RESUMO
Cell junctions, which are typically associated with dynamic cytoskeletons, are essential for a wide range of cellular activities, including cell migration, cell communication, barrier function and signal transduction. Observing cell junctions in real-time can help us understand the mechanisms by which they regulate these cellular activities. This study examined the binding capacity of a modified tridecapeptide from Connexin 43 (Cx43) to the cell junction protein zonula occludens-1 (ZO-1). The goal was to create a fluorescent peptide that can label cell junctions. A cell-penetrating peptide was linked to the modified tridecapeptide. The heterotrimeric peptide molecule was then synthesized. The binding of the modified tridecapeptide was tested using pulldown and immunoprecipitation assays. The ability of the peptide to label cell junctions was assessed by adding it to fixed or live Caco-2 cells. The testing assays revealed that the Cx43-derived peptide can bind to ZO-1. Additionally, the peptide was able to label cell junctions of fixed cells, although no obvious cell junction labeling was observed clearly in live cells, probably due to the inadequate affinity. These findings suggest that labeling cell junctions using a peptide-based strategy is feasible. Further efforts to improve its affinity are warranted in the future.
Assuntos
Conexina 43 , Junções Comunicantes , Humanos , Conexina 43/química , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Proteínas de Membrana/metabolismo , Células CACO-2 , Peptídeos/metabolismo , Fosfoproteínas/metabolismoRESUMO
Although inherited GJA1 (encoding Cx43) gene mutations most often lead to oculodentodigital dysplasia and related disorders, four variants have been linked to erythrokeratodermia variabilis et progressiva (EKVP), a skin disorder characterized by erythematous and hyperkeratotic lesions. While two autosomal-dominant EKVP-linked GJA1 mutations have been shown to lead to augmented hemichannels, the consequence(s) of keratinocytes harboring a de novo P283L variant alone or in combination with a de novo T290N variant remain unknown. Interestingly, these variants reside within or adjacent to a carboxy terminus polypeptide motif that has been shown to be important in regulating the internalization and degradation of Cx43. Cx43-rich rat epidermal keratinocytes (REKs) or Cx43-ablated REKs engineered to express fluorescent protein-tagged P283L and/or T290N variants formed prototypical gap junctions at cell-cell interfaces similar to wildtype Cx43. Dye coupling and dye uptake studies further revealed that each variant or a combination of both variants formed functional gap junction channels, with no evidence of augmented hemichannel function or induction of cell death. Tracking the fate of EKVP-associated variants in the presence of the protein secretion blocker brefeldin A, or an inhibitor of protein synthesis cycloheximide, revealed that P283L or the combination of P283L and T290N variants either significantly extended Cx43 residency on the cell surface of keratinocytes or delayed its degradation. However, caution is needed in concluding that this modest change in the Cx43 life cycle is sufficient to cause EKVP, or whether an additional underlying mechanism or another unidentified gene mutation is contributing to the pathogenesis found in patients. This question will be resolved if further patients are identified where whole exome sequencing reveals a Cx43 P283L variant alone or, in combination with a T290N variant, co-segregates with EKVP across several family generations.
