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1.
Cell Physiol Biochem ; 58(4): 431-444, 2024 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-39215550

RESUMO

BACKGROUND/AIMS: Over the years, the number of patients with neurodegenerative diseases is constantly rising illustrating the need for new neuroprotective drugs. A promising treatment approach is the reduction of excitotoxicity induced by rising (S)-glutamate levels and subsequent NMDA receptor overactivation. To facilitate the search for new NMDA receptor inhibitors neuronal cell models are needed. In this study, we evaluated the suitability of human SK-N-SH cells to serve as a cell model for neurodegeneration induced by NMDA receptor overstimulation. METHODS: The cytoprotective effect of the unselective NMDA receptor blocker ketamine as well as the GluN2B-selective inhibitor WMS14-10 was evaluated utilizing different cell viability assays, such as endpoint (LDH, CCK-8, DAPI/FACS) and time dependent methods (bioimpedance). RESULTS: Non-differentiated as well as differentiated SK-N-SH cells express GluN1 and GluN2B subunits. Furthermore, 50 mM (S)-glutamate led to an instantaneous decrease in cell survival. Only application of unselective channel blocker ketamine could protect differentiated cells against this effect, while the selective inhibitor WMS14-10 did not significantly increase cell survival. CONCLUSION: SK-N-SH cells show an increased sensitivity to (S)-glutamate mediated cytotoxicity with higher differentiation level, that is only partially induced by NMDA receptor overstimulation. Furthermore, we showed that only unselective NMDA receptor inhibition can partially reverse (S)-glutamate-induced toxicity.


Assuntos
Sobrevivência Celular , Ácido Glutâmico , Ketamina , Receptores de N-Metil-D-Aspartato , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Humanos , Sobrevivência Celular/efeitos dos fármacos , Ketamina/farmacologia , Linhagem Celular Tumoral , Ácido Glutâmico/metabolismo , Ácido Glutâmico/toxicidade , Diferenciação Celular/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/citologia , Neurônios/patologia , Proteínas do Tecido Nervoso
2.
Sci Rep ; 14(1): 19673, 2024 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-39187541

RESUMO

Several human diseases, including cancer and neurodegeneration, are associated with excessive mitochondrial fragmentation. In this context, mitochondrial division inhibitor (Mdivi-1) has been tested as a therapeutic to block the fission-related protein dynamin-like protein-1 (Drp1). Recent studies suggest that Mdivi-1 interferes with mitochondrial bioenergetics and complex I function. Here we show that the molecular mechanism of Mdivi-1 is based on inhibition of complex I at the IQ site. This leads to the destabilization of complex I, impairs the assembly of N- and Q-respirasomes, and is associated with increased ROS production and reduced efficiency of ATP generation. Second, the calcium homeostasis of cells is impaired, which for example affects the electrical activity of neurons. Given the results presented here, a potential therapeutic application of Mdivi-1 is challenging because of its potential impact on synaptic activity. Similar to the Complex I inhibitor rotenone, Mdivi-1 may lead to neurodegenerative effects in the long term.


Assuntos
Complexo I de Transporte de Elétrons , Mitocôndrias , Quinazolinonas , Complexo I de Transporte de Elétrons/metabolismo , Humanos , Quinazolinonas/farmacologia , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Animais , Espécies Reativas de Oxigênio/metabolismo , Cálcio/metabolismo , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Camundongos
3.
Biol Chem ; 404(4): 241-254, 2023 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-36809224

RESUMO

The Phosphatidylinositol 3-phosphate 5-kinase Type III PIKfyve is the main source for selectively generated phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a known regulator of membrane protein trafficking. PI(3,5)P2 facilitates the cardiac KCNQ1/KCNE1 channel plasma membrane abundance and therewith increases the macroscopic current amplitude. Functional-physical interaction of PI(3,5)P2 with membrane proteins and its structural impact is not sufficiently understood. This study aimed to identify molecular interaction sites and stimulatory mechanisms of the KCNQ1/KCNE1 channel via the PIKfyve-PI(3,5)P2 axis. Mutational scanning at the intracellular membrane leaflet and nuclear magnetic resonance (NMR) spectroscopy identified two PI(3,5)P2 binding sites, the known PIP2 site PS1 and the newly identified N-terminal α-helix S0 as relevant for functional PIKfyve effects. Cd2+ coordination to engineered cysteines and molecular modeling suggest that repositioning of S0 stabilizes the channel s open state, an effect strictly dependent on parallel binding of PI(3,5)P2 to both sites.


