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1.
Front Physiol ; 15: 1342761, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38505707

RESUMO

Cardiac arrhythmias cause significant morbidity and mortality and pose a major public health problem. They arise from disruptions in the normally orderly propagation of cardiac electrophysiological activation and recovery through successive cardiomyocytes in the heart. They reflect abnormalities in automaticity, initiation, conduction, or recovery in cardiomyocyte excitation. The latter properties are dependent on surface membrane electrophysiological mechanisms underlying the cardiac action potential. Their disruption results from spatial or temporal instabilities and heterogeneities in the generation and propagation of cellular excitation. These arise from abnormal function in their underlying surface membrane, ion channels, and transporters, as well as the interactions between them. The latter, in turn, form common regulatory targets for the hierarchical network of diverse signaling mechanisms reviewed here. In addition to direct molecular-level pharmacological or physiological actions on these surface membrane biomolecules, accessory, adhesion, signal transduction, and cytoskeletal anchoring proteins modify both their properties and localization. At the cellular level of excitation-contraction coupling processes, Ca2+ homeostatic and phosphorylation processes affect channel activity and membrane excitability directly or through intermediate signaling. Systems-level autonomic cellular signaling exerts both acute channel and longer-term actions on channel expression. Further upstream intermediaries from metabolic changes modulate the channels both themselves and through modifying Ca2+ homeostasis. Finally, longer-term organ-level inflammatory and structural changes, such as fibrotic and hypertrophic remodeling, similarly can influence all these physiological processes with potential pro-arrhythmic consequences. These normal physiological processes may target either individual or groups of ionic channel species and alter with particular pathological conditions. They are also potentially alterable by direct pharmacological action, or effects on longer-term targets modifying protein or cofactor structure, expression, or localization. Their participating specific biomolecules, often clarified in experimental genetically modified models, thus constitute potential therapeutic targets. The insights clarified by the physiological and pharmacological framework outlined here provide a basis for a recent modernized drug classification. Together, they offer a translational framework for current drug understanding. This would facilitate future mechanistically directed therapeutic advances, for which a number of examples are considered here. The latter are potentially useful for treating cardiac, in particular arrhythmic, disease.

2.
Biochem Biophys Res Commun ; 666: 61-67, 2023 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-37178506

RESUMO

The RGD motif on the SARS-CoV-2 spike protein has been suggested to interact with RGD-binding integrins αVß3 and α5ß1 to enhance viral cell entry and alter downstream signaling cascades. The D405N mutation on the Omicron subvariant spike proteins, resulting in an RGN motif, has recently been shown to inhibit binding to integrin αVß3. Deamidation of asparagines in protein ligand RGN motifs has been demonstrated to generate RGD and RGisoD motifs that permit binding to RGD-binding integrins. Two asparagines, N481 and N501, on the Wild-type spike receptor-binding domain have been previously shown to have deamidation half-lives of 16.5 and 123 days, respectively, which may occur during the viral life cycle. Deamidation of Omicron subvariant N405 may recover the ability to interact with RGD-binding integrins. Thus, herein, all-atom molecular dynamics simulations of the Wild-type and Omicron subvariant spike protein receptor-binding domains were conducted to investigate the potential for asparagines, the Omicron subvariant N405 in particular, to assume the optimized geometry for deamidation to occur. In summary, the Omicron subvariant N405 was primarily found to be stabilized in a state unfavourable for deamidation after hydrogen bonding with downstream E406. Nevertheless, a small number of RGD or RGisoD motifs on the Omicron subvariant spike proteins may restore the ability to interact with RGD-binding integrins. The simulations also provided structural clarification regarding the deamidation rates of Wild-type N481 and N501 and highlighted the utility of tertiary structure dynamics information in predicting asparagine deamidation. Further work is needed to characterize the effects of deamidation on spike-integrin interactions.