Assuntos
Conexina 43/química , Conexina 43/genética , Eritroceratodermia Variável/genética , Mutação/genética , Animais , Corantes , Retículo Endoplasmático/metabolismo , Junções Comunicantes/metabolismo , Células HeLa , Humanos , Proteínas Mutantes/metabolismo , Proteólise , RatosRESUMO
Identification of proteins that interact with Cx43 has been instrumental in the understanding of gap junction (GJ) regulation. An in vitro phosphorylation screen identified that Protein tyrosine kinase 2 beta (Pyk2) phosphorylated purified Cx43CT and this led us to characterize the impact of this phosphorylation on Cx43 function. Mass spectrometry identified Pyk2 phosphorylates Cx43 residues Y247, Y265, Y267, and Y313. Western blot and immunofluorescence staining using HeLaCx43 cells, HEK 293 T cells, and neonatal rat ventricular myocytes (NRVMs) revealed Pyk2 can be activated by Src and active Pyk2 interacts with Cx43 at the plasma membrane. Overexpression of Pyk2 increases Cx43 phosphorylation and knock-down of Pyk2 decreases Cx43 phosphorylation, without affecting the level of active Src. In HeLaCx43 cells treated with PMA to activate Pyk2, a decrease in Cx43 GJ intercellular communication (GJIC) was observed when assayed by dye transfer. Moreover, PMA activation of Pyk2 could be inhibited by the small molecule PF4618433. This partially restored GJIC, and when paired with a Src inhibitor, returned GJIC to the no PMA control-level. The ability of Pyk2 and Src inhibitors to restore Cx43 function in the presence of PMA was also observed in NRVMs. Additionally, an animal model of myocardial infarction induced heart failure showed a higher level of active Pyk2 activity and increased interaction with Cx43 in ventricular myocytes. Src inhibitors have been used to reverse Cx43 remodeling and improve heart function after myocardial infarction; however, they alone could not fully restore proper Cx43 function. Our data suggest that Pyk2 may need to be inhibited, in addition to Src, to further (if not completely) reverse Cx43 remodeling and improve intercellular communication.
Assuntos
Comunicação Celular , Conexina 43/metabolismo , Quinase 2 de Adesão Focal/antagonistas & inibidores , Junções Comunicantes/metabolismo , Quinases da Família src/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Linhagem Celular , Conexina 43/química , Modelos Animais de Doenças , Quinase 2 de Adesão Focal/metabolismo , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/patologia , Ventrículos do Coração/patologia , Humanos , Mutação/genética , Fosforilação , Ligação Proteica , Domínios Proteicos , Ratos , Acetato de Tetradecanoilforbol/farmacologia , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Connexin (Cx) gap junction channels comprise two hemichannels in neighboring cells, and their permeability is well-described, but permeabilities of the single Cx hemichannel remain largely unresolved. Moreover, determination of isoform-specific Cx hemichannel permeability is challenging because of concurrent expression of other channels with similar permeability profiles and inhibitor sensitivities. The mammalian Cx hemichannels Cx30 and Cx43 are gated by extracellular divalent cations, removal of which promotes fluorescent dye uptake in both channels but atomic ion conductance only through Cx30. To determine the molecular determinants of this difference, here we employed chimeras and mutagenesis of predicted pore-lining residues in Cx43. We expressed the mutated channels in Xenopus laevis oocytes to avoid background activity of alternative channels. Oocytes expressing a Cx43 hemichannel chimera containing the N terminus or the first extracellular loop from Cx30 displayed ethidium uptake and, unlike WT Cx43, ion conduction, an observation further supported by molecular dynamics simulations. Additional C-terminal truncation of the chimeric Cx43 hemichannel elicited an even greater ion conductance with a magnitude closer to that of Cx30. The inhibitory profile for the connexin hemichannels depended on the permeant, with conventional connexin hemichannel inhibitors having a higher potency toward the ion conductance pathway than toward fluorescent dye uptake. Our results demonstrate a permeant-dependent, isoform-specific inhibition of connexin hemichannels. They further reveal that the outer segments of the pore-lining region, including the N terminus and the first extracellular loop, together with the C terminus preclude ion conductance of the open Cx43 hemichannel.
Assuntos
Conexina 43/química , Conexina 43/metabolismo , Sequência de Aminoácidos , Membrana Celular/metabolismo , Fenômenos Eletrofisiológicos , Simulação de Dinâmica Molecular , Permeabilidade , Porosidade , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Especificidade por SubstratoRESUMO
Gap junctions (GJs) assembled from connexin (Cx) proteins allow direct cell-cell communication. While phosphorylation is known to regulate multiple GJ functions, much less is known about the role of ubiquitin in these processes. Using ubiquitylation-type-specific antibodies and Cx43 lysine-to-arginine mutants we show that â¼8% of a GJ, localized in central plaque domains, is K63-polyubiquitylated on K264 and K303. Levels and localization of ubiquitylation correlated well with: (1) the short turnover rate of Cxs and GJs; (2) removal of older channels from the plaque center; and (3) the fact that not all Cxs in an internalizing GJ channel need to be ubiquitylated. Connexins mutated at these two sites assembled significantly larger GJs, exhibited much longer protein half-lives and were internalization impaired. Interestingly, these ubiquitin-deficient Cx43 mutants accumulated as hyper-phosphorylated polypeptides in the plasma membrane, suggesting that K63-polyubiquitylation is triggered by phosphorylation. Phospho-specific anti-Cx43 antibodies revealed that upregulated phosphorylation affected serines 368, 279/282 and 255, which are well-known regulatory PKC and MAPK sites. Together, these novel findings suggest that the internalizing portion of channels in a GJ is K63-polyubiquitylated, ubiquitylation is critical for GJ internalization and that phosphorylation induces Cx K63-polyubiquitylation.This article has an associated First Person interview with the first author of the paper.