Assuntos
Canal de Potássio KCNQ1 , Fosfatidilinositol 4,5-Difosfato , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Sítios de Ligação , Mutação , Membrana Celular/metabolismo
4.
Cell Res ; 32(1): 72-88, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34702947

RESUMO

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that drive the cell fate decisions towards conventional (Tconv) or regulatory T cells (Treg). Following TCR activation, intracellular calcium (Ca2+) is the most important second messenger, for which the potassium channel K2P18.1 is a relevant regulator. Here, we identify K2P18.1 as a central translator of the TCR signal into the thymus-derived Treg (tTreg) selection process. TCR signal was coupled to NF-κB-mediated K2P18.1 upregulation in tTreg progenitors. K2P18.1 provided the driving force for sustained Ca2+ influx that facilitated NF-κB- and NFAT-dependent expression of FoxP3, the master transcription factor for Treg development and function. Loss of K2P18.1 ion-current function induced a mild lymphoproliferative phenotype in mice, with reduced Treg numbers that led to aggravated experimental autoimmune encephalomyelitis, while a gain-of-function mutation in K2P18.1 resulted in increased Treg numbers in mice. Our findings in human thymus, recent thymic emigrants and multiple sclerosis patients with a dominant-negative missense K2P18.1 variant that is associated with poor clinical outcomes indicate that K2P18.1 also plays a role in human Treg development. Pharmacological modulation of K2P18.1 specifically modulated Treg numbers in vitro and in vivo. Finally, we identified nitroxoline as a K2P18.1 activator that led to rapid and reversible Treg increase in patients with urinary tract infections. Conclusively, our findings reveal how K2P18.1 translates TCR signals into thymic T cell fate decisions and Treg development, and provide a basis for the therapeutic utilization of Treg in several human disorders.


Assuntos
Canais de Potássio , Receptores de Antígenos de Linfócitos T , Linfócitos T Reguladores , Animais , Diferenciação Celular , Fatores de Transcrição Forkhead , Humanos , Camundongos , NF-kappa B , Timócitos , Timo
5.
Cell Physiol Biochem ; 55(6): 679-703, 2021 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-34791861

RESUMO

Viral diseases are a major threat to modern society and the global health system. It is therefore of utter relevance to understand the way viruses affect the host as a basis to find new treatment solutions. The understanding of viral myocarditis (VMC) is incomplete and effective treatment options are lacking. This review will discuss the mechanism, effects, and treatment options of the most frequent myocarditis-causing viruses namely enteroviruses such as Coxsackievirus B3 (CVB3) and Parvovirus B19 (PVB19) on the human heart. Thereby, we focus on: 1. Viral entry: CVB3 use Coxsackievirus-Adenovirus-Receptor (CAR) and Decay Accelerating Factor (DAF) to enter cardiac myocytes while PVB19 use the receptor globoside (Gb4) to enter cardiac endothelial cells. 2. Immune system responses: The innate immune system mediated by activated cardiac toll-like receptors (TLRs) worsen inflammation in CVB3-infected mouse hearts. Different types of cells of the adaptive immune system are recruited to the site of inflammation that have either protective or adverse effects during VMC. 3. Autophagy: CVB3 evades autophagosomal degradation and misuses the autophasomal pathway for viral replication and release. 4. Viral replication sites: CVB3 promotes the formation of double membrane vesicles (DMVs), which it uses as replication sites. PVB19 uses the host cell nucleus as the replication site and uses the host cell DNA replication system. 5. Cell cycle manipulation: CVB3 attenuates the cell cycle at the G1/S phase, which promotes viral transcription and replication. PVB19 exerts cell cycle arrest in the S phase using its viral endonuclease activity. 6. Regulation of apoptosis: Enteroviruses prevent apoptosis during early stages of infection and promote cell death during later stages by using the viral proteases 2A and 3C, and viroporin 2B. PVB19 promotes apoptosis using the non-structural proteins NS1 and the 11 kDa protein. 7. Energy metabolism: Dysregulation of respiratory chain complex expression, activity and ROS production may be altered in CVB3- and PVB19-mediated myocarditis. 8. Ion channel modulation: CVB3-expression was indicated to alter calcium and potassium currents in Xenopus laevis oocytes and rodent cardiomyocytes. The phospholipase 2-like activity of PVB19 may alter several calcium, potassium and sodium channels. By understanding the general pathophysiological mechanisms of well-studied myocarditis-linked viruses, we might be provided with a guideline to handle other less-studied human viruses.