Assuntos
COVID-19 , Humanos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Asparagina , Integrina alfaVbeta3
3.
Philos Trans R Soc Lond B Biol Sci ; 378(1879): 20220162, 2023 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-37122213

RESUMO

Skeletal and cardiac muscle excitation-contraction coupling commences with Nav1.4/Nav1.5-mediated, surface and transverse (T-) tubular, action potential generation. This initiates feedforward, allosteric or Ca2+-mediated, T-sarcoplasmic reticular (SR) junctional, voltage sensor-Cav1.1/Cav1.2 and ryanodine receptor-RyR1/RyR2 interaction. We review recent structural, physiological and translational studies on possible feedback actions of the resulting SR Ca2+ release on Nav1.4/Nav1.5 function in native muscle. Finite-element modelling predicted potentially regulatory T-SR junctional [Ca2+]TSR domains. Nav1.4/Nav1.5, III-IV linker and C-terminal domain structures included Ca2+ and/or calmodulin-binding sites whose mutations corresponded to specific clinical conditions. Loose-patch-clamped native murine skeletal muscle fibres and cardiomyocytes showed reduced Na+ currents (INa) following SR Ca2+ release induced by the Epac and direct RyR1/RyR2 activators, 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphate and caffeine, abrogated by the RyR inhibitor dantrolene. Conversely, dantrolene and the Ca2+-ATPase inhibitor cyclopiazonic acid increased INa. Experimental, catecholaminergic polymorphic ventricular tachycardic RyR2-P2328S and metabolically deficient Pgc1ß-/- cardiomyocytes also showed reduced INa accompanying [Ca2+]i abnormalities rescued by dantrolene- and flecainide-mediated RyR block. Finally, hydroxychloroquine challenge implicated action potential (AP) prolongation in slowing AP conduction through modifying Ca2+ transients. The corresponding tissue/organ preparations each showed pro-arrhythmic, slowed AP upstrokes and conduction velocities. We finally extend discussion of possible Ca2+-mediated effects to further, Ca2+, K+ and Cl-, channel types. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.


Assuntos
Dantroleno , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Camundongos , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Dantroleno/farmacologia , Retroalimentação , Músculo Esquelético , Potenciais de Ação , Cálcio/metabolismo
4.
J Cell Physiol ; 238(6): 1354-1367, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37042220

RESUMO

The voltage-gated sodium channel NaV 1.7 is involved in various pain phenotypes and is physiologically regulated by the NaV -ß3-subunit. Venom toxins ProTx-II and OD1 modulate NaV 1.7 channel function and may be useful as therapeutic agents and/or research tools. Here, we use patch-clamp recordings to investigate how the ß3-subunit can influence and modulate the toxin-mediated effects on NaV 1.7 function, and we propose a putative binding mode of OD1 on NaV 1.7 to rationalise its activating effects. The inhibitor ProTx-II slowed the rate of NaV 1.7 activation, whilst the activator OD1 reduced the rate of fast inactivation and accelerated recovery from inactivation. The ß3-subunit partially abrogated these effects. OD1 induced a hyperpolarising shift in the V1/2 of steady-state activation, which was not observed in the presence of ß3. Consequently, OD1-treated NaV 1.7 exhibited an enhanced window current compared with OD1-treated NaV 1.7-ß3 complex. We identify candidate OD1 residues that are likely to prevent the upward movement of the DIV S4 helix and thus impede fast inactivation. The binding sites for each of the toxins and the predicted location of the ß3-subunit on the NaV 1.7 channel are distinct. Therefore, we infer that the ß3-subunit influences the interaction of toxins with NaV 1.7 via indirect allosteric mechanisms. The enhanced window current shown by OD1-treated NaV 1.7 compared with OD1-treated NaV 1.7-ß3 is discussed in the context of differing cellular expressions of NaV 1.7 and the ß3-subunit in dorsal root ganglion (DRG) neurons. We propose that ß3, as the native binding partner for NaV 1.7 in DRG neurons, should be included during screening of molecules against NaV 1.7 in relevant analgesic discovery campaigns.


Assuntos
Peçonhas , Canais de Sódio Disparados por Voltagem , Humanos , Peçonhas/uso terapêutico , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Analgésicos/uso terapêutico , Dor/tratamento farmacológico
5.
J Physiol ; 601(5): 923-940, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36354758

RESUMO

In cardiac myocytes, the voltage-gated sodium channel NaV 1.5 opens in response to membrane depolarisation and initiates the action potential. The NaV 1.5 channel is typically associated with regulatory ß-subunits that modify gating and trafficking behaviour. These ß-subunits contain a single extracellular immunoglobulin (Ig) domain, a single transmembrane α-helix and an intracellular region. Here we focus on the role of the ß1 and ß3 subunits in regulating NaV 1.5. We catalogue ß1 and ß3 domain specific mutations that have been associated with inherited cardiac arrhythmia, including Brugada syndrome, long QT syndrome, atrial fibrillation and sudden death. We discuss how new structural insights into these proteins raises new questions about physiological function.


