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Structural organization of gap junction channels
Abstract
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.
... However, and despite their electrophysiological differences, all connexins share a similar structure. They are oriented so that the amino-and carboxyterminal (CT) tails of the protein are located within the cell cytoplasm, and they include four transmembrane-spanning α-helix domains linked by two extracellular segments (E1 and E2) and one cytoplasmic loop ( Figure 1) [4]. Differences between connexin isoforms are mostly due to variations in the amino acid sequence at the CT domain, although also, to some extent, at the cytoplasmic loop [5]. ...
... All connexin isoforms are integral components of plasma membranes where they form channels around a central pore. These channels are formed by the oligomerization of six individual connexin molecules and are known as hemichannels or connexons ( Figure 1) [4,13]. Hundreds to thousands of these hemichannels gather in plaques, termed gap junctions, where they dock with opposing connexons from adjacent cells, forming intercellular channels ( Figure 1) [4,13]. ...
... These channels are formed by the oligomerization of six individual connexin molecules and are known as hemichannels or connexons ( Figure 1) [4,13]. Hundreds to thousands of these hemichannels gather in plaques, termed gap junctions, where they dock with opposing connexons from adjacent cells, forming intercellular channels ( Figure 1) [4,13]. In cardiomyocytes, gap junctions are mainly located at the cell poles (Figure 2), perpendicular to the long axis of the cell, within the intercalated discs, which are complex structures in which plasma membranes of neighboring cells are in close contact and that also include adherens junctions, hemichannels and ion channels [14,15]. ...
Connexins are a family of transmembrane proteins that play a key role in cardiac physiology. Gap junctional channels put into contact the cytoplasms of connected cardiomyocytes, allowing the existence of electrical coupling. However, in addition to this fundamental role, connexins are also involved in cardiomyocyte death and survival. Thus, chemical coupling through gap junctions plays a key role in the spreading of injury between connected cells. Moreover, in addition to their involvement in cell-to-cell communication, mounting evidence indicates that connexins have additional gap junction-independent functions. Opening of unopposed hemichannels, located at the lateral surface of cardiomyocytes, may compromise cell homeostasis and may be involved in ischemia/reperfusion injury. In addition, connexins located at non-canonical cell structures, including mitochondria and the nucleus, have been demonstrated to be involved in cardioprotection and in regulation of cell growth and differentiation. In this review, we will provide, first, an overview on connexin biology, including their synthesis and degradation, their regulation and their interactions. Then, we will conduct an in-depth examination of the role of connexins in cardiac pathophysiology, including new findings regarding their involvement in myocardial ischemia/reperfusion injury, cardiac fibrosis, gene transcription or signaling regulation.
... En effet, ils permettent respectivement la formation du pore et l'appariement entre deux connexons. La formation de la GJ repose sur l'interaction non covalente entre 3 résidus cystéine, situés sur chaque boucle extracellulaire, qui forment des ponts-disulfures (Sosinsky and Nicholson, 2005) (Figure 30). Le domaine Cter et la boucle cytoplasmique sont les parties les plus variables, ils forment des sites d'interaction avec des protéines cytoplamiques qui modulent les propriétés d'ouverture et de fermeture du canal (Sosinsky and Nicholson, 2005). ...
... La formation de la GJ repose sur l'interaction non covalente entre 3 résidus cystéine, situés sur chaque boucle extracellulaire, qui forment des ponts-disulfures (Sosinsky and Nicholson, 2005) (Figure 30). Le domaine Cter et la boucle cytoplasmique sont les parties les plus variables, ils forment des sites d'interaction avec des protéines cytoplamiques qui modulent les propriétés d'ouverture et de fermeture du canal (Sosinsky and Nicholson, 2005). Les connexons peuvent être formés de connexines de même nature (homomérique) ou non (hétéromérique), et peuvent faire face à des connexons identiques ou non, formant respectivement des GJ homotypique ou hétérotypiques (Sosinsky and Nicholson, 2005) (Figure 30). ...