Assuntos
Conexina 43/química , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Animais , Anticorpos , Arginina/química , Arginina/genética , Membrana Celular/metabolismo , Conexina 43/genética , Cães , Endocitose/genética , Endocitose/fisiologia , Células HeLa , Humanos , Lisina/química , Lisina/genética , Células Madin Darby de Rim Canino , Peso Molecular , Fosforilação/genética , Fosforilação/fisiologia , Ubiquitinação/genética , Ubiquitinação/fisiologiaRESUMO
Intracellular protons and calcium ions are two major chemical factors that regulate connexin43 (Cx43) gap junction communication and the synergism or antagonism between pH and Ca2+ has been questioned for decades. To assess the ability of Ca2+ ions to modulate Cx43 junctional conductance (gj) in the absence of pH-sensitivity, patch clamp experiments were performed on Neuroblastoma-2a (N2a) cells or neonatal mouse ventricular myocytes (NMVMs) expressing either full-length Cx43 or the Cx43-M257 (Cx43K258stop) mutant protein, a carboxyl-terminus (CT) truncated version of Cx43 lacking pH-sensitivity. The addition of 1â µM ionomycin to normal calcium saline reduced Cx43 or Cx43-M257 gj to zero within 15 min of perfusion. This response was prevented by Ca2+-free saline or addition of 100â nM calmodulin (CaM) inhibitory peptide to the internal pipette solution. Internal addition of a connexin50 cytoplasmic loop calmodulin-binding domain (CaMBD) mimetic peptide (200â nM) prevented the Ca2+/ionomycin-induced decrease in Cx43 gj, while 100â µM Gap19 peptide had minimal effect. The investigation of the transjunctional voltage (Vj) gating properties of NMVM Cx43-M257 gap junctions confirmed the loss of the fast inactivation of Cx43-M257 gj, but also noted the abolishment of the previously reported facilitated recovery of gj from inactivating potentials. We conclude that the distal CT domain of Cx43 contributes to the Vj-dependent fast inactivation and facilitated recovery of Cx43 gap junctions, but the Ca2+/CaM-dependent gating mechanism remains intact in its absence. Sequence-specific connexin CaMBD mimetic peptides act by binding Ca2+/CaM non-specifically and the Cx43 mimetic Gap19 peptide has negligible effect on this chemical gating mechanism.
Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Substituição de Aminoácidos , Animais , Sinalização do Cálcio , Linhagem Celular , Conexina 43/química , Conexina 43/genética , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Ativação do Canal Iônico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mimetismo Molecular , Mutagênese Sítio-Dirigida , Miócitos Cardíacos/metabolismo , Domínios ProteicosRESUMO
Cx43 hemichannels (HCs) are electrically and chemically gated transmembrane pores with low open probability and multiple conductance states, which makes kinetic studies of channel gating in large datasets challenging. Here, we developed open access software, named HemiGUI, to analyze HC gating transitions and investigated voltage-induced HC opening based on up to ≈4000 events recorded in HeLa-Cx43-overexpressing cells. We performed a detailed characterization of Cx43 HC gating profiles and specifically focused on the role of the C-terminal tail (CT) domain by recording the impact of adding an EGFP tag to the Cx43 CT end (Cx43-EGFP) or by supplying the Cx43 HC-inhibiting peptide Gap19 that interferes with CT interaction with the cytoplasmic loop (CL). We found that Gap19 not only decreased HC opening activity to the open state (≈217 pS) but also increased the propensity of subconductance (≈80 pS) transitions that additionally became slower as compared to the control. The work demonstrates that large sample transition analysis allows detailed investigations on Cx43 HC gating and shows that Gap19 acts as a HC gating modifier by interacting with the CT that forms a crucial gating element.
