RESUMEN
Despite significant advances in the development of therapeutic interventions targeting autoimmune diseases and chronic inflammatory conditions, lack of effective treatment still poses a high unmet need. Modulating chronically activated T cells through the blockade of the Kv1.3 potassium channel is a promising therapeutic approach; however, developing selective Kv1.3 inhibitors is still an arduous task. Phage display-based high throughput peptide library screening is a rapid and robust approach to develop promising drug candidates; however, it requires solid-phase immobilization of target proteins with their binding site preserved. Historically, the KcsA bacterial channel chimera harboring only the turret region of the human Kv1.3 channel was used for screening campaigns. Nevertheless, literature data suggest that binding to this type of chimera does not correlate well with blocking potency on the native Kv1.3 channels. Therefore, we designed and successfully produced advanced KcsA-Kv1.3, KcsA-Kv1.1, and KcsA-Kv1.2 chimeric proteins in which both the turret and part of the filter regions of the human Kv1.x channels were transferred. These T+F (turret-filter) chimeras showed superior peptide ligand-binding predictivity compared to their T-only versions in novel phage ELISA assays. Phage ELISA binding and competition results supported with electrophysiological data confirmed that the filter region of KcsA-Kv1.x is essential for establishing adequate relative affinity order among selected peptide toxins (Vm24 toxin, Hongotoxin-1, Kaliotoxin-1, Maurotoxin, Stichodactyla toxin) and consequently obtaining more reliable selectivity data. These new findings provide a better screening tool for future drug development efforts and offer insight into the target-ligand interactions of these therapeutically relevant ion channels.
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Canal de Potasio Kv1.3 , Bloqueadores de los Canales de Potasio , Proteínas Recombinantes de Fusión , Animales , Humanos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/antagonistas & inhibidores , Sitios de Unión , Canal de Potasio Kv1.3/metabolismo , Canal de Potasio Kv1.3/antagonistas & inhibidores , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/química , Ligandos , Biblioteca de Péptidos , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/metabolismo , Canales de Potasio/química , Canales de Potasio/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Línea CelularRESUMEN
Membrane lipids extensively modulate the activation gating of voltage-gated potassium channels (KV), however, much less is known about the mechanisms of ceramide and glucosylceramide actions including which structural element is the main intramolecular target and whether there is any contribution of indirect, membrane biophysics-related mechanisms to their actions. We used two-electrode voltage-clamp fluorometry capable of recording currents and fluorescence signals to simultaneously monitor movements of the pore domain (PD) and the voltage sensor domain (VSD) of the KV1.3 ion channel after attaching an MTS-TAMRA fluorophore to a cysteine introduced into the extracellular S3-S4 loop of the VSD. We observed rightward shifts in the conductance-voltage (G-V) relationship, slower current activation kinetics, and reduced current amplitudes in response to loading the membrane with C16-ceramide (Cer) or C16-glucosylceramide (GlcCer). When analyzing VSD movements, only Cer induced a rightward shift in the fluorescence signal-voltage (F-V) relationship and slowed fluorescence activation kinetics, whereas GlcCer exerted no such effects. These results point at a distinctive mechanism of action with Cer primarily targeting the VSD, while GlcCer only the PD of KV1.3. Using environment-sensitive probes and fluorescence-based approaches, we show that Cer and GlcCer similarly increase molecular order in the inner, hydrophobic regions of bilayers, however, Cer induces a robust molecular reorganization at the membrane-water interface. We propose that this unique ordering effect in the outermost membrane layer in which the main VSD rearrangement involving an outward sliding of the top of S4 occurs can explain the VSD targeting mechanism of Cer, which is unavailable for GlcCer.
