RESUMEN
Pannexin 1 (PANX1) is an ATP-permeable channel with critical roles in a variety of physiological functions such as blood pressure regulation1, apoptotic cell clearance2 and human oocyte development3. Here we present several structures of human PANX1 in a heptameric assembly at resolutions of up to 2.8 angström, including an apo state, a caspase-7-cleaved state and a carbenoxolone-bound state. We reveal a gating mechanism that involves two ion-conducting pathways. Under normal cellular conditions, the intracellular entry of the wide main pore is physically plugged by the C-terminal tail. Small anions are conducted through narrow tunnels in the intracellular domain. These tunnels connect to the main pore and are gated by a long linker between the N-terminal helix and the first transmembrane helix. During apoptosis, the C-terminal tail is cleaved by caspase, allowing the release of ATP through the main pore. We identified a carbenoxolone-binding site embraced by W74 in the extracellular entrance and a role for carbenoxolone as a channel blocker. We identified a gap-junction-like structure using a glycosylation-deficient mutant, N255A. Our studies provide a solid foundation for understanding the molecular mechanisms underlying the channel gating and inhibition of PANX1 and related large-pore channels.
Asunto(s)
Conexinas/química , Conexinas/metabolismo , Microscopía por Crioelectrón , Activación del Canal Iónico , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Técnicas de Placa-Clamp , Adenosina Trifosfato/metabolismo , Animales , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestructura , Apoptosis , Sitios de Unión/efectos de los fármacos , Carbenoxolona/química , Carbenoxolona/metabolismo , Carbenoxolona/farmacología , Caspasa 7/metabolismo , Línea Celular , Conexinas/ultraestructura , Uniones Comunicantes , Glicosilación , Humanos , Activación del Canal Iónico/efectos de los fármacos , Modelos Moleculares , Mutación , Proteínas del Tejido Nervioso/ultraestructura , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Células Sf9RESUMEN
Gap junctions establish direct pathways for cell-to-cell communication through the assembly of twelve connexin subunits that form intercellular channels connecting neighbouring cells. Co-assembly of different connexin isoforms produces channels with unique properties and enables communication across cell types. Here we used single-particle cryo-electron microscopy to investigate the structural basis of connexin co-assembly in native lens gap junction channels composed of connexin 46 and connexin 50 (Cx46/50). We provide the first comparative analysis to connexin 26 (Cx26), which-together with computational studies-elucidates key energetic features governing gap junction permselectivity. Cx46/50 adopts an open-state conformation that is distinct from the Cx26 crystal structure, yet it appears to be stabilized by a conserved set of hydrophobic anchoring residues. 'Hot spots' of genetic mutations linked to hereditary cataract formation map to the core structural-functional elements identified in Cx46/50, suggesting explanations for many of the disease-causing effects.
Asunto(s)
Conexinas/química , Conexinas/ultraestructura , Microscopía por Crioelectrón , Cristalino/citología , Cristalino/ultraestructura , Secuencia de Aminoácidos , Catarata/congénito , Catarata/genética , Conexina 26/química , Conexinas/genética , Uniones Comunicantes/química , Uniones Comunicantes/genética , Uniones Comunicantes/ultraestructura , Humanos , Cristalino/química , Modelos Moleculares , MutaciónRESUMEN
Cardiac connexin-43 (Cx43) creates gap junction channels (GJCs) at intercellular contacts and hemi-channels (HCs) at the peri-junctional plasma membrane and sarcolemmal caveolae/rafts compartments. GJCs are fundamental for the direct cardiac cell-to-cell transmission of electrical and molecular signals which ensures synchronous myocardial contraction. The HCs and structurally similar pannexin1 (Panx1) channels are active in stressful conditions. These channels are essential for paracrine and autocrine communication through the release of ions and signaling molecules to the extracellular environment, or for uptake from it. The HCs and Panx1 channel-opening profoundly affects intracellular ionic homeostasis and redox status and facilitates via purinergic signaling pro-inflammatory and pro-fibrotic processes. These conditions promote cardiac arrhythmogenesis due to the impairment of the GJCs and selective ion channel function. Crosstalk between GJCs and HCs/Panx1 channels could be crucial in the development of arrhythmogenic substrates, including fibrosis. Despite the knowledge gap in the regulation of these channels, current evidence indicates that HCs and Panx1 channel activation can enhance the risk of cardiac arrhythmias. It is extremely challenging to target HCs and Panx1 channels by inhibitory agents to hamper development of cardiac rhythm disorders. Progress in this field may contribute to novel therapeutic approaches for patients prone to develop atrial or ventricular fibrillation.