Assuntos
Conexina 43/química , Proteínas de Fluorescência Verde/química , Ativação do Canal Iônico/genética , Software , Conexina 43/antagonistas & inibidores , Junções Comunicantes , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Cinética , Peptídeos/químicaRESUMO
Phosphorylation regulates connexin43 (Cx43) function from assembly/disassembly to coupling at the plaque. Src is a tyrosine kinase known to both phosphorylate Cx43 (residues Y247 and Y265) and affect gap junction intercellular communication. However, the Cx43 carboxyl-terminal (CT) domain contains additional tyrosine residues and proteomic discovery mass spectrometry data identified Y313 as a potential phosphorylation target. Based upon the study of Lin et al. (2001) J. Cell Biol., which still observed tyrosine phosphorylation by Src when using a Cx43 Y247/Y265F mutant, we addressed the possibility of Y313 phosphorylation (pY313) by Src. In vitro Src phosphorylation of purified Cx43CT followed by mass spectroscopy revealed that Src also phosphorylates Y313. This observation was confirmed by repeating the in vitro phosphorylation using different combinations of Cx43CT Yâ¯ââ¯F mutants and a general anti-pTyr antibody. Next, a phospho-specific antibody was generated to help characterize the importance of pY313. We established an in cyto experimental system by stably expressing Cx43 WT and mutants (Y247F, Y265F, Y313F, Y247/265F, Y247/313F, Y265/313F, or Y247/265/313F) in Cx43-deficient HeLa cells. Cx43 WT and mutants, in the absence of v-Src, localized to the plasma membrane and formed gap junctions. When v-Src was over-expressed, Cx43 WT localized intracellularly, while all of the single and double mutants remained able to form plaques and transfer dye, albeit variable in number and amount, respectively. Complete Src-resistance was only achieved with the Cx43 Y247/265/313F mutant. Furthermore, Cx43 Y265F inhibited the ability of v-Src to phosphorylate Y247 and Y313 as well as phosphorylation at both Y265 and Y313 was necessary to inhibit the Cx43 interaction with Drebrin. Finally, we observed in diseased cardiac tissue, in which Src is active, an increase in intercalated disc and intracellular localized Cx43 pY313.
Assuntos
Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Neuropeptídeos/metabolismo , Fosfotirosina/metabolismo , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Especificidade de Anticorpos , Conexina 43/química , Células HeLa , Humanos , Miocárdio/metabolismo , Miocárdio/patologia , Fosforilação , Ligação Proteica , RatosRESUMO
In skeletal tissue, loss or mutation of the gap junction protein connexin 43 (Cx43, also known as GJA1) in cells of the osteoblast lineage leads to a profound cortical bone phenotype and defective tissue remodeling. There is mounting evidence in bone cells that the C-terminus (CT) of Cx43 is a docking platform for signaling effectors and is required for efficient downstream signaling. Here, we examined this function, using a mouse model of Cx43 CT-truncation (Gja1 K258Stop). Relative to Gja1+/- controls, male Gja1-/K258Stop mice have a cortical bone phenotype that is remarkably similar to those reported for deletion of the entire Cx43 gene in osteoblasts. Furthermore, we show that the Cx43 CT binds several signaling proteins that are required for optimal osteoblast function, including PKCδ, ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1, respectively) and ß-catenin. Deletion of the Cx43 CT domain affects these signaling cascades, impacting osteoblast proliferation, differentiation, and collagen processing and organization. These data imply that, at least in bone, Cx43 gap junctions not only exchange signals, but also recruit the appropriate effector molecules to the Cx43 CT in order to efficiently activate signaling cascades that affect cell function and bone acquisition.