RESUMEN
Diverse structural scaffolds have been described in peptides from sea anemones, with the ShKT domain being a common scaffold first identified in ShK toxin from Stichodactyla helianthus. ShK is a potent blocker of voltage-gated potassium channels (KV 1.x), and an analog, ShK-186 (dalazatide), has completed Phase 1 clinical trials in plaque psoriasis. The ShKT domain has been found in numerous other species, but only a tiny fraction of ShKT domains has been characterized functionally. Despite adopting the canonical ShK fold, some ShKT peptides from sea anemones inhibit KV 1.x, while others do not. Mutagenesis studies have shown that a Lys-Tyr (KY) dyad plays a key role in KV 1.x blockade, although a cationic residue followed by a hydrophobic residue may also suffice. Nevertheless, ShKT peptides displaying an ShK-like fold and containing a KY dyad do not necessarily block potassium channels, so additional criteria are needed to determine whether new ShKT peptides might show activity against potassium channels. In this study, we used a combination of NMR and molecular dynamics (MD) simulations to assess the potential activity of a new ShKT peptide. We determined the structure of ShKT-Ts1, from the sea anemone Telmatactis stephensoni, examined its tissue localization, and investigated its activity against a range of ion channels. As ShKT-Ts1 showed no activity against KV 1.x channels, we used MD simulations to investigate whether solvent exposure of the dyad residues may be informative in rationalizing and potentially predicting the ability of ShKT peptides to block KV 1.x channels. We show that either a buried dyad that does not become exposed during MD simulations, or a partially exposed dyad that becomes buried during MD simulations, correlates with weak or absent activity against KV 1.x channels. Therefore, structure determination coupled with MD simulations, may be used to predict whether new sequences belonging to the ShKT family may act as potassium channel blockers.
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Anémonas de Mar , Animales , Anémonas de Mar/química , Péptidos/farmacología , Péptidos/química , Canales de Potasio/metabolismo , Simulación de Dinámica Molecular , Relación Estructura-ActividadRESUMEN
BACKGROUND: The ShK toxin from Stichodactyla helianthus has established the therapeutic potential of sea anemone venom peptides, but many lineage-specific toxin families in Actiniarians remain uncharacterised. One such peptide family, sea anemone 8 (SA8), is present in all five sea anemone superfamilies. We explored the genomic arrangement and evolution of the SA8 gene family in Actinia tenebrosa and Telmatactis stephensoni, characterised the expression patterns of SA8 sequences, and examined the structure and function of SA8 from the venom of T. stephensoni. RESULTS: We identified ten SA8-family genes in two clusters and six SA8-family genes in five clusters for T. stephensoni and A. tenebrosa, respectively. Nine SA8 T. stephensoni genes were found in a single cluster, and an SA8 peptide encoded by an inverted SA8 gene from this cluster was recruited to venom. We show that SA8 genes in both species are expressed in a tissue-specific manner and the inverted SA8 gene has a unique tissue distribution. While the functional activity of the SA8 putative toxin encoded by the inverted gene was inconclusive, its tissue localisation is similar to toxins used for predator deterrence. We demonstrate that, although mature SA8 putative toxins have similar cysteine spacing to ShK, SA8 peptides are distinct from ShK peptides based on structure and disulfide connectivity. CONCLUSIONS: Our results provide the first demonstration that SA8 is a unique gene family in Actiniarians, evolving through a variety of structural changes including tandem and proximal gene duplication and an inversion event that together allowed SA8 to be recruited into the venom of T. stephensoni.
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Anémonas de Mar , Animales , Anémonas de Mar/genética , Genómica , Inversión Cromosómica , Cisteína , DisulfurosRESUMEN
CD8+ T cells play a role in the suppression of tumor growth and immunotherapy. Ion channels control the Ca2+-dependent function of CD8+ lymphocytes such as cytokine/granzyme production and tumor killing. Kv1.3 and KCa3.1 K+ channels stabilize the negative membrane potential of T cells to maintain Ca2+ influx through CRAC channels. We assessed the expression of Kv1.3, KCa3.1 and CRAC in CD8+ cells from ovarian cancer (OC) patients (n = 7). We found that the expression level of Kv1.3 was higher in patients with malignant tumors than in control or benign tumor groups while the KCa3.1 activity was lower in the malignant tumor group as compared to the others. We demonstrated that the Ca2+ response in malignant tumor patients is higher compared to control groups. We propose that altered Kv1.3 and KCa3.1 expression in CD8+ cells in OC could be a reporter and may serve as a biomarker in diagnostics and that increased Ca2+ response through CRAC may contribute to the impaired CD8+ function.