Assuntos
Infecções por Coxsackievirus/imunologia , Interações Hospedeiro-Patógeno/imunologia , Miocardite , Infecções por Parvoviridae/imunologia , Parvovirus B19 Humano/fisiologia , Replicação Viral , Infecções por Coxsackievirus/patologia , Humanos , Miocardite/imunologia , Miocardite/patologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/patologia , Miócitos Cardíacos/virologia , Infecções por Parvoviridae/patologia , Receptores Virais/imunologia
6.
Cell Physiol Biochem ; 55(S3): 108-130, 2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34043299

RESUMO

Transient receptor potential vanilloid (TRPV) channels are part of the TRP channel superfamily and named after the first identified member TRPV1, that is sensitive to the vanillylamide capsaicin. Their overall structure is similar to the structure of voltage gated potassium channels (Kv) built up as homotetramers from subunits with six transmembrane helices (S1-S6). Six TRPV channel subtypes (TRPV1-6) are known, that can be subdivided into the thermoTRPV (TRPV1-4) and the Ca2+-selective TRPV channels (TRPV5, TRPV6). Contrary to Kv channels, TRPV channels are not primary voltage gated. All six channels have distinct properties and react to several endogenous ligands as well as different gating stimuli such as heat, pH, mechanical stress, or osmotic changes. Their physiological functions are highly diverse and subtype as well as tissue specific. In many tissues they serve as sensors for different pain stimuli (heat, pressure, pH) and contribute to the homeostasis of electrolytes, the maintenance of barrier functions and the development of macrophages. Due to their fundamental role in manifold physiological and pathophysiological processes, TRPV channels are promising targets for drug development. However, drugs targeting specific TRPV channels, that are suitable for drug therapy, are rare. Moreover, selective and potent compounds for further research at TRPV channels are often lacking. In this review different aspects of the structure, the different gating stimuli, the expression pattern, the physiological and pathophysiological roles as well as the modulating mechanisms of synthetic, natural and endogenous ligands are summarized.


Assuntos
Analgésicos/farmacologia , Antineoplásicos/farmacologia , Fatores Imunológicos/farmacologia , Moduladores de Transporte de Membrana/farmacologia , Canais de Cátion TRPV/metabolismo , Analgésicos/química , Analgésicos/classificação , Antineoplásicos/química , Antineoplásicos/classificação , Sítios de Ligação , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/classificação , Ativação do Canal Iônico/efeitos dos fármacos , Ligantes , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Moduladores de Transporte de Membrana/química , Moduladores de Transporte de Membrana/classificação , Modelos Moleculares , Especificidade de Órgãos , Ligação Proteica , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/classificação , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Baço/citologia , Baço/efeitos dos fármacos , Baço/metabolismo , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/classificação
7.
J Med Chem ; 64(2): 1170-1179, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33426889

RESUMO

Ifenprodil (1) is a potent GluN2B-selective N-methyl-d-aspartate (NMDA) receptor antagonist that is used as a cerebral vasodilator and has been examined in clinical trials for the treatment of drug addiction, idiopathic pulmonary fibrosis, and COVID-19. To correlate biological data with configuration, all four ifenprodil stereoisomers were prepared by diastereoselective reduction and subsequent separation of enantiomers by chiral HPLC. The absolute configuration of ifenprodil stereoisomers was determined by X-ray crystal structure analysis of (1R,2S)-1a and (1S,2S)-1d. GluN2B affinity, ion channel inhibitory activity, and selectivity over α, σ, and 5-HT receptors were evaluated. (1R,2R)-Ifenprodil ((1R,2R)-1c) showed the highest affinity toward GluN2B-NMDA receptors (Ki = 5.8 nM) and high inhibition of ion flux in two-electrode voltage clamp experiments (IC50 = 223 nM). Whereas the configuration did not influence considerably the GluN2B-NMDA receptor binding, (1R)-configuration is crucial for elevated inhibitory activity. (1R,2R)-Configured ifenprodil (1R,2R)-1c exhibited high selectivity for GluN2B-NMDA receptors over adrenergic, serotonergic, and σ1 receptors.


Assuntos
Antifibrinolíticos/química , Antifibrinolíticos/farmacologia , Antivirais/química , Antivirais/farmacologia , Piperidinas/síntese química , Piperidinas/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Antifibrinolíticos/síntese química , Antivirais/síntese química , COVID-19/metabolismo , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/metabolismo , Modelos Moleculares , Estrutura Molecular , Piperidinas/química , Receptores de N-Metil-D-Aspartato/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Tratamento Farmacológico da COVID-19
8.
Adv Exp Med Biol ; 1349: 335-353, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35138621

