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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.
Biol Chem ; 404(4): 267-277, 2023 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-36630596

RESUMO

N-Methyl-D-aspartate receptors (NMDARs) are central for learning and information processing in the brain. Dysfunction of NMDARs can play a key role in the pathogenesis of neurodegeneration and drug addiction. The development of selective NMDAR modulators represents a promising strategy to target these diseases. Among such modulating compounds are ifenprodil and its 3-benzazepine derivatives. Classically, the effects of these NMDAR modulators have been tested by techniques like two-electrode voltage clamp (TEVC), patch clamp, or fluorescence-based assays. However, testing their functional effects in complex human systems requires more advanced approaches. Here, we established a human induced pluripotent stem cell-derived (hiPSC-derived) neural cell system and proved its eligibility as a test system for investigating NMDAR modulators and pharmaceutical effects on human neurons.


Assuntos
Células-Tronco Pluripotentes Induzidas , Receptores de N-Metil-D-Aspartato , Humanos , Neurônios
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 Mol Life Sci ; 79(8): 440, 2022 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-35864219

RESUMO

The enterovirus Coxsackievirus B3 (CVB3) is known to be a major source for the development of cardiac dysfunctions like viral myocarditis (VMC) and dilatative cardiomyopathy (DCM), but also results in bradycardia and fatal cardiac arrest. Besides clinical reports on bradycardia and sudden cardiac death, very little is known about the influence of CVB3 on the activity of human cardiac pacemaker cells. Here, we address this issue using the first human induced pluripotent stem cell (hiPSC)-derived pacemaker-like cells, in which the expression of a transgenic non-infectious variant of CVB3 can be controlled dose- and time-dependently. We found that CVB3 drastically changed hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) distribution and function in hiPSC-derived pacemaker-like tissue. In addition, using HCN4 cell expression systems, we found that HCN4 currents were decreased with altered voltage dependency of activation when CVB3 was expressed. Increased autophagosome formation and autophagosomal HCN4 insertion was observed in hiPSC-derived pacemaker-like cells under CVB3 expression as well. Individual effects of single, non-structural CVB3 proteins were analyzed and demonstrated that CVB3 proteins 2C and 3A had the most robust effect on HCN4 activity. Treatment of cells with the Rab7 inhibitor CID 106770 or the CVB3-3A inhibitor GW5074 led to the recovery of the cytoplasmatic HCN4 accumulation into a healthy appearing phenotype, indicating that malfunctioning Rab7-directed autophagosome transport is involved in the disturbed, cytoplasmatic HCN4 accumulation in CVB3-expressing human pacemaker-like cells. Summarizing, the enterovirus CVB3 inhibits human cardiac pacemaker function by reducing the pacemaker channel plasma membrane density, an effect that can be corrected by pharmacological intervention of endocytic vesicle trafficking.


Assuntos
Bradicardia , Células-Tronco Pluripotentes Induzidas , Bradicardia/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Musculares/genética , Canais de Potássio , Nó Sinoatrial/metabolismo
5.
Int J Mol Sci ; 24(17)2023 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-37686171

RESUMO

The human heart controls blood flow, and therewith enables the adequate supply of oxygen and nutrients to the body. The correct function of the heart is coordinated by the interplay of different cardiac cell types. Thereby, one can distinguish between cells of the working myocardium, the pace-making cells in the sinoatrial node (SAN) and the conduction system cells in the AV-node, the His-bundle or the Purkinje fibres. Tissue-engineering approaches aim to generate hiPSC-derived cardiac tissues for disease modelling and therapeutic usage with a significant improvement in the differentiation quality of myocardium and pace-making cells. The differentiation of cells with cardiac conduction system properties is still challenging, and the produced cell mass and quality is poor. Here, we describe the generation of cardiac cells with properties of the cardiac conduction system, called conduction system-like cells (CSLC). As a primary approach, we introduced a CrispR-Cas9-directed knockout of the NKX2-5 gene in hiPSC. NKX2-5-deficient hiPSC showed altered connexin expression patterns characteristic for the cardiac conduction system with strong connexin 40 and connexin 43 expression and suppressed connexin 45 expression. Application of differentiation protocols for ventricular- or SAN-like cells could not reverse this connexin expression pattern, indicating a stable regulation by NKX2-5 on connexin expression. The contraction behaviour of the hiPSC-derived CSLCs was compared to hiPSC-derived ventricular- and SAN-like cells. We found that the contraction speed of CSLCs resembled the expected contraction rate of human conduction system cells. Overall contraction was reduced in differentiated cells derived from NKX2-5 knockout hiPSC. Comparative transcriptomic data suggest a specification of the cardiac subtype of CSLC that is distinctly different from ventricular or pacemaker-like cells with reduced myocardial gene expression and enhanced extracellular matrix formation for improved electrical insulation. In summary, knockout of NKX2-5 in hiPSC leads to enhanced differentiation of cells with cardiac conduction system features, including connexin expression and contraction behaviour.