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Linfocitos T CD8-positivos , Neoplasias Ováricas , Humanos , Femenino , Linfocitos T CD8-positivos/metabolismo , Canales de Potasio/metabolismo , Pronóstico , Biomarcadores/metabolismo , Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/metabolismo , Canal de Potasio Kv1.3/metabolismoRESUMEN
OBJECTIVES: Cardiovascular (CV) morbidity and mortality, and perpetuated synovial angiogenesis have been associated with RA. In our study we evaluated angiogenic factors in relation to vascular inflammation and function, and clinical markers in RA patients undergoing 1-year tofacitinib therapy. METHODS: Thirty RA patients treated with either 5 mg or 10 mg twice daily tofacitinib were included in a 12-month follow-up study. Eventually, 26 patients completed the study and were included in data analysis. Levels of various angiogenic cytokines (TNF-α, IL-6), growth factors [VEGF, basic fibroblast (bFGF), epidermal (EGF), placental (PlGF)], cathepsin K (CathK), CXC chemokine ligand 8 (CXCL8), galectin-3 (Gal-3) and N-terminal prohormone brain natriuretic peptide (NT-proBNP) were determined at baseline, and at 6 and 12 months after initiating tofacitinib treatment. In order to assess flow-mediated vasodilation, common carotid intima-media thickness (ccIMT) and carotid-femoral pulse-wave velocity, ultrasonography was performed. Synovial and aortic inflammation was also assessed by 18F-fluorodeoxyglucose-PET/CT. RESULTS: One-year tofacitinib therapy significantly decreased IL-6, VEGF, bFGF, EGF, PlGF and CathK, while it increased Gal-3 production (P < 0.05). bFGF, PlGF and NT-proBNP levels were higher, while platelet-endothelial cell adhesion molecule 1 (PECAM-1) levels were lower in RF-seropositive patients (P < 0.05). TNF-α, bFGF and PlGF correlated with post-treatment synovial inflammation, while aortic inflammation was rather dependent on IL-6 and PECAM-1 as determined by PET/CT (P < 0.05). In the correlation analyses, NT-proBNP, CXCL8 and Cath variables correlated with ccIMT (P < 0.05). CONCLUSIONS: Decreasing production of bFGF, PlGF or IL-6 by 1-year tofacitinib therapy potentially inhibits synovial and aortic inflammation. Although NT-proBNP, CXCL8 and CathK were associated with ccIMT, their role in RA-associated atherosclerosis needs to be further evaluated.
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Artritis Reumatoide , Grosor Intima-Media Carotídeo , Embarazo , Humanos , Femenino , Factor de Necrosis Tumoral alfa , Estudios de Seguimiento , Interleucina-6 , Factor de Crecimiento Epidérmico/uso terapéutico , Molécula-1 de Adhesión Celular Endotelial de Plaqueta , Tomografía Computarizada por Tomografía de Emisión de Positrones , Factor A de Crecimiento Endotelial Vascular , Placenta/metabolismo , Artritis Reumatoide/complicaciones , Inflamación/complicaciones , BiomarcadoresRESUMEN
The voltage-gated potassium channel Kv1.3 regulates the pro-inflammatory function of microglia and is highly expressed in the post-mortem brains of individuals with Alzheimer's and Parkinson's diseases. HsTX1[R14A] is a selective and potent peptide inhibitor of the Kv1.3 channel (IC50 â¼ 45 pM) that has been shown to decrease cytokine levels in a lipopolysaccharide (LPS)-induced mouse model of inflammation. Central nervous system exposure to HsTX1[R14A] was previously detected in this mouse model using liquid chromatography with tandem mass spectrometry, but this technique does not report on the spatial distribution of the peptide in the different brain regions or peripheral organs. Herein, the in vivo distribution of a [64Cu]Cu-labeled DOTA conjugate of HsTX1[R14A] was observed for up to 48 h by positron emission tomography (PET) in mice. After subcutaneous administration to untreated C57BL/6J mice, considerable uptake of the radiolabeled peptide was observed in the kidney, but it was undetectable in the brain. Biodistribution of a [68Ga]Ga-DOTA conjugate of HsTX1[R14A] was then investigated in the LPS-induced mouse model of neuroinflammation to assess the effects of inflammation on uptake of the peptide in the brain. A control peptide with very weak Kv1.3 binding, [68Ga]Ga-DOTA-HsTX1[R14A,Y21A,K23A] (IC50 â¼ 6 µM), was also tested. Significantly increased uptake of [68Ga]Ga-DOTA-HsTX1[R14A] was observed in the brains of LPS-treated mice compared to mice treated with control peptide, implying that the enhanced uptake was due to increased Kv1.3 expression rather than simply increased blood-brain barrier disruption. PET imaging also showed accumulation of [68Ga]Ga-DOTA-HsTX1[R14A] in inflamed joints and decreased clearance from the kidneys in LPS-treated mice. These biodistribution data highlight the potential of HsTX1[R14A] as a therapeutic for the treatment of neuroinflammatory diseases mediated by overexpression of Kv1.3.