Asunto(s)
Conexina 43/metabolismo , Conexinas/metabolismo , Activación del Canal Iónico , Miocardio/metabolismo , Animales , Antiarrítmicos/farmacología , Arritmias Cardíacas/etiología , Arritmias Cardíacas/metabolismo , Conexina 43/ultraestructura , Conexinas/ultraestructura , Susceptibilidad a Enfermedades , Humanos , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/genética , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/ultraestructura , Miocardio/ultraestructuraRESUMEN
OBJECTIVE: This study investigated whether chondrocytes within the cartilage matrix have the capacity to communicate through intercellular connections mediated by voltage-gated gap junction (GJ) channels. METHODS: Frozen cartilage samples were used for immunofluorescence and immunohistochemistry assays. Samples were embedded in cacodylate buffer before dehydration for scanning electron microscopy. Co-immunoprecipitation experiments and mass spectrometry (MS) were performed to identify proteins that interact with the C-terminal end of Cx43. GJ communication was studied through in situ electroporation, electrophysiology and dye injection experiments. A transwell layered culture system and MS were used to identify and quantify transferred amino acids. RESULTS: Microscopic images revealed the presence of multiple cellular projections connecting chondrocytes within the matrix. These projections were between 5 and 150â µm in length. MS data analysis indicated that the C-terminus of Cx43 interacts with several cytoskeletal proteins implicated in Cx trafficking and GJ assembly, including α-tubulin and ß-tubulin, actin, and vinculin. Electrophysiology experiments demonstrated that 12-mer oligonucleotides could be transferred between chondrocytes within 12â min after injection. Glucose was homogeneously distributed within 22 and 35â min. No transfer was detected when glucose was electroporated into A549 cells, which have no GJs. Transwell layered culture systems coupled with MS analysis revealed connexins can mediate the transfer of L-lysine and L-arginine between chondrocytes. CONCLUSIONS: This study reveals that intercellular connections between chondrocytes contain GJs that play a key role in cell-cell communication and a metabolic function by exchange of nutrients including glucose and essential amino acids. A three-dimensional cellular network mediated through GJs might mediate metabolic and physiological homeostasis to maintain cartilage tissue.
Asunto(s)
Cartílago Articular/metabolismo , Comunicación Celular , Condrocitos/metabolismo , Conexinas/metabolismo , Uniones Comunicantes/metabolismo , Aminoácidos Esenciales/metabolismo , Animales , Cartílago Articular/ultraestructura , Condrocitos/ultraestructura , Conexinas/ultraestructura , Uniones Comunicantes/ultraestructura , Glucosa/metabolismo , Homeostasis , Humanos , Inmunohistoquímica , Inmunoprecipitación , Articulación de la Rodilla , Microscopía Electrónica de Rastreo , PorcinosRESUMEN
Gap junction channels connect the cytoplasms of adjacent cells through the end-to-end docking of single-membrane structures called connexons, formed by a ring of six connexin monomers. Each monomer contains four transmembrane alpha-helices, for a total of 24 alpha-helices in a connexon. The fundamental structure of the connexon pore is probably similar in unpaired connexons and junctional channels, and for channels formed by different connexin isoforms. Nevertheless, variability in results from structurally focused mutagenesis and electrophysiological studies raise uncertainty about the specific assignments of the transmembrane helices. Mapping of human mutations onto a suggested C(alpha) model predicts that mutations that disrupt helix-helix packing impair channel function. An experimentally determined structure at atomic resolution will be essential to confirm and resolve these concepts.