Assuntos
Proteína Homeobox Nkx-2.5 , Células de Purkinje , Fatores de Transcrição , Humanos , Doença do Sistema de Condução Cardíaco , Proteína Homeobox Nkx-2.5/genética , Ramos Subendocárdicos , Transdução de Sinais , Nó Sinoatrial , Células-Tronco , Fatores de Transcrição/genética , Células-Tronco Pluripotentes Induzidas/metabolismo
6.
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
7.
Cell Physiol Biochem ; 55(3): 301-310, 2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34148308

RESUMO

BACKGROUND/AIMS: Neanderthals, although well adapted to local environments, were rapidly replaced by anatomically modern humans (AMH) for unknown reasons. Genetic information on Neanderthals is limited restricting applicability of standard population genetics. METHODS: Here, we apply a novel combination of restricted genetic analyses on preselected physiological key players (ion channels), electrophysiological analyses of gene variants of unclear significance expressed in Xenopus laevis oocytes using two electrode voltage clamp and transfer of results to AMH genetics. Using genetic screening in infertile men identified a loss of CLC-2 associated with sperm deficiency. RESULTS: Increased genetic variation caused functionally impaired Neanderthals CLC-2 channels. CONCLUSION: Increased genetic variation could reflect an adaptation to different local salt supplies at the cost of reduced sperm density. Interestingly and consistent with this hypothesis, lack of CLC-2 protein in a patient associates with high blood K+ concentration and azoospermia.


Assuntos
Canais de Cloreto , Variação Genética , Infertilidade Masculina , Homem de Neandertal , Animais , Canais de Cloro CLC-2 , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Humanos , Masculino , Homem de Neandertal/genética , Homem de Neandertal/metabolismo , Oócitos/metabolismo , Xenopus laevis
8.
Cell Physiol Biochem ; 54(2): 321-332, 2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32259418

RESUMO

BACKGROUND/AIMS: The cardiac current IKs is carried by the KCNQ1/KCNE1-channel complex. Genetic aberrations that affect the activity of KCNQ1/KCNE1 can lead to the Long QT Syndrome 1 and 5 and, thereby, to a predisposition to sudden cardiac death. This might be prevented by pharmacological modulation of KCNQ1/KCNE1. The prototypic KCNQ1/KCNE1 activator 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) represents a candidate drug. Here, we study the mechanism of DIDS action on KCNQ1/KCNE1. METHODS: Channels were expressed in Xenopus oocytes and iPSC cardiomyocytes. The role of the central S6 region was investigated by alanin-screening of KCNQ1 residues 333-338. DIDS effects were measured by TEVC and MEA. RESULTS: DIDS-action is influenced by the presence of KCNE1 but not by KCNQ1/KCNE1 stochiometry. V334A produces a significant higher increase in current amplitude, whereas deactivation (slowdown) DIDS-sensitivity is affected by residues 334-338. CONCLUSION: We show that the central S6 region serves as a hub for allosteric channel activation by the drug and that DIDS shortens the pseudo QT interval in iPSC cardiomyocytes. The elucidation of the structural and mechanistic underpinnings of the DIDS action on KCNQ1/KCNE1 might allow for a targeted design of DIDS derivatives with improved potency and selectivity.


Assuntos
Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Potenciais de Ação/efeitos dos fármacos , Canal de Potássio KCNQ1/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/química , Regulação Alostérica , Animais , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/genética , Modelos Moleculares , Mutação , Oócitos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Domínios Proteicos , Xenopus laevis
9.
Cell Physiol Biochem ; 53(1): 121-140, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31230428