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Lipopolisacáridos , Enfermedades Neuroinflamatorias , Ratones , Animales , Distribución Tisular , Radioisótopos de Galio/metabolismo , Ratones Endogámicos C57BL , Péptidos/química , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Inflamación/metabolismo , Tomografía de Emisión de PositronesRESUMEN
Myeloid-derived suppressor cells (MDSCs) are key determinants of the immunosuppressive microenvironment in tumors. As ion channels play key roles in the physiology/pathophysiology of immune cells, we aimed at studying the ion channel repertoire in tumor-derived polymorphonuclear (PMN-MDSC) and monocytic (Mo-MDSC) MDSCs. Subcutaneous tumors in mice were induced by the Lewis lung carcinoma cell line (LLC). The presence of PMN-MDSC (CD11b+/Ly6G+) and Mo-MDSCs (CD11b+/Ly6C+) in the tumor tissue was confirmed using immunofluorescence microscopy and cells were identified as CD11b+/Ly6G+ PMN-MDSCs and CD11b+/Ly6C+/F4/80-/MHCII- Mo-MDSCs using flow cytometry and sorting. The majority of the myeloid cells infiltrating the LLC tumors were PMN-MDSC (~60%) as compared to ~10% being Mo-MDSCs. We showed that PMN- and Mo-MDSCs express the Hv1 H+ channel both at the mRNA and at the protein level and that the biophysical and pharmacological properties of the whole-cell currents recapitulate the hallmarks of Hv1 currents: ~40 mV shift in the activation threshold of the current per unit change in the extracellular pH, high H+ selectivity, and sensitivity to the Hv1 inhibitor ClGBI. As MDSCs exert immunosuppression mainly by producing reactive oxygen species which is coupled to Hv1-mediated H+ currents, Hv1 might be an attractive target for inhibition of MDSCs in tumors.
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Células Supresoras de Origen Mieloide , Neoplasias , Animales , Ratones , Línea Celular , Monocitos , Células Mieloides , Células Supresoras de Origen Mieloide/metabolismo , Neoplasias/metabolismo , Microambiente TumoralRESUMEN
Upregulation of the voltage-gated potassium channel KV1.3 is implicated in a range of autoimmune and neuroinflammatory diseases, including rheumatoid arthritis, psoriasis, multiple sclerosis, and type I diabetes. Understanding the expression, localization, and trafficking of KV1.3 in normal and disease states is key to developing targeted immunomodulatory therapies. HsTX1[R14A], an analogue of a 34-residue peptide toxin from the scorpion Heterometrus spinifer, binds KV1.3 with high affinity (IC50 of 45 pM) and selectivity (2000-fold for KV1.3 over KV1.1). We have synthesized a fluorescent analogue of HsTX1[R14A] by N-terminal conjugation of a Cy5 tag. Electrophysiology assays show that Cy5-HsTX1[R14A] retains activity against KV1.3 (IC50 â¼ 0.9 nM) and selectivity over a range of other potassium channels (KV1.2, KV1.4, KV1.5, KV1.6, KCa1.1 and KCa3.1), as well as selectivity against heteromeric channels assembled from KV1.3/KV1.5 tandem dimers. Live imaging of CHO cells expressing green fluorescent protein-tagged KV1.3 shows co-localization of Cy5-HsTX1[R14A] and KV1.3 fluorescence signals at the cell membrane. Moreover, flow cytometry demonstrated that Cy5-HsTX1[R14A] can detect KV1.3-expressing CHO cells. Stimulation of mouse microglia by lipopolysaccharide, which enhances membrane expression of KV1.3, was associated with increased staining by Cy5-HsTX1[R14A], demonstrating that it can be used to identify KV1.3 in disease-relevant models of inflammation. Furthermore, the biodistribution of Cy5-HsTX1[R14A] could be monitored using ex vivo fluorescence imaging of organs in mice dosed subcutaneously with the peptide. These results illustrate the utility of Cy5-HsTX1[R14A] as a tool for visualizing KV1.3, with broad applicability in fundamental investigations of KV1.3 biology, and the validation of novel disease indications where KV1.3 inhibition may be of therapeutic value.
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Canal de Potasio Kv1.3 , Venenos de Escorpión , Ratones , Animales , Cricetinae , Canal de Potasio Kv1.3/química , Canal de Potasio Kv1.3/metabolismo , Venenos de Escorpión/química , Venenos de Escorpión/metabolismo , Venenos de Escorpión/farmacología , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/farmacología , Cricetulus , Distribución Tisular , Péptidos/químicaRESUMEN
Voltage-dependent potassium channels (Kvs) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K+-selective pore. Animal toxins targeting Kvs are classified as pore blockers, which physically plug the ion conduction pathway, or as gating modifiers, which disrupt voltage sensor movements. A third group of toxins blocks K+ conduction by an unknown mechanism via binding to the channel turrets. Here, we show that Conkunitzin-S1 (Cs1), a peptide toxin isolated from cone snail venom, binds at the turrets of Kv1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K+ channels as a novel pharmacological target and provides a rational framework for drug design.