... Le domaine Cter et la boucle cytoplasmique sont les parties les plus variables, ils forment des sites d'interaction avec des protéines cytoplamiques qui modulent les propriétés d'ouverture et de fermeture du canal (Sosinsky and Nicholson, 2005). Les connexons peuvent être formés de connexines de même nature (homomérique) ou non (hétéromérique), et peuvent faire face à des connexons identiques ou non, formant respectivement des GJ homotypique ou hétérotypiques (Sosinsky and Nicholson, 2005) (Figure 30). Söhl et al., 2004 et Evans andMartin, 2002 Il existe plusieurs nomenclatures pour décrire les Cxs. ...
Numéro national (NNT) : 2012DIJOS097
... In terms of protein amino acid sequence features, the eutherian connexins were classified as 4TM α-helical transmembrane proteins including 4 transmembrane helices [5][6][7][8][9] . Morphologically, the gap junctions were described as "plaques" or "maculae" at intercellular interfaces including numerous intercellular channels that incorporated connexins 10,11 . Structurally, the eutherian connexins included 4 transmembrane α-helices traversing plasma membrane, cytoplasmic connexin regions including N-terminus, cytoplasmic loop that was positioned between second and third transmembrane helices and C-terminal domain, and, finally, extracellular connexin regions including two loops that were positioned between first and second transmembrane helices (region E1) and third and fourth transmembrane helices (region E2) [10][11][12][13][14][15][16][17][18] (see Protein molecular evolution analysis below). ...
... Morphologically, the gap junctions were described as "plaques" or "maculae" at intercellular interfaces including numerous intercellular channels that incorporated connexins 10,11 . Structurally, the eutherian connexins included 4 transmembrane α-helices traversing plasma membrane, cytoplasmic connexin regions including N-terminus, cytoplasmic loop that was positioned between second and third transmembrane helices and C-terminal domain, and, finally, extracellular connexin regions including two loops that were positioned between first and second transmembrane helices (region E1) and third and fourth transmembrane helices (region E2) [10][11][12][13][14][15][16][17][18] (see Protein molecular evolution analysis below). The connexin hexamers (connexons or hemichannels) that were located in adjacent cells were implicated in formation of gap junction channel connexon pores and intercellular docking [10][11][12][13][14][15][16][17][18] . ...
... Structurally, the eutherian connexins included 4 transmembrane α-helices traversing plasma membrane, cytoplasmic connexin regions including N-terminus, cytoplasmic loop that was positioned between second and third transmembrane helices and C-terminal domain, and, finally, extracellular connexin regions including two loops that were positioned between first and second transmembrane helices (region E1) and third and fourth transmembrane helices (region E2) [10][11][12][13][14][15][16][17][18] (see Protein molecular evolution analysis below). The connexin hexamers (connexons or hemichannels) that were located in adjacent cells were implicated in formation of gap junction channel connexon pores and intercellular docking [10][11][12][13][14][15][16][17][18] . The homomeric connexons included single connexins, and heteromeric connexons included multiple connexins that were encoded by about 20 connexin genes among eutherians. ...
The eutherian connexins were characterized as protein constituents of gap junctions implicated in cell-cell communications between adjoining cells in multiple cell types, regulation of major physiological processes and disease pathogeneses. However, conventional connexin gene and protein classifications could be regarded as unsuitable in descriptions of comprehensive eutherian connexin gene data sets, due to ambiguities and inconsistencies in connexin gene and protein nomenclatures. Using eutherian comparative genomic analysis protocol and 35 public eutherian reference genomic sequence data sets, the present analysis attempted to update and revise comprehensive eutherian connexin gene data sets, and address and resolve major discrepancies in their descriptions. Among 631 potential coding sequences, the tests of reliability of eutherian public genomic sequences annotated, in aggregate, 349 connexin complete coding sequences. The most comprehensive curated eutherian connexin gene data set described 21 major gene clusters, 4 of which included evidence of differential gene expansions. For example, the present gene annotations initially described human CXNK1 gene and annotated 22 human connexin genes. Phylogenetic tree calculations and calculations of pairwise nucleotide sequence identity patterns proposed revised and updated phylogenetic classification of eutherian connexin genes. Therefore, the present study integrating gene annotations, phylogenetic analysis and protein molecular evolution analysis proposed new nomenclature of eutherian connexin genes and proteins.