RESUMO

KCNQ1 (KV7.1) K+ channels are expressed in multiple tissues, including the heart, pancreas, colon, and inner ear. The gene encoding the KCNQ1 protein was discovered by a positional cloning effort to determine the genetic basis of long QT syndrome, an inherited ventricular arrhythmia that can cause sudden death. Mutations in KCNQ1 can also cause other types of arrhythmia (i.e., short QT syndrome, atrial fibrillation) and the gene may also have a role in diabetes and certain cancers. KCNQ1 α-subunits can partner with accessory ß-subunits (KCNE1-KCNE5) to form K+-selective channels that have divergent biophysical properties. In the heart, KCNQ1 α-subunits coassemble with KCNE1 ß-subunits to form channels that conduct IKs, a very slowly activating delayed rectifier K+ current. KV7.1 channels are highly regulated by PIP2, calmodulin, and phosphorylation, and rich pharmacology includes blockers and gating modulators. Recent biophysical studies and a cryo-EM structure of the KCNQ1-calmodulin complex have provided new insights into KV7.1 channel function, and how interactions between KCNQ1 and KCNE subunits alter the gating properties of heteromultimeric channels.


Assuntos
Síndrome do QT Longo , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Arritmias Cardíacas/genética , Humanos , Canais de Potássio KCNQ , Canal de Potássio KCNQ1/genética , Canais de Potássio , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética
9.
Structure ; 28(8): 933-942.e4, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32502382

RESUMO

The TSC complex is the cognate GTPase-activating protein (GAP) for the small GTPase Rheb and a crucial regulator of the mechanistic target of rapamycin complex 1 (mTORC1). Mutations in the TSC1 and TSC2 subunits of the complex cause tuberous sclerosis complex (TSC). We present the crystal structure of the catalytic asparagine-thumb GAP domain of TSC2. A model of the TSC2-Rheb complex and molecular dynamics simulations suggest that TSC2 Asn1643 and Rheb Tyr35 are key active site residues, while Rheb Arg15 and Asp65, previously proposed as catalytic residues, contribute to the TSC2-Rheb interface and indirectly aid catalysis. The TSC2 GAP domain is further stabilized by interactions with other TSC2 domains. We characterize TSC2 variants that partially affect TSC2 functionality and are associated with atypical symptoms in patients, suggesting that mutations in TSC1 and TSC2 might predispose to neurological and vascular disorders without fulfilling the clinical criteria for TSC.


Assuntos
Domínio Catalítico , Mutação de Sentido Incorreto , Proteína 2 do Complexo Esclerose Tuberosa/química , Esclerose Tuberosa/genética , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Proteína Enriquecida em Homólogo de Ras do Encéfalo/química , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo
10.
Cell Physiol Biochem ; 44(3): 1024-1037, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29179200

RESUMO

BACKGROUND/AIMS: TASK channels belong to the two-pore-domain potassium (K2P) channel family. TASK-1 is discussed to contribute to chronic atrial fibrillation (AFib) and has been together with uncoupling protein 1 found as a marker protein of brown adipose tissue (BAT) fat. In addition, TASK-1 was linked in a genome-wide association study to an increased body mass index. A recent study showed that TASK-1 inhibition is causing obesity in mice by a BAT whitening and that these effects are linked to the mineralocorticoid receptor pathway, albeit the mechanism remained elusive. Therefore, we aimed to probe whether K2P channels are regulated by serum- and glucocorticoid-inducible kinases (SGKs) which are known to modify many cellular functions by modulating ion channels. METHODS: To this end we used functional co-expression studies and chemiluminescence-assays in Xenopus oocytes, together with fluorescence imaging and quantitative PCR experiments. RESULTS: SGKs and proteinkinase B (PKB) induced a strong, dose- and time-dependent current reduction of TASK-1 and TASK-3. SGK co-expression reduced the surface expression of TASK-1/3, leading to a predominant localization of the channels into late endosomes. The down regulation of TASK-3 channels was abrogated by the dynamin inhibitor dynasore, confirming a role of SGKs in TASK-1/3 channel endocytosis. CONCLUSION: Stress-mediated changes in SGK expression pattern or activation is likely to alter TASK-1/3 expression at the surface membrane. The observed TASK-1 regulation might contribute to the pathogenesis of chronic AFib and provide a mechanistic link between increased mineralocorticoid levels and TASK-1 reduction, both linked to BAT whitening.


Assuntos
Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Animais , Células COS , Chlorocebus aethiops , Clatrina/metabolismo , Endocitose , Endossomos/metabolismo , Células HeLa , Humanos , Hidrazonas/farmacologia , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Medições Luminescentes , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/genética , Oócitos/química , Oócitos/fisiologia , Técnicas de Patch-Clamp , Plasmídeos/genética , Plasmídeos/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Imagem com Lapso de Tempo , Xenopus laevis/crescimento & desenvolvimento
11.
Int J Nanomedicine ; 11: 5221-5236, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27789942