Asunto(s)
Conexinas/ultraestructura , Uniones Comunicantes/ultraestructura , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Subunidades de Proteína/química , Comunicación Celular/fisiología , Conexinas/genética , Conexinas/metabolismo , Microscopía por Crioelectrón , Uniones Comunicantes/metabolismo , Humanos , Modelos Moleculares , Mutación , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
In the ovary, initiation of follicle growth is marked by cuboidalization of flattened granulosa cells (GCs). The regulation and cell biology of this shape change remains poorly understood. We propose that characterization of intercellular junctions and associated proteins is key to identifying as yet unknown regulators of this important transition. As GCs are conventionally described as epithelial cells, this study used mouse ovaries and isolated follicles to investigate epithelial junctional complexes (tight junctions [TJ], adherens junctions [AJ], and desmosomes) and associated molecules, as well as classic epithelial markers, by quantitative PCR and immunofluorescence. These junctions were further characterized using ultrastructural, calcium depletion and biotin tracer studies. Junctions observed by transmission electron microscopy between GCs and between GCs and oocyte were identified as AJs by expression of N-cadherin and nectin 2 and by the lack of TJ and desmosome-associated proteins. Follicles were also permeable to biotin, confirming a lack of functional TJs. Surprisingly, GCs lacked all epithelial markers analyzed, including E-cadherin, cytokeratin 8, and zonula occludens (ZO)-1alpha+. Furthermore, vimentin was expressed by GCs, suggesting a more mesenchymal phenotype. Under calcium-free conditions, small follicles maintained oocyte-GC contact, confirming the importance of calcium-independent nectin at this stage. However, in primary and multilayered follicles, lack of calcium resulted in loss of contact between GCs and oocyte, showing that nectin alone cannot maintain attachment between these two cell types. Lack of classic markers suggests that GCs are not epithelial. Identification of AJs during GC cuboidalization highlights the importance of AJs in regulating initiation of follicle growth.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Conexinas/fisiología , Folículo Ovárico/fisiología , Uniones Adherentes/fisiología , Uniones Adherentes/ultraestructura , Animales , Calcio/fisiología , Moléculas de Adhesión Celular/ultraestructura , Conexinas/ultraestructura , Células Epiteliales/fisiología , Células Epiteliales/ultraestructura , Femenino , Uniones Comunicantes/fisiología , Uniones Comunicantes/ultraestructura , Ratones , Folículo Ovárico/ultraestructura , Uniones Estrechas/fisiología , Uniones Estrechas/ultraestructuraRESUMEN
Despite the combination of light-microscopic immunocytochemistry, histochemical mRNA detection techniques and protein reporter systems, progress in identifying the protein composition of neuronal versus glial gap junctions, determination of the differential localization of their constituent connexin proteins in two apposing membranes and understanding human neurological diseases caused by connexin mutations has been problematic due to ambiguities introduced in the cellular and subcellular assignment of connexins. Misassignments occurred primarily because membranes and their constituent proteins are below the limit of resolution of light microscopic imaging techniques. Currently, only serial thin-section transmission electron microscopy and freeze-fracture replica immunogold labeling have sufficient resolution to assign connexin proteins to either or both sides of gap junction plaques. However, freeze-fracture replica immunogold labeling has been limited because conventional freeze fracturing allows retrieval of only one of the two membrane fracture faces within a gap junction, making it difficult to identify connexin coupling partners in hemiplaques removed by fracturing. We now summarize progress in ascertaining the connexin composition of two coupled hemiplaques using matched double-replicas that are labeled simultaneously for multiple connexins. This approach allows unambiguous identification of connexins and determination of the membrane "sidedness" and the identities of connexin coupling partners in homotypic and heterotypic gap junctions of vertebrate neurons.