... Intercellular channel formation occurs through an assembly of twelve connexin subunits 5 . Within the plasma membrane, six connexins are organized into a hemichannel structure. ...
... We were unable to resolve a specific pattern of Cx46/50 heteromeric or heterotypic co-assembly using 3D classification or refinement strategies (Methods, Extended Data Figs. 4,5). Nevertheless, high-resolution features corresponding to side-chain densities are observed throughout the reconstructions following 12-fold symmetry refinement (that is, by averaging signal contributed by both Cx46 and Cx50). ...
... Therefore, these two isoforms, which share about 80% core-sequence identity (88% similarity), also share a highly similar 3D structure (Fig. 1b, c, Extended Data Figs. [4][5][6], consistent with the ability of Cx46/50 to co-assemble in a variety of heteromeric and/or heterotypic states. ...
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.
... Une caractéristique bien connue de ces plaques, exception faite de celles entre cellules de fibres du cristallin, est leur insolubilité dans les détergents non ioniques malgré l'absence d'association détectable avec le cytosquelette (VanSlyke et al., 2000). Aujourd'hui, le terme gap junction se réfère à ces plaques contenant de multiples canaux intercellulaires, reliant deux membranes cytoplasmiques (Sosinsky et al., 2005). Les quelques cellules saines qui n'expriment pas de jonctions gap sont les cellules musculaires striées adultes, certains neurones, les spermatozoïdes matures et les cellules circulantes du sang (Meda, 1996). ...
... La microscopie électronique combinée au traitement d'images (Unger et al., 1999) et les analyses hydrodynamiques (Falk et al., 1997) ont montré que chaque connexon consiste en l'assemblage annulaire de 6 sous-unités protéiques transmembranaires polytopiques appelées connexines (Cx) (Fig. 14). Le demi-canal peut supposer être divisé en trois domaines fonctionnels : (i) les domaines transmembranaires qui forment le canal, (ii) les domaines extracellulaires qui contribuent à la reconnaissance cellule-cellule, à l'amarrage de deux Cxs, et à la formation de la portion extracellulaire du pore, (iii) les domaines cytoplasmiques qui influencent les propriétés physiologiques d'ouverture/fermeture du canal (Sosinsky et al., 2005). Cependant, il est connu que la séquence d'acides aminés primaires des domaines cytoplasmiques influe sur le mode de rencontre et de juxtaposition de deux connexons ( Fig. 15) . ...
Ce travail de recherche s'inscrit dans un axe de diagnostic du cancer via le développement d'une méthode optique pour la caractérisation fonctionnelle de tissus. La technique de gap- FRAP (Fluorescence Recovery After Photobleaching) permet l'étude quantitative de la fonctionnalité des jonctions gap. La majorité des cellules néoplasiques se caractérisent par une modification du niveau d'expression et/ou de la fonctionnalité des jonctions gap par comparaison à leurs homologues saines. La technique de gap-FRAP permet en conséquence de discriminer les cellules cancéreuses en fonction de la communication intercellulaire gap jonctionnelle (CIGJ). Particulièrement utilisée in vitro, cette technique restait cependant anecdotique ex vivo. Nous avons validé la faisabilité du transfert de cette méthode sur tissus et organes ex vivo. A partir de cellules de statuts différents en expression et en distribution des connexines, nous avons caractérisé la calcéine-AM comme étant une sonde fluorescente adaptée pour des mesures sur tissus. Puis nous avons développé un modèle d'ingénierie systéme pour l'analyse comparative des données de recouvrement de fluorescence sur des modèles bi et tridimensionnels. Nous avons transposé ces conditions préalablement définies sur organe entier ex vivo : la vessie de rat. Un marquage multiple a été optimisé avec une sonde fluorescente pour le tracking des cellules cancéreuses dans la vessie ex vivo, un marqueur pour l'identification histologique de l'urothélium et la calcéine-AM pour mesurer la CIGJ. Le gap-FRAP a été utilisé pour la première fois pour différencier le degré de communication intercellulaire gap jonctionnelle entre le tissu sain et néoplasique sur un organe entier ex vivo, ouvrant des perspectives pour le diagnostic du cancer de la vessie corrélé à la modification de la CIGJ.