RESUMO

Within the last years, progress has been made in the knowledge of the properties of medically used nanoparticles and their toxic effects, but still, little is known about their influence on cellular processes of immune cells. The aim of our comparative study was to present the influence of two different nanoparticle types on subcellular processes of primary monocytes and the leukemic monocyte cell line MM6. We used core-shell starch-coated superparamagnetic iron oxide nanoparticles (SPIONs) and matrix poly(lactic-co-glycolic acid) (PLGA) nanoparticles for our experiments. In addition to typical biocompatibility testing like the detection of necrosis or secretion of interleukins (ILs), we investigated the impact of these nanoparticles on the actin cytoskeleton and the two voltage-gated potassium channels Kv1.3 and Kv7.1. Induction of necrosis was not seen for PLGA nanoparticles and SPIONs in primary monocytes and MM6 cells. Likewise, no alteration in secretion of IL-1ß and IL-10 was detected under the same experimental conditions. In contrast, IL-6 secretion was exclusively downregulated in primary monocytes after contact with both nanoparticles. Two-electrode voltage clamp experiments revealed that both nanoparticles reduce currents of the aforementioned potassium channels. The two nanoparticles differed significantly in their impact on the actin cytoskeleton, demonstrated via atomic force microscopy elasticity measurement and phalloidin staining. While SPIONs led to the disruption of the respective cytoskeleton, PLGA did not show any influence in both experimental setups. The difference in the effects on ion channels and the actin cytoskeleton suggests that nanoparticles affect these subcellular components via different pathways. Our data indicate that the alteration of the cytoskeleton and the effect on ion channels are new parameters that describe the influence of nanoparticles on cells. The results are highly relevant for medical application and further evaluation of nanomaterial biosafety.


Assuntos
Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Compostos Férricos/química , Ácido Láctico/química , Monócitos/efeitos dos fármacos , Nanopartículas/química , Ácido Poliglicólico/química , Amido/química , Linhagem Celular Tumoral , Humanos , Interleucina-6/metabolismo , Monócitos/citologia , Monócitos/metabolismo , Copolímero de Ácido Poliláctico e Ácido Poliglicólico
12.
Cell Physiol Biochem ; 39(4): 1444-52, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27607061

RESUMO

BACKGROUND/AIMS: Inherited, autosomal dominant spinocerebellar ataxia type 11 (SCA11) is caused by loss of function mutations of TTBK2 (tau tubulin kinase 2). Mutations observed in patients with SCA11 include truncated TTBK2(450). The present study explored the possibility that TTBK2 influences the function of the glutamate receptor GluK2. METHODS: GluK2 was expressed in Xenopus oocytes without and with additional expression of wild type TTBK2, the truncated mutant TTBK2(450), or the kinase dead mutants TTBK2(KD) and TTBK2(450/KD). GluK2 current was determined by dual electrode voltage clamp and GluK2 protein abundance in the cell membrane utilizing confocal microscopy. RESULTS: Glutamate exposure of GluK2 expressing oocytes generated a current, which was significantly lower in oocytes expressing GluK2 together with TTBK2 wt or TTBK2(KD) than in oocytes expressing GluK2 alone or together with either TTBK2(450) or TTBK2(450/KD). According to confocal microscopy of EGFP-tagged GluK2, TTBK2 wt decreased the GluK2 protein abundance in the cell membrane. Overexpression of an inactive RAB5(N133I) mutant but not RAB5wt could reverse the TTBK2 effect on GluK2 suggesting that RAB5 function is required for the effect. CONCLUSIONS: TTBK2 down-regulates GluK2 activity by decreasing the receptor protein abundance in the cell membrane via RAB5-dependent endocytosis, an effect that may protect against neuroexcitotoxicity.


Assuntos
Ácido Glutâmico/metabolismo , Oócitos/metabolismo , Peptídeos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Ácido Caínico/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Transporte Biológico , Membrana Celular/metabolismo , Endocitose , Regulação da Expressão Gênica , Humanos , Microinjeções , Mutação , Oócitos/citologia , Técnicas de Patch-Clamp , Peptídeos/genética , Domínios Proteicos , Proteínas Serina-Treonina Quinases/genética , Receptores de Ácido Caínico/genética , Transdução de Sinais , Transgenes , Xenopus laevis , Proteínas rab5 de Ligação ao GTP/genética
13.
Europace ; 18(10): 1587-1592, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26705564