RESUMO

Infections with Coxsackievirus B3 and other members of the enterovirus genus are a common reason for myocarditis and sudden cardiac death in modern society. Despite intensive scientific efforts to cure enterovirus infections, there is still no standardized treatment option. The complexity of Coxsackievirus B3´s effects on the host cell make well defined studies on this topic very challenging. However, recent publications report newly found effects of CVB3´s structural and non-structural proteins on infected cells. For the first time, the viral capsid protein VP1 was shown to have direct influence on the viral life-cycle. By shortening the G0 and the G2 phase and simultaneously prolonging the G1 and G1-S phase, the translation of viral proteins is enhanced and the production of viable CVB3 particles is promoted. Coxsackievirus B3´s viroporin, protein 2B, was recently studied in more detail as well. Structural and physiological analyses identified two hydrophilic α-helices in the structure of 2B, enabling it to insert into cellular membranes of host cells. As main target of 2B the endoplasmatic reticulum was identified. The insertion of 2B into the ER membranes leads to an uncontrolled calcium outflow into the cytoplasm. Additional insertion of 2B into the cell membrane leads to host cell destabilization and in the end to release of viral progeny. The importance of the Coxsackievirus B3´s proteases 2A and 3C in pathogenicity is observed since years. Recently, DAP5 and eIf4G were identified as new cleavage targets for protease 2A. Cleavage of DAP-5 into DAP5-N and DAP5-C changes the gene expression of the host cell and promotes cell death. Additionally, protease 3C targets and cleaves procaspase 8 promoting the mitochondrial apoptosis pathway and cell death. Recent studies identified significant effects of CVB3 on mitochondria of infected cells. Mouse cardiomyocytes showed decreased activities of respiratory chain complexes I-III and changed transcription of important subunits of the complexes I-IV. A disrupted energy metabolism may be one of the main causes of cardiac insufficiency and death in CVB3 infected patients. In addition to a modified energy metabolism, CVB3 affects cardiac ion channels, KCNQ1 in particular. SGK1, which is an important mediator in KCNQ1 membrane insertions, is highly upregulated during CVB3 infections. This results in an increased insertion of KCNQ1 into the cell membrane of cardiac cells. Under stress conditions, this KCNQ1 overshoot may lead to a disturbed cardiac action potential and therefore to sudden cardiac death, as it is often observed in CVB3 infected persons.


Assuntos
Infecções por Coxsackievirus/patologia , Enterovirus Humano B/fisiologia , Animais , Proteínas do Capsídeo/metabolismo , Infecções por Coxsackievirus/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Enterovirus Humano B/patogenicidade , Humanos , Canal de Potássio KCNQ1/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/virologia , Proteínas não Estruturais Virais/metabolismo
10.
Cell Physiol Biochem ; 49(3): 1197-1207, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30196304

RESUMO

BACKGROUND/AIMS: The hyperpolarization-activated cyclic nucleotide-gated cation channel HCN4 contributes significantly to the generation of basic cardiac electrical activity in the sinus node and is a mediator of modulation by ß-adrenergic stimulation. Heterologous expression of sick sinus syndrome (SSS) and bradycardia associated mutations within the human HCN4 gene results in altered channel function. The main aim was to describe the functional characterization of three (two novel and one known) missense mutations of HCN4 identified in families with SSS. METHODS: Here, the two-electrode voltage clamp technique on Xenopus laevis oocytes and confocal imaging on transfected COS7 cells respectively, were used to analyze the functional effects of three HCN4 mutations; R378C, R550H, and E1193Q. Membrane surface expressions of wild type and the mutant channels were assessed by confocal microscopy, chemiluminescence assay, and Western blot in COS7 and HeLa cells. RESULTS: The homomeric mutant channels R550H and E1193Q showed loss of function through increased rates of deactivation and distinctly reduced surface expression in all three homomeric mutant channels. HCN4 channels containing R550H and E1193Q mutant subunits only showed minor effects on the voltage dependence and rates of activation/deactivation. In contrast, homomeric R378C exerted a left-shifted activation curve and slowed activation kinetics. These effects were reduced in heteromeric co-expression of R378C with wild-type (WT) channels. CONCLUSION: Dysfunction of homomeric/heteromeric mutant HCN4-R378C, R550H, and E1193Q channels in the present study was primarily caused by loss of function due to decreased channel surface expression.


Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Síndrome do Nó Sinusal/genética , Potenciais de Ação/fisiologia , Animais , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Microscopia Confocal , Simulação de Dinâmica Molecular , Proteínas Musculares/química , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/química , Estrutura Terciária de Proteína , Xenopus laevis
11.
Pflugers Arch ; 469(10): 1233-1243, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28573409

RESUMO

The possibility to generate induced pluripotent stem cells (iPSC) opens the way to generate virtually all cell types of our human body. In combination with modern gene editing techniques like CRISPR/CAS, a new set of powerful tools becomes available for life science. Scientific fields like genotype and cell type-specific pharmacology, disease modeling, stem cell biology, and developmental biology have been dramatically fostered and their faces have been changed. However, as golden as the age of iPSC-derived cells and their manipulation has started, the shine begins to tarnish. Researchers face more and more practical problems intrinsic to the system. These problems are related to the specific culturing conditions which are not yet sufficient to mimic the natural environment of native stem cells differentiating towards adult cells. However, researchers work hard to uncover these factors. Here, we review a common standard approach to generate iPSCs and transduce these to iPSC cardiomyocytes. Further, we review recent achievements and discuss their current limitations and future perspectives. We are on track, but the road is still under construction.


Assuntos
Diferenciação Celular/genética , Edição de Genes , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/metabolismo , Animais , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Humanos
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.
Cell Physiol Biochem ; 40(6): 1549-1558, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27997884

RESUMO

BACKGROUND/AIMS: Acquired as well as inherited channelopathies are disorders that are caused by altered ion channel function. A family of channels whose malfunction is associated with different channelopathies is the Kv7 K+ channel family; and restoration of normal Kv7 channel function by small molecule modulators is a promising approach for treatment of these often fatal diseases. METHODS: Here, we show the modulation of Kv7 channels by the natural compound Rottlerin heterologously expressed in Xenopus laevis oocytes and on iPSC cardiomyocytes overexpressing Kv7.1 channels. RESULTS: We show that currents carried by Kv7.1 (EC50 = 1.48 µM), Kv7.1/KCNE1 (EC50 = 4.9 µM), and Kv7.4 (EC50 = 0.148 µM) are strongly enhanced by the compound, whereas Kv7.2, Kv7.2/Kv7.3, and Kv7.5 are not sensitive to Rottlerin. Studies on Kv7.1/KCNE1 mutants and in silico modelling indicate that Rottlerin binds to the R-L3-activator site. Rottlerin mediated activation of Kv7.1/KCNE1 channels might be a promising approach in long QT syndrome. As a proof of concept, we show that Rottlerin shortens cardiac repolarisation in iPSC-derived cardiomyocytes expressing Kv7.1. CONCLUSION: Rottlerin or an optimized derivative holds a potential as QT interval correcting drug.


Assuntos
Acetofenonas/farmacologia , Benzopiranos/farmacologia , Produtos Biológicos/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Canal de Potássio KCNQ1/metabolismo , Acetofenonas/química , Animais , Benzopiranos/química , Produtos Biológicos/química , Simulação por Computador , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Canal de Potássio KCNQ1/química , Potenciais da Membrana/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Domínios Proteicos , Multimerização Proteica/efeitos dos fármacos , Xenopus laevis
14.
J Biol Chem ; 289(33): 22749-22758, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-24947509

RESUMO

Kv7.1 to Kv7.5 α-subunits belong to the family of voltage-gated potassium channels (Kv). Assembled with the ß-subunit KCNE1, Kv7.1 conducts the slowly activating potassium current IKs, which is one of the major currents underlying repolarization of the cardiac action potential. A known regulator of Kv7 channels is the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 increases the macroscopic current amplitude by stabilizing the open conformation of 7.1/KCNE1 channels. However, knowledge about the exact nature of the interaction is incomplete. The aim of this study was the identification of the amino acids responsible for the interaction between Kv7.1 and PIP2. We generated 13 charge neutralizing point mutations at the intracellular membrane border and characterized them electrophysiologically in complex with KCNE1 under the influence of diC8-PIP2. Electrophysiological analysis of corresponding long QT syndrome mutants suggested impaired PIP2 regulation as the cause for channel dysfunction. To clarify the underlying structural mechanism of PIP2 binding, molecular dynamics simulations of Kv7.1/KCNE1 complexes containing two PIP2 molecules in each subunit at specific sites were performed. Here, we identified a subset of nine residues participating in the interaction of PIP2 and Kv7.1/KCNE1. These residues may form at least two binding pockets per subunit, leading to the stabilization of channel conformations upon PIP2 binding.