Asunto(s)
Conexinas/metabolismo , Técnica de Fractura por Congelación/métodos , Uniones Comunicantes/metabolismo , Inmunohistoquímica/métodos , Animales , Astrocitos/metabolismo , Astrocitos/ultraestructura , Conexinas/ultraestructura , Uniones Comunicantes/ultraestructura , Humanos , Neuronas/metabolismo , Neuronas/ultraestructura , Oligodendroglía/metabolismo , Oligodendroglía/ultraestructuraRESUMEN
The coxsackievirus and adenovirus receptor (CAR) is a transmembrane protein that belongs to the family of adhesion molecules. In the postnatal heart, it is localized predominantly at the intercalated disc, where its function is not known. Here, we demonstrate that a first degree or complete block of atrioventricular (AV) conduction developed in the absence of CAR in the adult mouse heart and that prolongation of AV conduction occurred in the embryonic heart of the global CAR-KO mouse. In the cardiac-specific CAR-KO (CAR-cKO) mouse, we observed the loss of connexin 45 localization to the cell-cell junctions of the AV node but preservation of connexin 40 and 43 in contracting myocardial cells and connexin 30.2 in the AV node. There was also a marked decrease in beta-catenin and zonula occludens-1 (ZO-1) localization to the intercalated discs of CAR-cKO mouse hearts at 8 weeks before the mice developed cardiomyopathy at 21 weeks of age. We also found that CAR formed a complex with connexin 45 via its PSD-95/DigA/ZO-1-binding (PDZ-binding) motifs. We conclude that CAR expression is required for normal AV-node conduction and cardiac function. Furthermore, localization of connexin 45 at the AV-node cell-cell junction and of beta-catenin and ZO-1 at the ventricular intercalated disc are dependent on CAR.
Asunto(s)
Nodo Atrioventricular/metabolismo , Conexinas/metabolismo , Corazón , Miocardio/metabolismo , Receptores Virales/metabolismo , Animales , Conexinas/ultraestructura , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Electrocardiografía , Embrión de Mamíferos , Técnica del Anticuerpo Fluorescente Directa , Células HeLa , Ventrículos Cardíacos/ultraestructura , Humanos , Ratones , Ratones Noqueados , Miocardio/ultraestructura , Receptores Virales/ultraestructura , TelemetríaRESUMEN
In a genetic screen for active zone defective mutants in Caenorhabditis elegans, we isolated a loss-of-function allele of unc-7, a gene encoding an innexin/pannexin family gap junction protein. Innexin UNC-7 regulates the size and distribution of active zones at C. elegans neuromuscular junctions. Loss-of-function mutations in another innexin, UNC-9, cause similar active zone defects as unc-7 mutants. In addition to presumptive gap junction localizations, both UNC-7 and UNC-9 are also localized perisynaptically throughout development and required in presynaptic neurons to regulate active zone differentiation. Our mosaic analyses, electron microscopy, as well as expression studies suggest a novel and likely nonjunctional role of specific innexins in active zone differentiation in addition to gap junction formations.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Diferenciación Celular/fisiología , Proteínas de la Membrana/fisiología , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/ultraestructura , Diferenciación Celular/genética , Conexinas/genética , Conexinas/fisiología , Conexinas/ultraestructura , Uniones Comunicantes/genética , Uniones Comunicantes/fisiología , Uniones Comunicantes/ultraestructura , Regulación del Desarrollo de la Expresión Génica/fisiología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/ultraestructura , Organismos Modificados Genéticamente , Terminales Presinápticos/fisiología , Terminales Presinápticos/ultraestructuraRESUMEN
Pannexins are large-pore forming channels responsible for ATP release under a variety of physiological and pathological conditions. Although predicted to share similar membrane topology with other large-pore forming proteins such as connexins, innexins, and LRRC8, pannexins have minimal sequence similarity to these protein families. Here, we present the cryo-EM structure of a frog pannexin 1 (Panx1) channel at 3.0 Å. We find that Panx1 protomers harbor four transmembrane helices similar in arrangement to other large-pore forming proteins but assemble as a heptameric channel with a unique constriction formed by Trp74 in the first extracellular loop. Mutating Trp74 or the nearby Arg75 disrupt ion selectivity, whereas altering residues in the hydrophobic groove formed by the two extracellular loops abrogates channel inhibition by carbenoxolone. Our structural and functional study establishes the extracellular loops as important structural motifs for ion selectivity and channel inhibition in Panx1.