RESUMO

AIMS: The early repolarization pattern (ERP) has been shown to be associated with arrhythmias in patients with short QT syndrome, Brugada syndrome, and ischaemic heart disease. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome and related to malignant ventricular tachyarrhythmias in a structurally normal heart. The aim of this study was to evaluate the prevalence of ERP and clinical events in patients with CPVT. METHODS AND RESULTS: Digitalized resting 12-lead ECGs of patients were analysed for ERP and for repolarization markers (QT and Tpeak-Tend interval). The ERP was diagnosed as 'notching' or 'slurring' at the terminal portion of QRS with ≥0.1 mV elevation in at least two consecutive inferior (II, III, aVF) and/or lateral leads (V4-V6, I, aVL). Among 51 CPVT patients (mean age 36 ± 15 years, 11 males), the ERP was present in 23 (45%): strictly in the inferior leads in 9 (18%) patients, in the lateral leads in 9 (18%) patients, and in infero-lateral leads in 5 (10%) patients. All patients with ERP were symptomatic at presentation (23 of 23 patients with ERP vs. 19 of 28 patients without ERP, P = 0.003). Syncope was also more frequent in patients with ERP (18 of 23 patients with ERP vs. 11 of 28 patients without ERP, P = 0.005). CONCLUSION: A pathologic ERP is present in an unexpected large proportion (45%) of patients and is associated with an increased frequency of syncope. In patients with unexplained syncope and ERP at baseline, exercise testing should be performed to detect CPVT.


Assuntos
Sistema de Condução Cardíaco/fisiopatologia , Síncope/epidemiologia , Taquicardia Ventricular/diagnóstico , Taquicardia Ventricular/fisiopatologia , Adolescente , Antagonistas de Receptores Adrenérgicos beta 1/uso terapêutico , Adulto , Idoso , Criança , Desfibriladores Implantáveis , Eletrocardiografia , Feminino , Testes Genéticos , Alemanha , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Síncope/etiologia , Taquicardia Ventricular/terapia , Adulto Jovem
14.
FEBS Lett ; 587(11): 1663-8, 2013 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-23603386

RESUMO

Klotho, a hormone and enzyme, is a powerful regulator of ageing and life span. Klotho deficiency leads to cardiac arrythmia and sudden cardiac death. We thus explored whether klotho modifies cardiac K(+)-channel hERG. Current was determined utilizing dual electrode voltage clamp and hERG protein abundance utilizing immunohistochemistry and chemiluminescence in Xenopus oocytes expressing hERG with or without klotho. Coexpression of klotho increased cell membrane hERG-protein abundance and hERG current at any given voltage without significantly modifying the voltage required to activate the channel. The effect of klotho coexpression was mimicked by recombinant klotho protein and reversed by ß-glucuronidase-inhibitor D-saccharic acid-1,4-lactone.


Assuntos
Canais de Potássio Éter-A-Go-Go/fisiologia , Glucuronidase/metabolismo , Animais , Células Cultivadas , Canal de Potássio ERG1 , Ácido Glucárico/farmacologia , Glucuronidase/antagonistas & inibidores , Glucuronidase/genética , Ativação do Canal Iônico , Proteínas Klotho , Lactonas/farmacologia , Potenciais da Membrana , Oócitos/fisiologia , Técnicas de Patch-Clamp , RNA Complementar/genética , Xenopus
15.
Cell Physiol Biochem ; 30(6): 1538-46, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23234856

RESUMO

BACKGROUND: The amino acid transporter B0AT1 (SLC6A19) accomplishes concentrative cellular uptake of neutral amino acids. SLC6A19 is stimulated by serum- & glucocorticoid-inducible kinase (SGK) isoforms. SGKs are related to PKB/Akt isoforms, which also stimulate several amino acid transporters. PKB/Akt modulates glucose transport in part by phosphorylating and thus activating phosphatidylinositol-3-phosphate-5-kinase (PIKfyve), which fosters carrier protein insertion into the cell membrane. The present study explored whether PKB/Akt and/or PIKfyve stimulate SLC6A19. METHODS: SLC6A19 was expressed in Xenopus oocytes with or without wild-type PKB/Akt or inactive (T308A/S473A)PKB/Akt without or with additional expression of wild-type PIKfyve or PKB/Akt-resistant (S318A)PIKfyve. Electrogenic amino acid transport was determined by dual electrode voltage clamping. RESULTS: In SLC6A19-expressing oocytes but not in water-injected oocytes, the addition of the neutral amino acid L-leucine (2 mM) to the bath generated a current (I(le)), which was significantly increased following coexpression of PKB/Akt, but not by coexpression of (T308A/S473A)PKB/Akt. The effect of PKB/Akt was augmented by additional coexpression of PIKfyve but not of (S318A)PIKfyve. Coexpression of PKB/Akt enhanced the maximal transport rate without significantly modifying the affinity of the carrier. The decline of I(le) following inhibition of carrier insertion by brefeldin A (5 µM) was similar in the absence and presence of PKB/Akt indicating that PKB/Akt stimulated carrier insertion into rather than inhibiting carrier retrieval from the cell membrane. CONCLUSION: PKB/Akt up-regulates SLC6A19 activity, which may foster amino acid uptake into PKB/Akt-expressing epithelial and tumor cells.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regulação para Cima , Sistemas de Transporte de Aminoácidos Neutros/genética , Animais , Membrana Celular/metabolismo , Expressão Gênica , Humanos , Cinética , Leucina/metabolismo , Transporte Proteico , Xenopus
16.
PLoS One ; 7(8): e43353, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22905262