Assuntos
Canal de Potássio KCNQ1/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Potenciais de Ação/fisiologia , Substituição de Aminoácidos , Animais , Sítios de Ligação , Humanos , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/genética , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/genética , Mutação Puntual , Ligação Proteica , Xenopus laevis
15.
Pflugers Arch ; 466(10): 1885-97, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24389605

RESUMO

Ionotropic glutamate receptors are the most important excitatory receptors in the central nervous system, and their impairment can lead to multiple neuronal diseases. Here, we show that glutamate-induced currents in oocytes expressing GluA1 are increased by coexpression of the schizophrenia-associated phosphoinositide kinase PIP5K2A. This effect was due to enhanced membrane abundance and was blunted by a point mutation (N251S) in PIP5K2A. An increase in GluA1 currents was also observed upon acute injection of PI(4,5)P2, the main product of PIP5K2A. By expression of wild-type and mutant PIP5K2A in human embryonic kidney cells, we were able to provide evidence of impaired kinase activity of the mutant PIP5K2A. We defined the region K813-K823 of GluA1 as critical for the PI(4,5)P2 effect by performing an alanine scan that suggested PI(4,5)P2 binding to this area. A PIP strip assay revealed PI(4,5)P2 binding to the C-terminal GluA1 peptide. The present observations disclose a novel mechanism in the regulation of GluA1.


Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/química , Receptores de AMPA/química , Alanina/química , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células HEK293 , Humanos , Dados de Sequência Molecular , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Xenopus
16.
FASEB J ; 27(10): 4108-21, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23825229

RESUMO

Infections with coxsackieviruses of type B (CVBs), which are known to induce severe forms of acute and chronic myocarditis, are often accompanied by ventricular arrhythmias and sudden cardiac death. The mechanisms underlying the development of virus-induced, life-threatening arrhythmias, which are phenotypically similar to those observed in patients having functionally impaired cardiac ion channels, remain, however, enigmatic. In the present study, we show, for the first time, modulating time-dependent effects of CVB3 on the cardiac ion channels KCNQ1, hERG1, and Cav1.2 in heterologous expression. Channel protein abundance in cellular plasma membrane and patterns of their subcellular distribution were altered in infected murine hearts. The antiviral compound AG7088 did not prevent these effects on channels. In silico analyses of infected human myocytes suggest pronounced alterations of electrical and calcium signaling and increased risk of arrhythmogenesis. These modifications are attenuated by the common Asian polymorphism KCNQ1 P448R, a genetic determinant preventing coxsackievirus-induced effects in vitro. This study provides a previously unknown explanation for the development of arrhythmias in enteroviral myocarditis, which will help to develop therapeutic strategies for arrhythmia treatment.


Assuntos
Enterovirus Humano B/classificação , Enterovirus Humano B/fisiologia , Regulação da Expressão Gênica/fisiologia , Canais Iônicos/metabolismo , Miócitos Cardíacos/metabolismo , Transporte Proteico/fisiologia , Animais , Simulação por Computador , Células HEK293 , Humanos , Camundongos , Modelos Biológicos , Miócitos Cardíacos/virologia , Oócitos , Polimorfismo Genético , Xenopus
17.
Cell Physiol Biochem ; 31(6): 968-80, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23839156

RESUMO

BACKGROUND/AIMS: Potassium channels are tetrameric proteins providing potassium selective passage through lipid embedded proteinaceous pores with highest fidelity. The selectivity results from binding to discrete potassium binding sites and stabilization of a hydrated potassium ion in a central internal cavity. The four potassium binding sites, generated by the conserved TTxGYGD signature sequence are formed by the backbone carbonyls of the amino acids TXGYG. Residues KV1.5-Val481, KV4.3-Leu368 and KV7.1- Ile 313 represent the amino acids in the X position of the respective channels. METHODS: Here, we study the impact of these residues on ion selectivity, permeation and inactivation kinetics as well as the modulation by ß-subunits using site-specific mutagenesis, electrophysiological analyses and molecular dynamics simulations. RESULTS: We identify this position as key in modulation of slow inactivation by structurally dissimilar ß-subunits in different KV channels. CONCLUSION: We propose a model in which structural changes accompanying activation and ß-subunit modulation allosterically constrain the backbone carbonyl oxygen atoms via the side chain of the respective X-residue in the signature sequence to reduce conductance during slow inactivation.