Asunto(s)
Conexinas/ultraestructura , Proteínas de Xenopus/ultraestructura , Secuencia de Aminoácidos , Animales , Carbenoxolona/farmacología , Conexinas/antagonistas & inhibidores , Conexinas/química , Conexinas/metabolismo , Microscopía por Crioelectrón , Células HEK293 , Humanos , Estructura Terciaria de Proteína , Proteínas de Xenopus/antagonistas & inhibidores , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
The plasma membrane adenosine triphosphate (ATP) release channel pannexin 1 (PANX1) has been implicated in many physiological and pathophysiological processes associated with purinergic signaling, including cancer progression, apoptotic cell clearance, inflammation, blood pressure regulation, oocyte development, epilepsy and neuropathic pain. Here we present near-atomic-resolution structures of human and frog PANX1 determined by cryo-electron microscopy that revealed a heptameric channel architecture. Compatible with ATP permeation, the transmembrane pore and cytoplasmic vestibule were exceptionally wide. An extracellular tryptophan ring located at the outer pore created a constriction site, potentially functioning as a molecular sieve that restricts the size of permeable substrates. The amino and carboxyl termini, not resolved in the density map, appeared to be structurally dynamic and might contribute to narrowing of the pore during channel gating. In combination with functional characterization, this work elucidates the previously unknown architecture of pannexin channels and establishes a foundation for understanding their unique channel properties.
Asunto(s)
Adenosina Trifosfato/química , Membrana Celular/ultraestructura , Conexinas/ultraestructura , Microscopía por Crioelectrón , Proteínas del Tejido Nervioso/ultraestructura , Adenosina Trifosfato/genética , Animales , Anuros/genética , Membrana Celular/química , Membrana Celular/genética , Conexinas/química , Conexinas/genética , Humanos , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Conformación Proteica , Transducción de Señal/genéticaRESUMEN
Gap junctions form intercellular conduits with a large pore size whose closed and open states regulate communication between adjacent cells. The structural basis of the mechanism by which gap junctions close, however, remains uncertain. Here, we show the cryo-electron microscopy structures of Caenorhabditis elegans innexin-6 (INX-6) gap junction proteins in an undocked hemichannel form. In the nanodisc-reconstituted structure of the wild-type INX-6 hemichannel, flat double-layer densities obstruct the channel pore. Comparison of the hemichannel structures of a wild-type INX-6 in detergent and nanodisc-reconstituted amino-terminal deletion mutant reveals that lipid-mediated amino-terminal rearrangement and pore obstruction occur upon nanodisc reconstitution. Together with molecular dynamics simulations and electrophysiology functional assays, our results provide insight into the closure of the INX-6 hemichannel in a lipid bilayer before docking of two hemichannels.