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Membrana Celular/efectos de los fármacos , Proteínas de Drosophila/antagonistas & inhibidores , Activación del Canal Iónico/efectos de los fármacos , Canal de Potasio Kv.1.2/antagonistas & inhibidores , Venenos de Moluscos/toxicidad , Canales de Potasio de la Superfamilia Shaker/antagonistas & inhibidores , Animales , Membrana Celular/metabolismo , Cristalografía por Rayos X , Proteínas de Drosophila/genética , Proteínas de Drosophila/aislamiento & purificación , Proteínas de Drosophila/metabolismo , Diseño de Fármacos , Femenino , Enlace de Hidrógeno/efectos de los fármacos , Canal de Potasio Kv.1.2/genética , Canal de Potasio Kv.1.2/aislamiento & purificación , Canal de Potasio Kv.1.2/metabolismo , Dosificación Letal Mediana , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Venenos de Moluscos/química , Mutación , Oocitos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Canales de Potasio de la Superfamilia Shaker/genética , Canales de Potasio de la Superfamilia Shaker/aislamiento & purificación , Canales de Potasio de la Superfamilia Shaker/metabolismo , Agua/química , Agua/metabolismo , Xenopus laevisRESUMEN
The hEag1 (Kv10.1) K+ channel is normally found in the brain, but it is ectopically expressed in tumor cells, including osteosarcoma. Based on the pivotal role of ion channels in osteogenesis, we tested whether pharmacological modulation of hEag1 may affect osteogenic differentiation of osteosarcoma cell lines. Using molecular biology (RT-PCR), electrophysiology (patch-clamp) and pharmacology (astemizole sensitivity, IC50 = 0.135 µM) we demonstrated that SaOS-2 osteosarcoma cells also express hEag1 channels. SaOS-2 cells also express to KCa1.1 K+ channels as shown by mRNA expression and paxilline sensitivity of the current. The inhibition of hEag1 (2 µM astemizole) or KCa1.1 (1 mM TEA) alone did not induce Ca2+ deposition in SaOS-2 cultures, however, these inhibitors, at identical concentrations, increased Ca2+ deposition evoked by the classical or pathological (inorganic phosphate, Pi) induction pathway without causing cytotoxicity, as reported by three completer assays (LDH release, MTT assay and SRB protein assay). We observed a similar effect of astemizole on Ca2+ deposition in MG-63 osteosarcoma cultures as well. We propose that the increase in the osteogenic stimuli-induced mineral matrix formation of osteosarcoma cell lines by inhibiting hEag1 may be a useful tool to drive terminal differentiation of osteosarcoma.
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Neoplasias Óseas , Osteosarcoma , Astemizol/farmacología , Línea Celular Tumoral , Canales de Potasio Éter-A-Go-Go , Humanos , Osteogénesis , Osteosarcoma/tratamiento farmacológico , Fosfatos/metabolismo , ARN Mensajero/genéticaRESUMEN
Voltage-gated Kv1.3 potassium channels are essential for maintaining negative membrane potential during T-cell activation. They interact with membrane-associated guanylate kinases (MAGUK-s) via their C-terminus and with TCR/CD3, leading to enrichment at the immunological synapse (IS). Molecular interactions and mobility may impact each other and the function of these proteins. We aimed to identify molecular determinants of Kv1.3 mobility, applying fluorescence correlation spectroscopy on human Jurkat T-cells expressing WT, C-terminally truncated (ΔC), and non-conducting mutants of mGFP-Kv1.3. ΔC cannot interact with MAGUK-s and is not enriched at the IS, whereas cells expressing the non-conducting mutant are depolarized. Here, we found that in standalone cells, mobility of ΔC increased relative to the WT, likely due to abrogation of interactions, whereas mobility of the non-conducting mutant decreased, similar to our previous observations on other membrane proteins in depolarized cells. At the IS formed with Raji B-cells, mobility of WT and non-conducting channels, unlike ΔC, was lower than outside the IS. The Kv1.3 variants possessing an intact C-terminus had lower mobility in standalone cells than in IS-engaged cells. This may be related to the observed segregation of F-actin into a ring-like structure at the periphery of the IS, leaving much of the cell almost void of F-actin. Upon depolarizing treatment, mobility of WT and ΔC channels decreased both in standalone and IS-engaged cells, contrary to non-conducting channels, which themselves caused depolarization. Our results support that Kv1.3 is enriched at the IS via its C-terminal region regardless of conductivity, and that depolarization decreases channel mobility.