Assuntos
Remodelação Óssea , Conexina 43/química , Conexina 43/metabolismo , Osteoblastos/metabolismo , Osteogênese , Transdução de Sinais , Animais , Reabsorção Óssea/patologia , Calcificação Fisiológica , Diferenciação Celular , Proliferação de Células , Colágeno/metabolismo , Osso Cortical/metabolismo , Matriz Extracelular/metabolismo , Masculino , Camundongos , Modelos Animais , Fenótipo , Porosidade , Ligação Proteica , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Connexin 43 (Cx43) phosphorylation plays a pivotal role in cardiac electrical and contractile performance. In a previous study we have found that Cx43 phosphorylation at serine 282 (pS282) regulates cardiomyocyte survival. Considering that both sites are altered simultaneously in many studies, we designed this study to identify the status of S279 phosphorylation upon pS282 manipulation. In heterozygous mice with S282 gene substituted with alanine (S282A), we found ventricular arrhythmias with inhibition of Cx43 phosphorylation at both S282 and S279 in the hearts. In cultured neonatal rat ventricular myocytes (NRVMs), transfection of virus carrying S282A mutant also blocked Cx43 phosphorylation at both S279/282 and gap junction coupling, while expression of wild-type Cx43 or S279A did not. Further, NRVMs transfected with S282 phospho-mimicking mutant substituted with aspartate or treated with ATP exhibited promotions of Cx43 phosphorylation at S279/282 and intercellular communication. Therefore, this study demonstrated a regulatory role of Cx43-S282 on S279 phosphorylation in cardiomyocytes, and suggested an involvement of S279 in the Cx43-S282 mediated cardiomyocyte homeostasis.
Assuntos
Conexina 43/metabolismo , Miócitos Cardíacos/metabolismo , Serina/metabolismo , Animais , Comunicação Celular , Células Cultivadas , Conexina 43/química , Conexina 43/genética , Doxorrubicina/farmacologia , Junções Comunicantes/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mutação , Fosforilação/efeitos dos fármacos , Ratos Sprague-DawleyRESUMO
Most mammalian cells can intercommunicate via connexin-assembled, gap-junctional channels. To regulate signal transmission, connexin (Cx) channel permeability must respond dynamically to physiological and pathophysiological stimuli. One key stimulus is intracellular pH (pHi), which is modulated by a tissue's metabolic and perfusion status. Our understanding of the molecular mechanism of H+ gating of Cx43 channels-the major isoform in the heart and brain-is incomplete. To interrogate the effects of acidic and alkaline pHi on Cx43 channels, we combined voltage-clamp electrophysiology with pHi imaging and photolytic H+ uncaging, performed over a range of pHi values. We demonstrate that Cx43 channels expressed in HeLa or N2a cell pairs are gated biphasically by pHi via a process that consists of activation by H+ ions at alkaline pHi and inhibition at more acidic pHi. For Cx43 channel-mediated solute/ion transmission, the ensemble of these effects produces a pHi optimum, near resting pHi. By using Cx43 mutants, we demonstrate that alkaline gating involves cysteine residues of the C terminus and is independent of motifs previously implicated in acidic gating. Thus, we present a molecular mechanism by which cytoplasmic acid-base chemistry fine tunes intercellular communication and establishes conditions for the optimal transmission of solutes and signals in tissues, such as the heart and brain.-Garciarena, C. D., Malik, A., Swietach, P., Moreno, A. P., Vaughan-Jones, R. D. Distinct moieties underlie biphasic H+ gating of connexin43 channels, producing a pH optimum for intercellular communication.