Assuntos
Arritmias Cardíacas , Síndrome do QT Longo , Humanos , Potenciais de Ação/fisiologia , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canais de Sódio/metabolismo , Subunidades Proteicas
6.
Front Physiol ; 14: 1280151, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38235384

RESUMO

Introduction: In addition to gap junction conduction, recent reports implicate possible ephaptic coupling contributions to action potential (AP) propagation between successive adjacent cardiomyocytes. Here, AP generation in an active cell, withdraws Na+ from, creating a negative potential within, ephaptic spaces between the participating membranes, activating the initially quiescent neighbouring cardiomyocyte. However, sustainable ephaptic transmission requires subsequent complete recovery of the ephaptic charge difference. We explore physical contributions of passive electrodiffusive ion exchange with the remaining extracellular space to this recovery for the first time. Materials and Methods: Computational, finite element, analysis examined limiting, temporal and spatial, ephaptic [Na+], [Cl-], and the consequent Gaussian charge differences and membrane potential recovery patterns following a ΔV∼130 mV AP upstroke at physiological (37°C) temperatures. This incorporated Nernst-Planck formalisms into equations for the time-dependent spatial concentration gradient profiles. Results: Mammalian atrial, ventricular and purkinje cardiomyocyte ephaptic junctions were modelled by closely apposed circularly symmetric membranes, specific capacitance 1 µF cm-2, experimentally reported radii a = 8,000, 12,000 and 40,000 nm respectively and ephaptic axial distance w = 20 nm. This enclosed an ephaptic space containing principal ions initially at normal extracellular [Na+] = 153.1 mM and [Cl-] = 145.8 mM, respective diffusion coefficients D Na = 1.3 × 109 and D Cl = 2 × 109 nm2s-1. Stable, concordant computational solutions were confirmed exploring ≤1,600 nm mesh sizes and Δt≤0.08 ms stepsize intervals. The corresponding membrane voltage profile changes across the initially quiescent membrane were obtainable from computed, graphically represented a and w-dependent ionic concentration differences adapting Gauss's flux theorem. Further simulations explored biological variations in ephaptic dimensions, membrane anatomy, and diffusion restrictions within the ephaptic space. Atrial, ventricular and Purkinje cardiomyocytes gave 40, 180 and 2000 ms 99.9% recovery times, with 720 or 360 ms high limits from doubling ventricular radius or halving diffusion coefficient. Varying a, and D Na and D Cl markedly affected recovery time-courses with logarithmic and double-logarithmic relationships, Varying w exerted minimal effects. Conclusion: We thereby characterise the properties of, and through comparing atrial, ventricular and purkinje recovery times with interspecies in vivo background cardiac cycle duration data, (blue whale ∼2000, human∼90, Etruscan shrew, ∼40 ms) can determine physical limits to, electrodiffusive contributions to ephaptic recovery.

7.
J Med Virol ; 94(9): 4181-4192, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35575289

RESUMO

Cleavage of the severe respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein has been demonstrated to contribute to viral-cell fusion and syncytia formation. Studies have shown that variants of concern (VOC) and variants of interest (VOI) show differing membrane fusion capacity. Mutations near cleavage motifs, such as the S1/S2 and S2' sites, may alter interactions with host proteases and, thus, the potential for fusion. The biochemical basis for the differences in interactions with host proteases for the VOC/VOI spike proteins has not yet been explored. Using sequence and structure-based bioinformatics, mutations near the VOC/VOI spike protein cleavage sites were inspected for their structural effects. All mutations found at the S1/S2 sites were predicted to increase affinity to the furin protease but not TMPRSS2. Mutations at the spike residue P681 in several strains, such P681R in the Delta strain, resulted in the disruption of a proline-directed kinase phosphorylation motif at the S1/S2 site, which may lessen the impact of phosphorylation for these variants. However, the unique N679K mutation in the Omicron strain was found to increase the propensity for O-linked glycosylation at the S1/S2 cleavage site, which may prevent recognition by proteases. Such glycosylation in the Omicron strain may hinder entry at the cell surface and, thus, decrease syncytia formation and induce cell entry through the endocytic pathway as has been shown in previous studies. Further experimental work is needed to confirm the effect of mutations and posttranslational modifications on SARS-CoV-2 spike protein cleavage sites.