Asunto(s)
Proteínas de Caenorhabditis elegans/ultraestructura , Caenorhabditis elegans/metabolismo , Conexinas/ultraestructura , Microscopía por Crioelectrón , Simulación del Acoplamiento Molecular , Fosfolípidos/química , Animales , Proteínas de Caenorhabditis elegans/química , Conexinas/química , Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Nanopartículas/química , Oocitos/metabolismo , Xenopus/metabolismoRESUMEN
The importance of electrical and molecular signaling through connexin (Cx) channels is now widely recognized. The transfer of ions and other small molecules between adjacent cells is regulated by multiple stimuli, including voltage. Indeed, Cx channels typically exhibit complex voltage sensitivity. Most channels are sensitive to the voltage difference between the cell interiors (or transjunctional voltage, V(j)), while other channels are also sensitive to absolute inside-outside voltage (i.e., the membrane potential, V(m)). The first part of this review is focused on the description of the distinct forms of voltage sensitivity and the gating mechanisms that regulate hemichannel activity, both individually and as components of homotypic and heterotypic gap junctions. We then provide an up to date and precise picture of the molecular and structural aspects of how V(j) and V(m) are sensed, and how they, therefore, control channel opening and closing. Mutagenic strategies coupled with structural, biochemical and electrophysical studies are providing significant insights into how distinct forms of voltage dependence are brought about. The emerging picture indicates that Cx channels can undergo transitions between multiple conductance states driven by distinct voltage-gating mechanisms. Each hemichannel may contain a set of two V(j) gates, one fast and one slow, which mediate the transitions between the main open state to the residual state and to the fully closed state, respectively. Eventually, a V(m) gate regulates channel transitions between the open and closed states. Clusters of charged residues within separate domains of the Cx molecule have been identified as integral parts of the V(j) and V(m) sensors. The charges at the first positions of the amino terminal cytoplasmic domain determine the magnitude and polarity of the sensitivity to fast V(j)-gating, as well as contributing to the V(j)-rectifying properties of ion permeation. Additionally, important advances have been made in identifying the conformational rearrangements responsible for fast V(j)-gating transitions to the residual state in the Cx43 channel. These changes involve an intramolecular particle-receptor interaction between the carboxy terminal domain and the cytoplasmic loop.
Asunto(s)
Conexinas/química , Conexinas/ultraestructura , Uniones Comunicantes/metabolismo , Activación del Canal Iónico , Potenciales de la Membrana , Modelos Químicos , Modelos Moleculares , Comunicación Celular , Simulación por Computador , Campos Electromagnéticos , Conformación ProteicaRESUMEN
Gap junction channels are essential for mediating intercellular communication in most multicellular organisms. Two gene families encode gap junction channels, innexin and connexin. Although the sequence similarity between these two families based on bioinformatics is not conclusively determined, the gap junction channels encoded by these two gene families are structurally and functionally analogous. We recently reported an atomic structure of an invertebrate innexin gap junction channel using single-particle cryo-electron microscopy. Our findings revealed that connexin and innexin families share several structural properties with regard to their monomeric and oligomeric structures, while simultaneously suggesting a diversity of gap junction channels in nature. This review summarizes cutting-edge progress toward determining an innexin gap junction channel structure, as well as essential tips for preparing cryo-electron microscopy samples for high-resolution structural analysis of an innexin gap junction channel.
Asunto(s)
Conexinas/ultraestructura , Microscopía por Crioelectrón/métodos , Uniones Comunicantes/ultraestructura , Manejo de Especímenes/métodos , Animales , Transporte Biológico , Comunicación Celular , Conexinas/química , Uniones Comunicantes/química , Manejo de Especímenes/instrumentaciónRESUMEN
Pannexin 1 (PANX1) subunits form oligomeric plasma membrane channels that mediate nucleotide release for purinergic signalling, which is involved in diverse physiological processes such as apoptosis, inflammation, blood pressure regulation, and cancer progression and metastasis. Here we explore the mechanistic basis for PANX1 activation by using wild type and engineered concatemeric channels. We find that PANX1 activation involves sequential stepwise sojourns through multiple discrete open states, each with unique channel gating and conductance properties that reflect contributions of the individual subunits of the hexamer. Progressive PANX1 channel opening is directly linked to permeation of ions and large molecules (ATP and fluorescent dyes) and occurs during both irreversible (caspase cleavage-mediated) and reversible (α1 adrenoceptor-mediated) forms of channel activation. This unique, quantized activation process enables fine tuning of PANX1 channel activity and may be a generalized regulatory mechanism for other related multimeric channels.
Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Membrana Celular/metabolismo , Conexinas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Multimerización de Proteína/fisiología , Adenosina Trifosfato/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Carbenoxolona/farmacología , Inhibidores de Caspasas/farmacología , Caspasas/metabolismo , Membrana Celular/ultraestructura , Permeabilidad de la Membrana Celular/efectos de los fármacos , Conexinas/antagonistas & inhibidores , Conexinas/ultraestructura , Colorantes Fluorescentes/farmacocinética , Fluoroquinolonas/farmacología , Células HEK293 , Humanos , Iones/metabolismo , Células Jurkat , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Microscopía Electrónica , Naftiridinas/farmacología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/ultraestructura , Técnicas de Placa-Clamp , Quinolinas/farmacología , Receptores Adrenérgicos alfa 1/metabolismoRESUMEN
Gap junctions were initially described morphologically, and identified as semi-crystalline arrays of channels linking two cells. This suggested that they may represent an amenable target for electron and X-ray crystallographic studies in much the same way that bacteriorhodopsin has. Over 30 years later, however, an atomic resolution structural solution of these unique intercellular pores is still lacking due to many challenges faced in obtaining high expression levels and purification of these structures. A variety of microscopic techniques, as well as NMR structure determination of fragments of the protein, have now provided clearer and correlated views of how these structures are assembled and function as intercellular conduits. As a complement to these structural approaches, a variety of mutagenic studies linking structure and function have now allowed molecular details to be superimposed on these lower resolution structures, so that a clearer image of pore architecture and its modes of regulation are beginning to emerge.
Asunto(s)
Conexinas/ultraestructura , Uniones Comunicantes/ultraestructura , Canales Iónicos/fisiología , Animales , Conexinas/genética , Humanos , Activación del Canal Iónico/fisiología , Microscopía de Fuerza Atómica , Microscopía Electrónica , Mutagénesis , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína , Resonancia por Plasmón de Superficie , Difracción de Rayos XRESUMEN
Innexins, a large protein family comprising invertebrate gap junction channels, play an essential role in nervous system development and electrical synapse formation. Here we report the cryo-electron microscopy structures of Caenorhabditis elegans innexin-6 (INX-6) gap junction channels at atomic resolution. We find that the arrangements of the transmembrane helices and extracellular loops of the INX-6 monomeric structure are highly similar to those of connexin-26 (Cx26), despite the lack of significant sequence similarity. The INX-6 gap junction channel comprises hexadecameric subunits but reveals the N-terminal pore funnel, consistent with Cx26. The helix-rich cytoplasmic loop and C-terminus are intercalated one-by-one through an octameric hemichannel, forming a dome-like entrance that interacts with N-terminal loops in the pore. These observations suggest that the INX-6 cytoplasmic domains are cooperatively associated with the N-terminal funnel conformation, and an essential linkage of the N-terminal with channel activity is presumably preserved across gap junction families.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/ultraestructura , Caenorhabditis elegans/metabolismo , Conexinas/metabolismo , Conexinas/ultraestructura , Microscopía por Crioelectrón , Uniones Comunicantes/metabolismo , Uniones Comunicantes/ultraestructura , Animales , Proteínas de Caenorhabditis elegans/química , Conexinas/química , Modelos Moleculares , Dominios Proteicos , Homología Estructural de ProteínaRESUMEN
The purification of membrane proteins in a form and amount suitable for structural or biochemical studies still remains a great challenge. Gap junctions have long been studied using electron microscopy and X-ray diffraction. However, only a limited number of proteins in the connexin family have been amenable to protein or membrane purification techniques. Molecular biology techniques for expressing large gap junctions in tissue culture cells combined with improvements in electron crystallography have shown great promise for determining the channel structure to better than 10 A resolution. Here, we have isolated two-dimensional (2D) gap junction crystals from HeLa Cx26 transfectants. This isoform has never been isolated in large fractions from tissues. We characterize these preparations by SDS-PAGE, Western blotting, negative stain electron microscopy and atomic force microscopy. In our preparations, the Cx26 is easily detected in the Western blots and we have increased expression levels so that connexin bands are visible on SDS-PAGE gels. Preliminary assessment of the samples by electron cryo-microscopy shows that these 2D crystals diffract to at least 22 A. Atomic force microscopy of these Cx26 gap junctions show exquisite surface modulation at the extracellular surface in force dissected gap junctions. We also applied our protocol to cell lines such as NRK cells that express endogenous Cx43 and NRK and HeLa cell lines transfected with exogenous connexins. While the gap junction membrane channels are recognizable in negatively stained electron micrographs, these lattices are disordered and the gap junction plaques are smaller. SDS-PAGE and Western blotting revealed expression of connexins, but at a lower level than with our HeLa Cx26 transfectants. Therefore, the purity and morphology of the gap junction plaques depends the size and abundance of the gap junctions in the cell line itself.