RESUMO

The multifunctional protein ß-catenin governs as transcription factor the expression of a wide variety of genes relevant for cell proliferation and cell survival. In addition, ß-catenin is localized at the cell membrane and may influence the function of channels. The present study explored the possibility that ß-catenin participates in the regulation of the HERG K(+) channel. To this end, HERG was expressed in Xenopus oocytes with or without ß-catenin and the voltage-gated current determined utilizing the dual electrode voltage clamp. As a result, expression of ß-catenin markedly upregulated HERG channel activity, an effect not sensitive to inhibition of transcription with actinomycin D (10 µM). According to chemiluminescence, ß-catenin may increase HERG channel abundance within the oocyte cell membrane. Following inhibition of channel insertion into the cell membrane by brefeldin A (5 µM) the decay of current was similar in oocytes expressing HERG together with ß-catenin to oocytes expressing HERG alone. The experiments uncover a novel function of APC/ß-catenin, i.e. the regulation of HERG channels.


Assuntos
Canais de Potássio Éter-A-Go-Go/metabolismo , beta Catenina/metabolismo , Animais , Caderinas/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células , Dactinomicina/farmacologia , Canal de Potássio ERG1 , Humanos , Mutação , Oócitos/citologia , Técnicas de Patch-Clamp , Xenopus
17.
Cell Physiol Biochem ; 29(5-6): 809-18, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22613981

RESUMO

BACKGROUND: Cardiac action potential repolarisation is determined by K(+) currents including I(Ks). I(Ks) channels are heteromeric channels composed of KCNQ1 and KCNE E-subunits. Mutations in KCNQ1 are associated with sinus bradycardia, familial atrial fibrillation (fAF) and/or short QT syndrome as a result of gain-of-function, and long QT syndrome (LQTS) due to loss-of-function in the ventricles. Here, we report that the missense mutation R231C located in S4 voltage sensor domain is associated with a combined clinical phenotype of sinus bradycardia, fAF and LQTS. We aim to understand the molecular basis of the complex clinical phenotype. METHODS: We expressed and functionally analyzed the respective channels kinetics in Xenopus laevis oocytes. The molecular nature of the residue R231 was studied by homology modeling and molecular dynamics simulation. RESULTS: As a result, the mutation reduced voltage sensitivity of channels, possibly due to neutralization of the positive charge of the arginine side chain substituted by cysteine. Modeling suggested that the charge carrying side chain of R231 is positioned suitably to transfer transmembrane voltages into conformational energy. Further, the mutation altered the functional interactions with KCNE subunits. CONCLUSION: The mutation acted in a E-subunit dependent manner, suggesting I(Ks) function altered by the presence of different KCNE subunits in sinus node, atria and ventricles as the molecular basis of sinus bradycardia, fAF and LQTS in mutation carriers.


Assuntos
Canal de Potássio KCNQ1/genética , Mutação , Adulto , Sequência de Aminoácidos , Animais , Criança , Eletrocardiografia , Feminino , Humanos , Lactente , Recém-Nascido , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/fisiologia , Masculino , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Linhagem , Fenótipo , Homologia de Sequência de Aminoácidos , Xenopus laevis
18.
PLoS One ; 7(3): e33889, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22470488

RESUMO

We previously showed that the serum- and glucocorticoid-inducible kinase 3 (SGK3) increases the AMPA-type glutamate receptor GluA1 protein in the plasma membrane. The activation of AMPA receptors by NMDA-type glutamate receptors eventually leads to postsynaptic neuronal plasticity. Here, we show that SGK3 mRNA is upregulated in the hippocampus of new-born wild type Wistar rats after NMDA receptor activation. We further demonstrate in the Xenopus oocyte expression system that delivery of GluA1 protein to the plasma membrane depends on the small GTPase RAB11. This RAB-dependent GluA1 trafficking requires phosphorylation and activation of phosphoinositol-3-phosphate-5-kinase (PIKfyve) and the generation of PI(3,5)P(2). In line with this mechanism we could show PIKfyve mRNA expression in the hippocampus of wild type C57/BL6 mice and phosphorylation of PIKfyve by SGK3. Incubation of hippocampal slices with the PIKfyve inhibitor YM201636 revealed reduced CA1 basal synaptic activity. Furthermore, treatment of primary hippocampal neurons with YM201636 altered the GluA1 expression pattern towards reduced synaptic expression of GluA1. Our findings demonstrate for the first time an involvement of PIKfyve and PI(3,5)P(2) in NMDA receptor-triggered synaptic GluA1 trafficking. This new regulatory pathway of GluA1 may contribute to synaptic plasticity and memory.