Assuntos
SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Glicosilação , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética
8.
Biochem J ; 479(3): 225-243, 2022 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-35015072

RESUMO

The ability of the cellular immune system to discriminate self from foreign antigens depends on the appropriate calibration of the T cell receptor (TCR) signalling threshold. The lymphocyte homeostatic cytokine interleukin 7 (IL-7) is known to affect TCR thresholding, but the molecular mechanism is not fully elucidated. A better understanding of this process is highly relevant in the context of autoimmune disease therapy and cancer immunotherapy. We sought to characterise the early signalling events attributable to IL-7 priming; in particular, the altered phosphorylation of signal transduction proteins and their molecular localisation to the TCR. By integrating high-resolution proximity- phospho-proteomic and imaging approaches using primary T cells, rather than engineered cell lines or an in vitro expanded T cell population, we uncovered transduction events previously not linked to IL-7. We show that IL-7 leads to dephosphorylation of cytohesin interacting protein (CYTIP) at a hitherto undescribed phosphorylation site (pThr280) and alters the co-localisation of cytohesin-1 with the TCR and LFA-1 integrin. These results show that IL-7, acting via CYTIP and cytohesin-1, may impact TCR activation thresholds by enhancing the co-clustering of TCR and LFA-1 integrin.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Interleucina-7/farmacologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/imunologia , Fatores de Transcrição/metabolismo , Citoesqueleto de Actina/metabolismo , Doadores de Sangue , Células Cultivadas , Humanos , Ativação Linfocitária/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Treonina/metabolismo
9.
Front Cell Infect Microbiol ; 11: 765300, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34869067

RESUMO

The RGD motif in the Severe Acute Syndrome Coronavirus 2 (SARS-CoV-2) spike protein has been predicted to bind RGD-recognizing integrins. Recent studies have shown that the spike protein does, indeed, interact with αVß3 and α5ß1 integrins, both of which bind to RGD-containing ligands. However, computational studies have suggested that binding between the spike RGD motif and integrins is not favourable, even when unfolding occurs after conformational changes induced by binding to the canonical host entry receptor, angiotensin-converting enzyme 2 (ACE2). Furthermore, non-RGD-binding integrins, such as αx, have been suggested to interact with the SARS-CoV-2 spike protein. Other viral pathogens, such as rotaviruses, have been recorded to bind integrins in an RGD-independent manner to initiate host cell entry. Thus, in order to consider the potential for the SARS-CoV-2 spike protein to bind integrins independent of the RGD sequence, we investigate several factors related to the involvement of integrins in SARS-CoV-2 infection. First, we review changes in integrin expression during SARS-CoV-2 infection to identify which integrins might be of interest. Then, all known non-RGD integrin-binding motifs are collected and mapped to the spike protein receptor-binding domain and analyzed for their 3D availability. Several integrin-binding motifs are shown to exhibit high sequence similarity with solvent accessible regions of the spike receptor-binding domain. Comparisons of these motifs with other betacoronavirus spike proteins, such as SARS-CoV and RaTG13, reveal that some have recently evolved while others are more conserved throughout phylogenetically similar betacoronaviruses. Interestingly, all of the potential integrin-binding motifs, including the RGD sequence, are conserved in one of the known pangolin coronavirus strains. Of note, the most recently recorded mutations in the spike protein receptor-binding domain were found outside of the putative integrin-binding sequences, although several mutations formed inside and close to one motif, in particular, may potentially enhance binding. These data suggest that the SARS-CoV-2 spike protein may interact with integrins independent of the RGD sequence and may help further explain how SARS-CoV-2 and other viruses can evolve to bind to integrins.


Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Linhagem Celular , Humanos , Integrinas , Glicoproteínas de Membrana , Oligopeptídeos , Peptidil Dipeptidase A , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Proteínas do Envelope Viral
10.
Biochem Soc Trans ; 49(5): 1941-1961, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34643236