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Canal de Potasio Kv1.3/metabolismo , Linfocitos T , Actinas/metabolismo , Humanos , Canal de Potasio Kv1.3/genética , Potenciales de la Membrana , Sinapsis/metabolismo , Linfocitos T/metabolismoRESUMEN
Acrorhagin I (U-AITX-Aeq5a) is a disulfide-rich peptide identified in the aggressive organs (acrorhagi) of the sea anemone Actinia equina. Previous studies (Toxicon 2005, 46:768-74) found that the peptide is toxic in crabs, although the structural and functional properties of acrorhagin I have not been reported. In this work, an Escherichia coli (BL21 strain) expression system was established for the preparation of 13C,15N-labelled acrorhagin I, and the solution structure was determined using NMR spectroscopy. Structurally, acrorhagin I is similar to B-IV toxin from the marine worm Cerebratulus lacteus (PDB id 1VIB), with a well-defined helical hairpin structure stabilised by four intramolecular disulfide bonds. The recombinant peptide was tested in patch-clamp electrophysiology assays against voltage-gated potassium and sodium channels, and in bacterial and fungal growth inhibitory assays and haemolytic assays. Acrorhagin I was not active against any of the ion channels tested and showed no activity in functional assays, indicating that this peptide may possess a different biological function. Metal ion interaction studies using NMR spectroscopy showed that acrorhagin I bound zinc and nickel, suggesting that its function might be modulated by metal ions or that it may be involved in regulating metal ion levels and their transport. The similarity between the structure of acrorhagin I and that of B-IV toxin from a marine worm suggests that this fold may prove to be a recurring motif in disulfide-rich peptides from marine organisms.
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Venenos de Cnidarios/química , Péptidos/química , Péptidos/farmacología , Animales , Células CHO , Células Cultivadas , Cricetulus , Disulfuros/química , Evolución Molecular , Hemólisis/efectos de los fármacos , Humanos , Espectroscopía de Resonancia Magnética , Metales/química , Metales/metabolismo , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Técnicas de Placa-Clamp , Péptidos/genética , Péptidos/metabolismo , Pliegue de Proteína , Anémonas de Mar/química , Homología Estructural de Proteína , Linfocitos T/efectos de los fármacosRESUMEN
Dravet syndrome is a catastrophic, pharmacoresistant epileptic encephalopathy. Disease onset occurs in the first year of life, followed by developmental delay with cognitive and behavioral dysfunction and substantially elevated risk of premature death. The majority of affected individuals harbor a loss-of-function mutation in one allele of SCN1A, which encodes the voltage-gated sodium channel NaV1.1. Brain NaV1.1 is primarily localized to fast-spiking inhibitory interneurons; thus the mechanism of epileptogenesis in Dravet syndrome is hypothesized to be reduced inhibitory neurotransmission leading to brain hyperexcitability. We show that selective activation of NaV1.1 by venom peptide Hm1a restores the function of inhibitory interneurons from Dravet syndrome mice without affecting the firing of excitatory neurons. Intracerebroventricular infusion of Hm1a rescues Dravet syndrome mice from seizures and premature death. This precision medicine approach, which specifically targets the molecular deficit in Dravet syndrome, presents an opportunity for treatment of this intractable epilepsy.