Assuntos
Receptores de AMPA/metabolismo , Transdução de Sinais , Aminopiridinas/farmacologia , Animais , Animais Recém-Nascidos , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Regulação para Cima , Xenopus/crescimento & desenvolvimento , Xenopus/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
19.
Biochem Biophys Res Commun ; 417(2): 692-6, 2012 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-22166221

RESUMO

Voltage-gated Kv1.5 channels are expressed in a wide variety of tissues including cardiac myocytes, smooth muscle and tumor cells. Kv1.5 channel activity is modified by N-cadherin, which in turn binds the multifunctional oncogenic protein ß-catenin. The present experiments explored the effect of ß-catenin on Kv1.5 channel activity. To this end, Kv1.5 was expressed in Xenopus oocytes with or without ß-catenin and the voltage-gated Kv current determined by dual electrode voltage clamp. As a result, expression of ß-catenin significantly increased the voltage-gated Kv current at positive potentials. The stimulating effect of ß-catenin on Kv1.5 was not dependent on the stimulation of transcription since it was observed even in the presence of the transcription inhibitor actinomycin D. Specific antibody binding to surface Kv1.5 in Xenopus oocytes revealed that ß-catenin enhances the membrane abundance of Kv1.5. Further experiments with brefeldin A showed that ß-catenin fosters the insertion of Kv1.5 into rather than delaying the retrieval from the plasma membrane. According to electrophysiological recordings with mutant ß-catenin, the effect on Kv1.5 requires the same protein domains that are required for association of ß-catenin with cadherin. The experiments disclose a completely novel function of ß-catenin, i.e. the regulation of Kv1.5 channel activity.


Assuntos
Membrana Celular/metabolismo , Canal de Potássio Kv1.5/metabolismo , beta Catenina/metabolismo , Animais , Células Cultivadas , Dactinomicina/farmacologia , Humanos , Canal de Potássio Kv1.5/agonistas , Canal de Potássio Kv1.5/genética , Oócitos , Transcrição Gênica/efeitos dos fármacos , Xenopus , beta Catenina/genética
20.
Cell Physiol Biochem ; 26(4-5): 503-12, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-21063088

RESUMO

RATIONALE: The plateau phase of the ventricular action potential is the result of balanced Ca(2+) influx and K(+) efflux. The action potential is terminated by repolarizing K(+) currents. Under ß-adrenergic stimulation, both the Ca(2+)-influx and the delayed rectifier K(+) currents I(K) are stimulated to adjust the cardiac action potential duration to the enhanced heart rate and to ascertain adequate increase in net Ca(2+) influx. Intracellularly, a Calsequestrin2 (CASQ2)-ryanodine receptor complex serves as the most effective Ca(2+) reservoir/release system to aid the control of intracellular Ca(2+) levels. Currently, it is unclear if disease-associated CASQ2 gene variants alter intracellular free Ca(2+) concentrations and if cardiac ion channels are affected by it. OBJECTIVE: The goal of this study is to test if CASQ2 determines intracellular free Ca(2+) concentrations and to identify cardiac ion channels that are affected by it. Further, we aim to study disease-associated CASQ2 gene variants in this context. METHODS AND RESULTS: Here, we study the effects of the CASQ2 mutations R33Q, F189L, and D307H, located in highly conserved regions, on the functions of cardiac potassium channels in Xenopus oocytes using two electrode voltage clamp. As a result, CASQ2 wild type and CASQ2-mutants modulated hERG functions differently. Free Ca(2+) measurements and molecular dynamics simulations imply alterations in Ca(2+) buffer capacity paralled by changes in the dynamic behavior of the CASQ2-mutants compared to CASQ2 wild type. CONCLUSIONS: These in vitro and in silico data suggest a regulatory role of CASQ2 on cytosolic Ca(2+) and hERG channels which may contribute to the etiology of CPVT.


Assuntos
Calsequestrina/fisiologia , Canais de Potássio Éter-A-Go-Go/metabolismo , Potenciais de Ação/fisiologia , Substituição de Aminoácidos , Animais , Cálcio/metabolismo , Calsequestrina/genética , Calsequestrina/metabolismo , Humanos , Simulação de Dinâmica Molecular , Mutação , Oócitos/metabolismo , Potássio/metabolismo , Estrutura Terciária de Proteína , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Taquicardia Ventricular/etiologia , Xenopus/crescimento & desenvolvimento
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