RESUMO

Voltage-dependent Na+ channel activation underlies action potential generation fundamental to cellular excitability. In skeletal and cardiac muscle this triggers contraction via ryanodine-receptor (RyR)-mediated sarcoplasmic reticular (SR) Ca2+ release. We here review potential feedback actions of intracellular [Ca2+] ([Ca2+]i) on Na+ channel activity, surveying their structural, genetic and cellular and functional implications, translating these to their possible clinical importance. In addition to phosphorylation sites, both Nav1.4 and Nav1.5 possess potentially regulatory binding sites for Ca2+ and/or the Ca2+-sensor calmodulin in their inactivating III-IV linker and C-terminal domains (CTD), where mutations are associated with a range of skeletal and cardiac muscle diseases. We summarize in vitro cell-attached patch clamp studies reporting correspondingly diverse, direct and indirect, Ca2+ effects upon maximal Nav1.4 and Nav1.5 currents (Imax) and their half-maximal voltages (V1/2) characterizing channel gating, in cellular expression systems and isolated myocytes. Interventions increasing cytoplasmic [Ca2+]i down-regulated Imax leaving V1/2 constant in native loose patch clamped, wild-type murine skeletal and cardiac myocytes. They correspondingly reduced action potential upstroke rates and conduction velocities, causing pro-arrhythmic effects in intact perfused hearts. Genetically modified murine RyR2-P2328S hearts modelling catecholaminergic polymorphic ventricular tachycardia (CPVT), recapitulated clinical ventricular and atrial pro-arrhythmic phenotypes following catecholaminergic challenge. These accompanied reductions in action potential conduction velocities. The latter were reversed by flecainide at RyR-blocking concentrations specifically in RyR2-P2328S as opposed to wild-type hearts, suggesting a basis for its recent therapeutic application in CPVT. We finally explore the relevance of these mechanisms in further genetic paradigms for commoner metabolic and structural cardiac disease.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Ativação do Canal Iônico , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Sódio/metabolismo , Potenciais de Ação , Animais , Sítios de Ligação , Modelos Animais de Doenças , Flecainida/uso terapêutico , Humanos , Camundongos , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Taquicardia Ventricular/tratamento farmacológico , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismo , Resultado do Tratamento , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêutico
11.
Biomolecules ; 10(7)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32630316

RESUMO

Voltage-gated sodium (Nav) channels drive the rising phase of the action potential, essential for electrical signalling in nerves and muscles. The Nav channel α-subunit contains the ion-selective pore. In the cardiomyocyte, Nav1.5 is the main Nav channel α-subunit isoform, with a smaller expression of neuronal Nav channels. Four distinct regulatory ß-subunits (ß1-4) bind to the Nav channel α-subunits. Previous work has emphasised the ß-subunits as direct Nav channel gating modulators. However, there is now increasing appreciation of additional roles played by these subunits. In this review, we focus on ß-subunits as homophilic and heterophilic cell-adhesion molecules and the implications for cardiomyocyte function. Based on recent cryogenic electron microscopy (cryo-EM) data, we suggest that the ß-subunits interact with Nav1.5 in a different way from their binding to other Nav channel isoforms. We believe this feature may facilitate trans-cell-adhesion between ß1-associated Nav1.5 subunits on the intercalated disc and promote ephaptic conduction between cardiomyocytes.


Assuntos
Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Subunidades beta do Canal de Sódio Disparado por Voltagem/metabolismo , Potenciais de Ação , Animais , Adesão Celular , Humanos , Modelos Moleculares , Canal de Sódio Disparado por Voltagem NAV1.5/química , Subunidades Proteicas/metabolismo , Subunidades beta do Canal de Sódio Disparado por Voltagem/química
12.
Int J Oncol ; 57(1): 87-99, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32319587

RESUMO

The immune checkpoint protein B7­H4 plays an important role in the positive as well as the negative regulation of immune T­cell responses. When expressed on cancer cells, B7­H4 inhibits T­cell activity, and numerous types of cancer cells use upregulation of B7­H4 as a survival strategy. Thus, B7­H4 is a potential target for anticancer drug therapy. Unfortunately, the cell biology of this molecule has yet to be fully elucidated. Even basic properties, such as the nature of B7­H4 interactors, are controversial. In particular, the cis­interactors of B7­H4 on cancer cell plasma membranes have not been investigated to date. The present study used a proteomic proximity­labelling assay to investigate the molecular neighbours of B7­H4 on the surface of the human breast cancer cells SK­BR­3. By comparison to a comprehensive proteome analysis of SK­BR­3 cells, the proximity method detected a relatively small number of low abundance plasma membrane proteins highly enriched for proteins known to modulate cell adhesion and immune recognition. It may be inferred that these molecules contribute to the immunosuppressive behaviour that is characteristic of B7­H4 on cancer cells.