Assuntos
Conexina 43/metabolismo , Ativação do Canal Iônico , Prótons , Animais , Comunicação Celular , Conexina 43/química , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , CamundongosRESUMO
Connexin 43 (Cx43) hemichannels establish local signaling networks via the release of ATP and other molecules, but their excessive opening may result in cell death. Hence, the activity of Cx43-hemichannels ought to be critically controlled. This involves interactions between the C-terminal tail (CT) and the cytoplasmic loop (CL), more particularly the L2 domain within CL. Previous work revealed an important role for the last nine amino acids of the Cx43 CT by targeting the L2 domain, as these nine amino acids were sufficient to restore the activity of CT-truncated Cx43-hemichannels. However, we discovered that deletion of the last 19 amino acids of the CT only partially lowered the binding to the L2 domain, indicating that a second L2-binding region is present in the CT. We here provide evidence that the SH3-binding domain is another CT region that targets the L2 domain. At the functional level, the SH3-binding domain was able to restore the activity of CT-truncated Cx43-hemichannels and alleviate the inhibition of full-length Cx43-hemichannels by high intracellular Ca2+ concentration ([Ca2+]i) as demonstrated by various approaches including patch clamp studies of unitary Cx43-hemichannel activity. Finally, we show that in full-length Cx43-hemichannels, deletion of either the SH3-binding domain or the CT9 region suppresses the hemichannel activity, while deletion of both domains completely annihilates the hemichannel activity. These results demonstrate that the Cx43 SH3-binding domain, in addition to the CT9 region, critically controls hemichannel activity at high [Ca2+]i, which may be involved in pathological hemichannel opening.
Assuntos
Conexina 43/metabolismo , Domínios de Homologia de src , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cálcio/metabolismo , Bovinos , Células Cultivadas , Conexina 43/química , Células HeLa , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre ProteínasRESUMO
Gap junctions are highly ordered plasma membrane domains that are constantly assembled, remodeled and turned over due to the short half-life of connexins, the integral membrane proteins that form gap junctions. Connexin 43 (Cx43), by far the most widely expressed connexin, is phosphorylated at multiple serine residues in the cytoplasmic, C-terminal region allowing for exquisite cellular control over gap junctional communication. This is evident during epidermal wounding where spatiotemporal changes in connexin expression occur as cells are instructed whether to die, proliferate or migrate to promote repair. Early gap junctional communication is required for initiation of keratinocyte migration, but accelerated Cx43 turnover is also critical for proper wound healing at later stages. These events are controlled via a "kinase program" where sequential phosphorylation of Cx43 leads to reductions in Cx43's half-life and significant depletion of gap junctions from the plasma membrane within several hours. The complex regulation of gap junction assembly and turnover affords several steps where intervention might speed wound healing.
Assuntos
Junções Comunicantes/metabolismo , Proteínas Quinases/metabolismo , Cicatrização , Animais , Conexina 43/química , Conexina 43/metabolismo , Humanos , Modelos Biológicos , FosforilaçãoRESUMO
Connexins are chordate gap junction channel proteins that, by enabling direct communication between the cytosols of adjacent cells, create a unique cell signalling network. Gap junctional intercellular communication (GJIC) has important roles in controlling cell growth and differentiation and in tissue development and homeostasis. Moreover, several non-canonical connexin functions unrelated to GJIC have been discovered. Of the 21 members of the human connexin family, connexin 43 (Cx43) is the most widely expressed and studied. The long cytosolic C-terminus (CT) of Cx43 is subject to extensive post-translational modifications that modulate its intracellular trafficking and gap junction channel gating. Moreover, the Cx43 CT contains multiple domains involved in protein interactions that permit crosstalk between Cx43 and cytoskeletal and regulatory proteins. These domains endow Cx43 with the capacity to affect cell growth and differentiation independently of GJIC. Here, we review the current understanding of the regulation and unique functions of the Cx43 CT, both as an essential component of full-length Cx43 and as an independent signalling hub. We highlight the complex regulatory and signalling networks controlled by the Cx43 CT, including the extensive protein interactome that underlies both gap junction channel-dependent and -independent functions. We discuss these data in relation to the recent discovery of the direct translation of specific truncated forms of Cx43. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.