Asunto(s)
Conexinas/aislamiento & purificación , Conexinas/ultraestructura , Uniones Comunicantes/química , Uniones Comunicantes/ultraestructura , Animales , Western Blotting , Línea Celular , Conexina 26 , Conexina 43/química , Conexina 43/genética , Conexina 43/metabolismo , Conexina 43/ultraestructura , Conexinas/química , Conexinas/genética , Cristalización , Electroforesis en Gel de Poliacrilamida , Uniones Comunicantes/genética , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Proteínas Luminiscentes , Microscopía de Fuerza Atómica , Microscopía Electrónica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/ultraestructura , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/ultraestructura , TransfecciónRESUMEN
Due to its non-invasive character, fluorescence correlation spectroscopy (FCS) is particularly suited for the investigation of diffusion behavior of proteins in living cells. In this study we have investigated the diffusion properties of CFP-labeled gap junction hemichannels in the plasma membrane of living HeLa cells. Gap junction hemichannels or connexons are the precursors for the cell-cell- or gap junction channels that form large plaques at the contact areas between two adjacent cells. It has been proposed that new channels are recruited into a gap junction structure from a pool of hemichannels that can freely diffuse over the entire plasma membrane. The statistical approach shows that the geometry of the membrane within the focus is the most important property for the form of the autocorrelation curve and in turn for the determination of the diffusion coefficient. On the other hand binding-unbinding events which lead to anomalous diffusion have only a minor effect to the position and shape of the correlation curve compared to the geometry of the membrane.
Asunto(s)
Conexinas/química , Conexinas/metabolismo , Uniones Comunicantes/química , Uniones Comunicantes/metabolismo , Microscopía Fluorescente/métodos , Transporte de Proteínas/fisiología , Espectrometría de Fluorescencia/métodos , Membrana Celular/química , Membrana Celular/metabolismo , Conexinas/ultraestructura , Difusión , Uniones Comunicantes/ultraestructura , Células HeLa , Humanos , Interpretación de Imagen Asistida por Computador/métodosRESUMEN
Trypanosoma musculi, a protozoan parasite specific to mouse, was cultured in vitro in the presence of spleen-derived adherent cells. T. musculi co-cultured with adherent cells survived and proliferated indefinitely as long as cellular contact was retained. Scanning and transmission electron microscopy confirmed intimate membrane-to-membrane contact between the adherent cells and parasites. Cellular contact, therefore, seemed to be essential for trypanosomal survival and growth. Immunocytochemical studies demonstrated intense fibroblast growth factor (FGF) activity in adherent cells, and FGFR-2 in associated trypanosomes. BioPorter Lucifer yellow protein delivery reagent studies demonstrated that Lucifer yellow transfected into fibroblast was incorporated into associated trypanosomes. The results suggest the existence of viable channels reminiscent of gap junctions between associated cells. Such transfer of low molecular weight molecules might represent antiapoptotic metabolic factors that support survival of adherent trypanosomes in vitro. Immunocytochemical studies also detected connexin-32 and connexin-43 in the cytoplasm of fibroblasts and associated trypanosomes, however, restriction of connexons to trypanosome/fibroblast adherent sites was not observed. Western blots confirmed the presence of connexin protein molecules in trypanosomes.