Assuntos
Neoplasias da Mama/imunologia , Mapeamento de Interação de Proteínas , Inibidor 1 da Ativação de Células T com Domínio V-Set/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/imunologia , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Feminino , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Ativação Linfocitária/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Mapas de Interação de Proteínas/imunologia , Proteômica/métodos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , Inibidor 1 da Ativação de Células T com Domínio V-Set/antagonistas & inibidores , Inibidor 1 da Ativação de Células T com Domínio V-Set/imunologia
13.
FASEB J ; 34(3): 3537-3553, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31950564

RESUMO

Voltage-gated sodium channels comprise an ion-selective α-subunit and one or more associated ß-subunits. The ß3-subunit (encoded by the SCN3B gene) is an important physiological regulator of the heart-specific sodium channel, Nav1.5. We have previously shown that when expressed alone in HEK293F cells, the full-length ß3-subunit forms trimers in the plasma membrane. We extend this result with biochemical assays and use the proximity ligation assay (PLA) to identify oligomeric ß3-subunits, not just at the plasma membrane, but throughout the secretory pathway. We then investigate the corresponding clustering properties of the α-subunit and the effects upon these of the ß3-subunits. The oligomeric status of the Nav1.5 α-subunit in vivo, with or without the ß3-subunit, has not been previously investigated. Using super-resolution fluorescence imaging, we show that under conditions typically used in electrophysiological studies, the Nav1.5 α-subunit assembles on the plasma membrane of HEK293F cells into spatially localized clusters rather than individual and randomly dispersed molecules. Quantitative analysis indicates that the ß3-subunit is not required for this clustering but ß3 does significantly change the distribution of cluster sizes and nearest-neighbor distances between Nav1.5 α-subunits. However, when assayed by PLA, the ß3-subunit increases the number of PLA-positive signals generated by anti-(Nav1.5 α-subunit) antibodies, mainly at the plasma membrane. Since PLA can be sensitive to the orientation of proteins within a cluster, we suggest that the ß3-subunit introduces a significant change in the relative alignment of individual Nav1.5 α-subunits, but the clustering itself depends on other factors. We also show that these structural and higher-order changes induced by the ß3-subunit do not alter the degree of electrophysiological gating cooperativity between Nav1.5 α-subunits. Our data provide new insights into the role of the ß3-subunit and the supramolecular organization of sodium channels, in an important model cell system that is widely used to study Nav channel behavior.


Assuntos
Membrana Celular/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Subunidades Proteicas/metabolismo , Eletrofisiologia , Células HEK293 , Humanos , Imunoprecipitação , Cinética , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Técnicas de Patch-Clamp , Subunidades Proteicas/química , Subunidades Proteicas/genética
14.
J Biol Chem ; 294(51): 19752-19763, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31659116

RESUMO

The auxiliary ß3-subunit is an important functional regulator of the cardiac sodium channel Nav1.5, and some ß3 mutations predispose individuals to cardiac arrhythmias. The ß3-subunit uses its transmembrane α-helix and extracellular domain to bind to Nav1.5. Here, we investigated the role of an unusually located and highly conserved glutamic acid (Glu-176) within the ß3 transmembrane region and its potential for functionally synergizing with the ß3 extracellular domain (ECD). We substituted Glu-176 with lysine (E176K) in the WT ß3-subunit and in a ß3-subunit lacking the ECD. Patch-clamp experiments indicated that the E176K substitution does not affect the previously observed ß3-dependent depolarizing shift of V½ of steady-state inactivation but does attenuate the accelerated recovery from inactivation conferred by the WT ß3-subunit. Removal of the ß3-ECD abrogated both the depolarizing shift of steady-state inactivation and the accelerated recovery, irrespective of the presence or absence of the Glu-176 residue. We found that steady-state inactivation and recovery from inactivation involve movements of the S4 helices within the DIII and DIV voltage sensors in response to membrane potential changes. Voltage-clamp fluorometry revealed that the E176K substitution alters DIII voltage sensor dynamics without affecting DIV. In contrast, removal of the ECD significantly altered the dynamics of both DIII and DIV. These results imply distinct roles for the ß3-Glu-176 residue and the ß3-ECD in regulating the conformational changes of the voltage sensors that determine channel inactivation and recovery from inactivation.


