvs) which underlie the shape, duration and frequency of action potentials. In this review, we gather recent evidence on the expression, trafficking and maintenance mechanisms which control the surface density of Kvs in different subcellular compartments of neurons and how these may be regulated to control cell excitability.