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Epilepsias Mioclónicas/tratamiento farmacológico , Interneuronas/metabolismo , Mutación , Canal de Sodio Activado por Voltaje NAV1.1/metabolismo , Venenos de Araña/farmacología , Transmisión Sináptica/efectos de los fármacos , Animales , Células CHO , Cricetulus , Epilepsias Mioclónicas/genética , Epilepsias Mioclónicas/metabolismo , Epilepsias Mioclónicas/patología , Células HEK293 , Humanos , Interneuronas/patología , Ratones , Ratones Mutantes , Canal de Sodio Activado por Voltaje NAV1.1/genéticaRESUMEN
CRAC, which plays important role in Ca2+-dependent T-lymphocyte activation, is composed of the ER-resident STIM1 and the plasma membrane Orai1 pore-forming subunit. Both accumulate at the immunological synapse (IS) between a T cell and an antigen-presenting cell (APC). We hypothesized that adapter/interacting proteins regulate Orai1 residence in the IS. We could show that mGFP-tagged Orai1-Full channels expressed in Jurkat cells had a biphasic IS-accumulation kinetics peaked at 15 min. To understand the background of Orai1 IS-redistribution we knocked down STIM1 and SAP97 (adaptor protein with a short IS-residency (15 min) and ability to bind Orai1 N-terminus): the mGFP-Orai1-Full channels kept on accumulating in the IS up to the 60th minute in the STIM1- and SAP97-lacking Jurkat cells. Deletion of Orai1 N terminus (mGFP-Orai1-Δ72) resulted in the same time course as described for STIM1/SAP97 knock-down cells. Ca2+-imaging of IS-engaged T-cells revealed that of Orai1 residency modifies the Ca2+-response: cells expressing mGFP-Orai1-Δ72 construct or mGFP-Orai1-Full in SAP-97 knock-down cells showed higher number of Ca2+-oscillation up to the 90th minute after IS formation. Overall, these data suggest that SAP97 may contribute to the short-lived IS-residency of Orai1 and binding of STIM1 to Orai1 N-terminus is necessary for SAP97-Orai1 interaction.
Asunto(s)
Señalización del Calcio/inmunología , Sinapsis Inmunológicas/metabolismo , Proteína ORAI1/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Inmunidad Adaptativa , Homólogo 1 de la Proteína Discs Large/antagonistas & inhibidores , Homólogo 1 de la Proteína Discs Large/genética , Homólogo 1 de la Proteína Discs Large/metabolismo , Retículo Endoplásmico/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Sinapsis Inmunológicas/genética , Sinapsis Inmunológicas/inmunología , Células Jurkat , Cinética , Activación de Linfocitos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/química , Proteína ORAI1/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Molécula de Interacción Estromal 1/antagonistas & inhibidores , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismoRESUMEN
The immunological synapse (IS) is a specialized contact area formed between a T cell and an antigen presenting cell (APC). Besides molecules directly involved in antigen recognition such as the TCR/CD3 complex, ion channels important in the membrane potential and intracellular free Ca2+ concentration control of T cells are also recruited into the IS. These are the voltage-gated Kv1.3 and Ca2+-activated KCa3.1 K+ channels and the calcium release-activated Ca2+ channel (CRAC). However, the consequence of this recruitment on membrane potential and Ca2+ level control is not known. Here we demonstrate that the membrane potential (MP) of murine T cells conjugated with APCs in an IS shows characteristic oscillations. We found that depolarization of the membrane by current injection or by increased extracellular K+ concentration produced membrane potential oscillations (MPO) significantly more frequently in conjugated T cells than in lone T cells. Furthermore, oscillation of the free intracellular Ca2+ concentration could also be observed more frequently in cells forming an IS than in lone cells. We suggest that in the IS the special arrangement of channels and the constrained space between the interacting cells creates a favorable environment for these oscillations, which may enhance the signaling process leading to T cell activation.
Asunto(s)
Señalización del Calcio , Sinapsis Inmunológicas/metabolismo , Potenciales de la Membrana , Linfocitos T/metabolismo , Animales , Células Presentadoras de Antígenos/metabolismo , Células Presentadoras de Antígenos/fisiología , Calcio/metabolismo , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Línea Celular , Sinapsis Inmunológicas/fisiología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Canal de Potasio Kv1.3/metabolismo , Ratones , Potasio/metabolismo , Linfocitos T/fisiologíaRESUMEN
BACKGROUND: In vitro chondrogenesis depends on the concerted action of numerous signalling pathways, many of which are sensitive to the changes of intracellular Ca2+ concentration. N-methyl-D-aspartate (NMDA) glutamate receptor is a cation channel with high permeability for Ca2+. Whilst there is now accumulating evidence for the expression and function of NMDA receptors in non-neural tissues including mature cartilage and bone, the contribution of glutamate signalling to the regulation of chondrogenesis is yet to be elucidated. METHODS: We studied the role of glutamatergic signalling during the course of in vitro chondrogenesis in high density chondrifying cell cultures using single cell fluorescent calcium imaging, patch clamp, transient gene silencing, and western blotting. RESULTS: Here we show that key components of the glutamatergic signalling pathways are functional during in vitro chondrogenesis in a primary chicken chondrogenic model system. We also present the full glutamate receptor subunit mRNA and protein expression profile of these cultures. This is the first study to report that NMDA-mediated signalling may act as a key factor in embryonic limb bud-derived chondrogenic cultures as it evokes intracellular Ca2+ transients, which are abolished by the GluN2B subunit-specific inhibitor ifenprodil. The function of NMDARs is essential for chondrogenesis as their functional knock-down using either ifenprodil or GRIN1 siRNA temporarily blocks the differentiation of chondroprogenitor cells. Cartilage formation was fully restored with the re-expression of the GluN1 protein. CONCLUSIONS: We propose a key role for NMDARs during the transition of chondroprogenitor cells to cartilage matrix-producing chondroblasts.