Assuntos
Regulação da Expressão Gênica , Ácido Glutâmico/química , Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Animais , Humanos , Ativação do Canal Iônico , Cinética , Lisina/química , Potenciais da Membrana , Mutagênese , Mutação , Oócitos/metabolismo , Técnicas de Patch-Clamp , Domínios Proteicos , Estrutura Secundária de Proteína , Xenopus
15.
Br J Pharmacol ; 175(8): 1260-1278, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28369767

RESUMO

Flecainide suppresses cardiac tachyarrhythmias including paroxysmal atrial fibrillation, supraventricular tachycardia and arrhythmic long QT syndromes (LQTS), as well as the Ca2+ -mediated, catecholaminergic polymorphic ventricular tachycardia (CPVT). However, flecainide can also exert pro-arrhythmic effects most notably following myocardial infarction and when used to diagnose Brugada syndrome (BrS). These divergent actions result from its physiological and pharmacological actions at multiple, interacting levels of cellular organization. These were studied in murine genetic models with modified Nav channel or intracellular ryanodine receptor (RyR2)-Ca2+ channel function. Flecainide accesses its transmembrane Nav 1.5 channel binding site during activated, open, states producing a use-dependent antagonism. Closing either activation or inactivation gates traps flecainide within the pore. An early peak INa related to activation of Nav channels followed by rapid de-activation, drives action potential (AP) upstrokes and their propagation. This is diminished in pro-arrhythmic conditions reflecting loss of function of Nav 1.5 channels, such as BrS, accordingly exacerbated by flecainide challenge. Contrastingly, pro-arrhythmic effects attributed to prolonged AP recovery by abnormal late INaL following gain-of-function modifications of Nav 1.5 channels in LQTS3 are reduced by flecainide. Anti-arrhythmic effects of flecainide that reduce triggering in CPVT models mediated by sarcoplasmic reticular Ca2+ release could arise from its primary actions on Nav channels indirectly decreasing [Ca2+ ]i through a reduced [Na+ ]i and/or direct open-state RyR2-Ca2+ channel antagonism. The consequent [Ca2+ ]i alterations could also modify AP propagation velocity and therefore arrhythmic substrate through its actions on Nav 1.5 channel function. This is consistent with the paradoxical differences between flecainide actions upon Na+ currents, AP conduction and arrhythmogenesis under circumstances of normal and increased RyR2 function. LINKED ARTICLES: This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.


Assuntos
Antiarrítmicos/farmacologia , Arritmias Cardíacas/fisiopatologia , Flecainida/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Animais , Antiarrítmicos/uso terapêutico , Arritmias Cardíacas/tratamento farmacológico , Cálcio/fisiologia , Flecainida/uso terapêutico , Humanos , Bloqueadores dos Canais de Potássio/uso terapêutico , Canais de Potássio/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia
16.
Curr Protoc Protein Sci ; 88: 19.27.1-19.27.18, 2017 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-28369668

RESUMO

This manuscript describes a new and general method to identify proteins localized into spatially restricted membrane microenvironments. Horseradish peroxidase (HRP) is brought into contact with a target protein by being covalently linked to a primary or secondary antibody, an antigen or substrate, a drug, or a toxin. A biotinylated tyramide-based reagent is then added. In the presence of HRP and hydrogen peroxide, the reagent is converted into a free radical that only diffuses a short distance before covalently labeling proteins within a few tens to hundreds of nanometers from the target. The biotinylated proteins can then be isolated by standard affinity chromatography and identified by liquid chromatography (LC) and mass spectrometry (MS). The assay can be made quantitative by using stable isotope labeling with amino acids in cell culture (SILAC) or isobaric tagging at the peptide level. © 2017 by John Wiley & Sons, Inc.


Assuntos
Biotina/análogos & derivados , Membrana Celular/química , Proteínas de Membrana/análise , Proteômica/métodos , Tiramina/análogos & derivados , Aminoácidos , Animais , Anticorpos/imunologia , Linhagem Celular , Cromatografia Líquida , Peroxidase do Rábano Silvestre , Humanos , Marcação por Isótopo , Proteínas de Membrana/química , Peptídeos , Espectrometria de Massas em Tandem
17.
Mol Cell Proteomics ; 14(11): 2848-56, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26355100

RESUMO

Within cells, proteins can co-assemble into functionally integrated and spatially restricted multicomponent complexes. Often, the affinities between individual proteins are relatively weak, and proteins within such clusters may interact only indirectly with many of their other protein neighbors. This makes proteomic characterization difficult using methods such as immunoprecipitation or cross-linking. Recently, several groups have described the use of enzyme-catalyzed proximity labeling reagents that covalently tag the neighbors of a targeted protein with a small molecule such as fluorescein or biotin. The modified proteins can then be isolated by standard pulldown methods and identified by mass spectrometry. Here we will describe the techniques as well as their similarities and differences. We discuss their applications both to study protein assemblies and to provide a new way for characterizing organelle proteomes. We stress the importance of proteomic quantitation and independent target validation in such experiments. Furthermore, we suggest that there are biophysical and cell-biological principles that dictate the appropriateness of enzyme-catalyzed proximity labeling methods to address particular biological questions of interest.