... Gap junction intercellular communication is facilitated by a unique macromolecular architecture, where intercellular channels directly couple the cytoplasms of two neighboring cells. The lipid bilayers of opposing cells are separated by a characteristic gap of 3.5 nm 8 , a feature for which these structures were first recognized in electron micrographs of cell sections 9,10 . Furthermore, large-scale gap junctional plaque formation is dependent upon a dense mosaic of protein-lipid interactions. ...
... Furthermore, large-scale gap junctional plaque formation is dependent upon a dense mosaic of protein-lipid interactions. In vitro reconstitution studies have established that plaque assembly and intercellular channel function are dependent on the lipid environment [10][11][12][13][14][15] . However, the molecular basis for these effects remain largely unknown, due to the lack of high-resolution structural information within a lipid bilayer. ...
Gap junctions establish direct pathways for cells to transfer metabolic and electrical messages. The local lipid environment is known to affect the structure, stability and intercellular channel activity of gap junctions; however, the molecular basis for these effects remains unknown. Here, we incorporate native connexin-46/50 (Cx46/50) intercellular channels into a dual lipid nanodisc system, mimicking a native cell-to-cell junction. Structural characterization by CryoEM reveals a lipid-induced stabilization to the channel, resulting in a 3D reconstruction at 1.9 Å resolution. Together with all-atom molecular dynamics simulations, it is shown that Cx46/50 in turn imparts long-range stabilization to the dynamic local lipid environment that is specific to the extracellular lipid leaflet. In addition, ~400 water molecules are resolved in the CryoEM map, localized throughout the intercellular permeation pathway and contributing to the channel architecture. These results illustrate how the aqueous-lipid environment is integrated with the architectural stability, structure and function of gap junction communication channels. The local lipid environment is known to affect the structure, stability and intercellular channel activity of gap junctions, however, the molecular basis for these effects remains unknown. Here authors report the CryoEM structure of Cx46/50 lipid-embedded channels, by which they reveal a lipid-induced stabilization to the channel.
... However, such simplification is imprecise given that conduction through gap junctions present a nonlinear behavior [48][49][50]. Experimental observations provide evidence that gap junctions are seen as pores distributed over the cellular membrane [51][52][53]. Thus, the membrane surface can be regarded as a porous medium, through which, the intercellular communication occurs when an AP is propagating. ...
... Using the relation β = 2π log ξ , the values obtained for β = β 2 ( Table 2) generate characteristic scales ζ = 1/ξ in the range of 8Å < ζ < 66Å. Such values are in agreement with the estimated sizes for cardiac intercellular channels [48,53,101,102]. From the obtained results the simulations in which γ = α + jβ 2 generate significant modulations on the microscopic or mesoscopic scale, suggesting an influence of this characteristic scale on the propagation dynamics. ...
Cardiac tissue is characterized by structural and cellular heterogeneities that play an important role in the cardiac conduction system. Under persistent atrial fibrillation (persAF), electrical and structural remodeling occur simultaneously. The classical mathematical models of cardiac electrophysiological showed remarkable progress during recent years. Among those models, it is of relevance the standard diffusion mathematical equation, that considers the myocardium as a continuum. However, the modeling of structural properties and their influence on electrical propagation still reveal several limitations. In this paper, a model of cardiac electrical propagation is proposed based on complex order derivatives. By assuming that the myocardium has an underlying fractal process, the complex order dynamics emerges as an important modeling option. In this perspective, the real part of the order corresponds to the fractal dimension, while the imaginary part represents the log-periodic corrections of the fractal dimension. Indeed, the imaginary part in the derivative implies characteristic scales within the cardiac tissue. The analytical and numerical procedures for solving the related equation are presented. The sinus rhythm and persAF conditions are implemented using the Courtemanche formalism. The electrophysiological properties are measured and analyzed on different scales of observation. The results indicate that the complex order modulates the electrophysiology of the atrial system, through the variation of its real and imaginary parts. The combined effect of the two components yields a broad range of electrophysiological conditions. Therefore, the proposed model can be a useful tool for modeling electrical and structural properties during cardiac conduction.