Assuntos
Conexina 43 , Junções Comunicantes , Ativação do Canal Iônico , Canais Iônicos , Processamento de Proteína Pós-Traducional , Animais , Conexina 43/química , Conexina 43/genética , Conexina 43/metabolismo , Citoesqueleto/química , Citoesqueleto/genética , Citoesqueleto/metabolismo , Junções Comunicantes/química , Junções Comunicantes/genética , Junções Comunicantes/metabolismo , Humanos , Canais Iônicos/química , Canais Iônicos/genética , Canais Iônicos/metabolismo , Domínios Proteicos , Transporte Proteico , Relação Estrutura-AtividadeRESUMO
Propofol is widely used as a general anesthetic and is generally considered to exert its action by regulating neuronal firing via facilitation of GABAA receptors. However, accumulating evidence suggests that propofol also acts on astrocytes, including inhibitory effects on gap junctional coupling, but the underlying molecular mechanisms remain largely unknown. Here, using acute cortical brain slices prepared from mice, we characterize propofol-induced molecular changes in astrocytic gap junction protein connexin 43 (Cx43). Propofol does not change the protein expression level of Cx43 or its incorporation into gap junctional plaques, according to biochemical and immunohistochemical analyses. However, propofol alters migration pattern of Cx43 on western blot, suggesting changes in its posttranslational modifications. Indeed, this change is accompanied by an increase in the phosphorylation of Cx43â¯at serine 368, which is known to reduce permeability of Cx43 gap junctions. Finally, we show that this change occurs in the absence of neuronal firing or glutamatergic transmissions. Overall, these results show that propofol induces posttranslational modification of Cx43 directly on astrocytes at the site of gap junctional plaques, exerting direct pharmacological action on astrocytes in parallel with its action on neurons.
Assuntos
Anestésicos Gerais/farmacologia , Astrócitos/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Conexina 43/metabolismo , Propofol/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Animais , Astrócitos/metabolismo , Córtex Cerebral/metabolismo , Conexina 43/química , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacosRESUMO
Activation of Wnt signaling induces Connexin43 (Cx43) expression via the transcriptional activity of ß-catenin, and results in the enhanced accumulation of the Cx43 protein and the formation of gap junction channels. In response to Wnt signaling, ß-catenin co-localizes with the Cx43 protein itself as part of a complex at the gap junction plaque. Work from several labs have also shown indirect evidence of this interaction via reciprocal co-immunoprecipitation. Our goal for the current study was to identify whether ß-catenin directly interacts with Cx43, and if so, the location of that direct interaction. Identifying residues involved in direct proteinâ»protein interaction is of importance when they are correlated to the phosphorylation of Cx43, as phosphorylation can modify the binding affinities of Cx43 regulatory protein partners. Therefore, combining the location of a protein partner interaction on Cx43 along with the phosphorylation pattern under different homeostatic and pathological conditions will be crucial information for any potential therapeutic intervention. Here, we identified that ß-catenin directly interacts with the Cx43 carboxyl-terminal domain, and that this interaction would be inhibited by the Src phosphorylation of Cx43CT residues Y265 and Y313.
Assuntos
Conexina 43/química , Conexina 43/metabolismo , beta Catenina/metabolismo , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Humanos , Espectroscopia de Ressonância Magnética , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Ratos , Ressonância de Plasmônio de Superfície , beta Catenina/químicaRESUMO
Connexin 43 (Cx43), a gap junction protein seemingly fit to support cardiac impulse propagation and synchronic contraction, is phosphorylated in normoxia by casein kinase 1 (CK1). However, during cardiac ischemia or pressure overload hypertrophy, this phosphorylation fades, Cx43 abundance decreases at intercalated disks and increases at myocytes' lateral borders, and the risk of arrhythmia rises. Studies in wild-type and transgenic mice indicate that enhanced CK1-phosphorylation of Cx43 protects from arrhythmia, while dephosphorylation precedes arrhythmia vulnerability. The mechanistic bases of these Cx43 (de)phosphoform-linked cardiac phenotypes are unknown. We used patch-clamp and dye injection techniques to study the channel function (gating, permeability) of Cx43 mutants wherein CK1-targeted serines were replaced by aspartate (Cx43-CK1-D) or alanine (Cx43-CK1-A) to emulate phosphorylation and dephosphorylation, respectively. Cx43-CK1-D, but not Cx43-CK1-A, displayed high Voltage-sensitivity and variable permselectivity. Both mutants showed multiple channel open states with overall increased conductivity, resistance to acidification-induced junctional uncoupling, and hemichannel openings in normal external calcium. Modest differences in the mutant channels' function and regulation imply the involvement of dissimilar structural conformations of the interacting domains of Cx43 in electrical and chemical gating that may contribute to the divergent phenotypes of CK1-(de)phospho-mimicking Cx43 transgenic mice and that may bear significance in arrhythmogenesis.