Assuntos
Marcação por Isótopo/métodos , Proteoma/análise , Proteômica/métodos , Coloração e Rotulagem/métodos , Biotina/química , Biotina/metabolismo , Carbono-Nitrogênio Ligases/química , Carbono-Nitrogênio Ligases/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Escherichia coli/química , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Fluoresceína/química , Fluoresceína/metabolismo , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/metabolismo , Espectrometria de Massas , Proteoma/química , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Tiramina/análogos & derivados
18.
Data Brief ; 3: 29-33, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26217713

RESUMO

In developing a new quantitative AP-MS method for exploring interactomes in the chicken B-cell line DT40, we also surveyed the most abundant proteins in this organism and explored the likely contaminants that bind to a variety of affinity resins that would later be confirmed quantitatively [1]. We present the 'Top 150 abundant DT40 proteins list', the DT40 beadomes as well as protein interaction lists for the Phosphatidyl inositol 5-phosphate 4-kinase 2ß and Fanconi anaemia protein complexes.

19.
Curr Protoc Protein Sci ; 80: 19.27.1-19.27.18, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25829300

RESUMO

This manuscript describes a new and general method to identify proteins localized into spatially restricted membrane microenvironments. Horseradish peroxidase (HRP) is brought into contact with a target protein by being covalently linked to a primary or secondary antibody, an antigen or substrate, a drug, or a toxin. A biotinylated tyramide-based reagent is then added. In the presence of HRP and hydrogen peroxide, the reagent is converted into a free radical that only diffuses a short distance before covalently labeling proteins within a few tens to hundreds of nanometers from the target. The biotinylated proteins can then be isolated by standard affinity chromatography and identified by liquid chromatography (LC) and mass spectrometry (MS). The assay can be made quantitative by using stable isotope labeling with amino acids in cell culture (SILAC) or isobaric tagging at the peptide level.


Assuntos
Biotina/análogos & derivados , Microdomínios da Membrana/química , Proteínas de Membrana/análise , Proteínas de Membrana/química , Proteômica/métodos , Tiramina/análogos & derivados , Marcadores de Afinidade , Animais , Biotina/química , Biotina/metabolismo , Linhagem Celular , Galinhas , Cromatografia de Afinidade , Marcação por Isótopo , Espectrometria de Massas , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Tiramina/química , Tiramina/metabolismo
20.
Open Biol ; 5(1): 140192, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25567098

RESUMO

Voltage-gated sodium (Nav) channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are a major focus of research in neurobiology, structural biology, membrane biology and pharmacology. Mutations in Nav channels are implicated in a wide variety of inherited pathologies, including cardiac conduction diseases, myotonic conditions, epilepsy and chronic pain syndromes. Drugs active against Nav channels are used as local anaesthetics, anti-arrhythmics, analgesics and anti-convulsants. The Nav channels are composed of a pore-forming α subunit and associated ß subunits. The ß subunits are members of the immunoglobulin (Ig) domain family of cell-adhesion molecules. They modulate multiple aspects of Nav channel behaviour and play critical roles in controlling neuronal excitability. The recently published atomic resolution structures of the human ß3 and ß4 subunit Ig domains open a new chapter in the study of these molecules. In particular, the discovery that ß3 subunits form trimers suggests that Nav channel oligomerization may contribute to the functional properties of some ß subunits.


Assuntos
Subunidades beta do Canal de Sódio Disparado por Voltagem/química , Potenciais de Ação , Sequência de Aminoácidos , Animais , Evolução Molecular , Humanos , Ativação do Canal Iônico , Dados de Sequência Molecular , Subunidades beta do Canal de Sódio Disparado por Voltagem/genética , Subunidades beta do Canal de Sódio Disparado por Voltagem/metabolismo
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