Assuntos
Canal de Potássio KCNQ1/metabolismo , Canal de Potássio Kv1.5/metabolismo , Canais de Potássio Shal/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/genética , Canal de Potássio Kv1.5/química , Canal de Potássio Kv1.5/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Potássio/metabolismo , Estrutura Quaternária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Alinhamento de Sequência , Canais de Potássio Shal/química , Canais de Potássio Shal/genética , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismo
18.
FASEB J ; 26(2): 513-22, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22002906

RESUMO

Inward rectifier potassium channels of the Kir2 subfamily are important determinants of the electrical activity of brain and muscle cells. Genetic mutations in Kir2.1 associate with Andersen-Tawil syndrome (ATS), a familial disorder leading to stress-triggered periodic paralysis and ventricular arrhythmia. To identify the molecular mechanisms of this stress trigger, we analyze Kir channel function and localization electrophysiologically and by time-resolved confocal microscopy. Furthermore, we employ a mathematical model of muscular membrane potential. We identify a novel corticoid signaling pathway that, when activated by glucocorticoids, leads to enrichment of Kir2 channels in the plasma membranes of mammalian cell lines and isolated cardiac and skeletal muscle cells. We further demonstrate that activation of this pathway can either partly restore (40% of cases) or further impair (20% of cases) the function of mutant ATS channels, depending on the particular Kir2.1 mutation. This means that glucocorticoid treatment might either alleviate or deteriorate symptoms of ATS depending on the patient's individual Kir2.1 genotype. Thus, our findings provide a possible explanation for the contradictory effects of glucocorticoid treatment on symptoms in patients with ATS and may open new pathways for the design of personalized medicines in ATS therapy.


Assuntos
Síndrome de Andersen/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Síndrome de Andersen/tratamento farmacológico , Síndrome de Andersen/genética , Animais , Feminino , Glucocorticoides/uso terapêutico , Cobaias , Células HEK293 , Células HeLa , Humanos , Proteínas Imediatamente Precoces/metabolismo , Técnicas In Vitro , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Miócitos Cardíacos/metabolismo , Oócitos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Estresse Fisiológico , Xenopus laevis
19.
Biomedicines ; 11(5)2023 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-37239037

RESUMO

The number of N-Methyl-D-aspartate receptor (NMDAR) linked neurodegenerative diseases such as Alzheimer's disease and dementia is constantly increasing. This is partly due to demographic change and presents new challenges to societies. To date, there are no effective treatment options. Current medications are nonselective and can lead to unwanted side effects in patients. A promising therapeutic approach is the targeted inhibition of NMDARs in the brain. NMDARs containing different subunits and splice variants display different physiological properties and play a crucial role in learning and memory, as well as in inflammatory or injury processes. They become overactivated during the course of the disease, leading to nerve cell death. Until now, there has been a lack of understanding of the general functions of the receptor and the mechanism of inhibition, which need to be understood in order to develop inhibitors. Ideal compounds should be highly targeted and even splice-variant-selective. However, a potent and splice-variant-selective NMDAR-targeting drug has yet to be developed. Recently developed 3-benzazepines are promising inhibitors for further drug development. The NMDAR splice variants GluN1-1b-4b carry a 21-amino-acid-long, flexible exon 5. Exon 5 lowers the NMDAR's sensitivity to allosteric modulators by probably acting as an NMDAR modulator itself. The role of exon 5 in NMDAR modulation is still poorly understood. In this review, we summarize the structure and pharmacological relevance of tetrahydro-3-benzazepines.

20.
Commun Biol ; 6(1): 745, 2023 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-37464013

RESUMO

The TWIK-related spinal cord K+ channel (TRESK, K2P18.1) is a K2P channel contributing to the maintenance of membrane potentials in various cells. Recently, physiological TRESK function was identified as a key player in T-cell differentiation rendering the channel a new pharmacological target for treatment of autoimmune diseases. The channel activator cloxyquin represents a promising lead compound for the development of a new class of immunomodulators. Identification of cloxyquin binding site and characterization of the molecular activation mechanism can foster the future drug development. Here, we identify the cloxyquin binding site at the M2/M4 interface by mutational scan and analyze the molecular mechanism of action by protein modeling as well as in silico and in vitro electrophysiology using different permeating ion species (K+ / Rb+). In combination with kinetic analyses of channel inactivation, our results suggest that cloxyquin allosterically stabilizes the inner selectivity filter facilitating the conduction process subsequently activating hTRESK.


Assuntos
Cloroquinolinóis , Canais de Potássio , Canais de Potássio/química , Sítios de Ligação , Cloroquinolinóis/química , Cloroquinolinóis/farmacologia , Potenciais da Membrana
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