Assuntos
Conexina 43/metabolismo , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Animais , Arritmias Cardíacas/metabolismo , Caseína Quinase I/metabolismo , Linhagem Celular Tumoral , Conexina 43/química , Conexina 43/genética , Conexina 43/fisiologia , Mutação , Fosforilação , RatosRESUMO
Connexins are integral membrane building blocks that form gap junctions, enabling direct cytoplasmic exchange of ions and low-molecular-mass metabolites between adjacent cells. In the heart, gap junctions mediate the propagation of cardiac action potentials and the maintenance of a regular beating rhythm. A number of connexin interacting proteins have been described and are known gap junction regulators either through direct effects (e.g., kinases) or the formation of larger multifunctional complexes (e.g., cytoskeleton scaffold proteins). Most connexin partners can be categorized as either proteins promoting coupling by stimulating forward trafficking and channel opening or inhibiting coupling by inducing channel closure, internalization, and degradation. While some interactions have only been implied through co-localization using immunohistochemistry, others have been confirmed by biophysical methods that allow detection of a direct interaction. Our understanding of these interactions is, by far, most well developed for connexin 43 (Cx43) and the scope of this review is to summarize our current knowledge of their functional and regulatory roles. The significance of these interactions is further exemplified by demonstrating their importance at the intercalated disc, a major hub for Cx43 regulation and Cx43 mediated effects.
Assuntos
Conexina 43/genética , Citoesqueleto/genética , Junções Comunicantes/genética , Mapas de Interação de Proteínas/genética , Fenômenos Biofísicos , Comunicação Celular/genética , Conexina 43/química , Citoesqueleto/química , Junções Comunicantes/química , Humanos , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/genéticaRESUMO
The most ubiquitous gap junction protein within the body, connexin 43 (Cx43), is a target of interest for modulating the dermal wound healing response. Observational studies found associations between Cx43 at the wound edge and poor healing response, and subsequent studies utilizing local knockdown of Cx43 found improvements in wound closure rate and final scar appearance. Further preclinical work conducted using Cx43-based peptide therapeutics, including alpha connexin carboxyl terminus 1 (αCT1), a peptide mimetic of the Cx43 carboxyl terminus, reported similar improvements in wound healing and scar formation. Clinical trials and further study into the mode of action have since been conducted on αCT1, and Phase III testing for treatment of diabetic foot ulcers is currently underway. Therapeutics targeting connexin activity show promise in beneficially modulating the human body's natural healing response for improved patient outcomes across a variety of injuries.
Assuntos
Cicatriz/metabolismo , Conexina 43/metabolismo , Pé Diabético/tratamento farmacológico , Pele/metabolismo , Animais , Cicatriz/tratamento farmacológico , Conexina 43/química , Conexina 43/genética , Pé Diabético/metabolismo , Humanos , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/uso terapêutico , Pele/efeitos dos fármacosRESUMO
It is becoming clear that in addition to gap junctions playing a role in cellâ»cell communication, gap junction proteins (connexins) located in cytoplasmic compartments may have other important functions. Mitochondrial connexin 43 (Cx43) is increased after ischemic preconditioning and has been suggested to play a protective role in the heart. How Cx43 traffics to the mitochondria and the interactions of mitochondria with other Cx43-containing structures are unclear. In this study, immunocytochemical, super-resolution, and transmission electron microscopy were used to detect cytoplasmic Cx43-containing structures and to demonstrate their interactions with other cytoplasmic organelles. The most prominent cytoplasmic Cx43-containing structures-annular gap junctions-were demonstrated to form intimate associations with lysosomes as well as with mitochondria. Surprisingly, the frequency of associations between mitochondria and annular gap junctions was greater than that between lysosomes and annular gap junctions. The benefits of annular gap junction/mitochondrial associations are not known. However, it is tempting to suggest, among other possibilities, that the contact between annular gap junction vesicles and mitochondria facilitates Cx43 delivery to the mitochondria. Furthermore, it points to the need for investigating annular gap junctions as more than only vesicles destined for degradation.