Asunto(s)
Condrogénesis/genética , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Calcio/análisis , Calcio/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Pollos , Condrogénesis/efectos de los fármacos , Ácido Glutámico/análisis , N-Metilaspartato/farmacología , Receptores de N-Metil-D-Aspartato/agonistas , Transducción de Señal/efectos de los fármacosRESUMEN
The high electric field across the plasma membrane might influence the conformation and behavior of transmembrane proteins that have uneven charge distributions in or near their transmembrane regions. Membrane depolarization of T cells occurs in the tumor microenvironment and in inflamed tissues because of K+ release from necrotic cells and hypoxia affecting the expression of K+ channels. However, little attention has been given to the effect of membrane potential (MP) changes on membrane receptor function. Therefore, we studied the influence of membrane de- and hyperpolarization on the biophysical properties and signaling of interleukin-2 (IL-2) and interleukin-15 (IL-15) receptors, which play important roles in T cell function. We investigated the mobility, clustering, and signaling of these receptors and major histocompatibility complex (MHC) I/II glycoproteins forming coclusters in lipid rafts of T cells. Depolarization by high K+ buffer or K+ channel blockers resulted in a decrease in the mobility of IL-2Rα and MHC glycoproteins, as shown by fluorescence correlation spectroscopy, whereas hyperpolarization by the K+ ionophore valinomycin increased their mobility. Contrary to this, the mobility of IL-15Rα decreased upon both de- and hyperpolarization. These changes in protein mobility are not due to an alteration of membrane fluidity, as evidenced by fluorescence anisotropy measurements. Förster resonance energy transfer measurements showed that most homo- or heteroassociations of IL-2R, IL-15R, and MHC I did not change considerably, either. MP changes modulated signaling by the two cytokines in distinct ways: depolarization caused a significant increase in the IL-2-induced phosphorylation of signal transducer and activator of transcription 5, whereas hyperpolarization evoked a decrease only in the IL-15-induced signal. Our data imply that the MP may be an important modulator of interleukin receptor signaling and dynamics. Enhanced IL-2 signaling in depolarized Treg cells highly expressing IL-2R may contribute to suppression of antitumor immune surveillance.
Asunto(s)
Potenciales de la Membrana , Receptores de Interleucina-15/metabolismo , Receptores de Interleucina-2/metabolismo , Transducción de Señal , Linfocitos T/citología , Linfocitos T/metabolismo , Línea Celular Tumoral , Humanos , Fluidez de la Membrana , Microambiente TumoralRESUMEN
The article describes the development of Buchwald-Hartwig amination of different bromoflavones with amino acid and peptide derivatives as nitrogen source giving unique structures. The previously observed racemization, which occurred during the synthesis of flavone-amino acid hybrids, was successfully prevented in most cases. The biological assays of these novel structures showed cytotoxic effects on different cancer cell lines.
Asunto(s)
Aminoácidos/química , Dipéptidos/química , Flavonas/química , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Cinética , Pruebas de Sensibilidad Microbiana , Análisis Espectral/métodosRESUMEN
Cholesterol is an essential lipid building block of the cellular plasma membrane. In addition to its structural role, it regulates the fluidity and raft structure of the membrane and influences the course of numerous membrane-linked signaling pathways and the function of transmembrane proteins, including ion channels. This is supported by a vast body of scientific data, which demonstrates the modulation of ion channels with a great variety of ion selectivity, gating, and tissue distribution by changes in membrane cholesterol. Here, we review what is currently known about the modulation of voltage-gated K+ (Kv) channels by changes in membrane cholesterol content, considering raft association of the channels, the roles of cholesterol recognition sites, and those of adaptor proteins in cholesterol-Kv channel interactions. We specifically focus on Kv1.3, the dominant K+ channel of human T cells. Effects of cholesterol depletion and enrichment and 7-dehydrocholesterol enrichment on Kv1.3 gating are discussed in the context of the immunological synapse and the comparison of the in vitro effects of sterol modifications on Kv1.3 function with ex vivo effects on cells from hypercholesterolemic and Smith-Lemli-Opitz patients.