... Structurally, both loops possess a highly conserved amino acid sequence among connexins (except Cx31), with a [C-X6-C-X3-C] pattern for E1 and [C-X5-C-X5-C] pattern for E2 [18,42]. Therefore, docking specificity is not thought to arise from sequence-specific coupling, but from a complex arrangement of antiparallel β sheets connected by disulfide bonds into concentric β barrels [42,43]. This structural hypothesis translates accordingly into innexins, where β-sheet "hairpins" accumulate around E2, while E1 creates a constriction ring around the axial pore through a small α-helix [44]. ...
... Throughout the hemichannel configuration, the orientation of α-helices surrounding the axial pore aligns predominantly in a clockwise fashion, with only a couple of right-handed segments lining the pore into a crisscrossed, tilted pattern [42,45]. Several models have aspired to predict an ion permeability mechanism based on this tilting, where structural occlusion of the pore would occur through the twisting constriction of these helices in a manner similar to that of an iris diaphragm in a camera [43,45,46] However, recent X-ray analysis performed on connexin-mediated GJs revealed no structural variation between Ca 2+ -bound and Ca 2+ -free channels, suggesting the existence of a cation exclusion mechanism based on electrostatic interactions instead [47]. ...
Gap junction (GJ) channels in invertebrates have been used to understand cell-to-cell communication in vertebrates. GJs are a common form of intercellular communication channels which connect the cytoplasm of adjacent cells. Dysregulation and structural alteration of the gap junction-mediated communication have been proven to be associated with a myriad of symptoms and tissue-specific pathologies. Animal models relying on the invertebrate nervous system have exposed a relationship between GJs and the formation of electrical synapses during embryogenesis and adulthood. The modulation of GJs as a therapeutic and clinical tool may eventually provide an alternative for treating tissue formation-related diseases and cell propagation. This review concerns the similarities between Hirudo medicinalis innexins and human connexins from nucleotide and protein sequence level perspectives. It also sets forth evidence of computational techniques applied to the study of proteins, sequences, and molecular dynamics. Furthermore, we propose machine learning techniques as a method that could be used to study protein structure, gap junction inhibition, metabolism, and drug development.
... More recently, gap junction fractions were also isolated from cultured cells (Hand, Muller, Nicholson, Engel, & Sosinsky, 2002;Yeager & Unger, 2001). For reviews on gap junction's molecular structure, see studies by Maeda et al. (2009), Meckes et al. (2014, Sosinsky and Nicholson (2005), Veenstra and Lin (2006). ...
... in;Peracchia, 1980Peracchia, , 1985. Note that the discovery of the hexameric structure of connexons is often erroneously attributed (Sosinsky & Nicholson, 2005) to Goodenough and Stoeckenius (1972). ...
Evidence that neighboring cells uncouple from each other as one dies surfaced in 1877, but it took almost a century for scientists to start understanding the uncoupling (gating) mechanism. The role of intracellular calcium (Cai) in cell-cell channel gating was first reported in the mid-sixties. In these studies, only micromolar [Ca2+]i were believed to affect gating - concentrations reachable only in cell death, which would discard Ca2+i as a fine modulator of cell coupling. More recently, however, numerous researchers, including our team, have reported the effectiveness of nanomolar [Ca2+]i. Since connexins do not have high affinity calcium sites, the effectiveness of nanomolar Ca2+i suggests the role of Ca-modulated proteins, calmodulin (CaM) being most obvious. Indeed, in 1981 we first reported that a CaM-inhibitor prevents chemical gating. Since then, the CaM role has been confirmed by experiments testing it with a variety of approaches including: treatments with CaM-inhibitors, inhibition of CaM expression, expression of CaM mutants, immunofluorescent co-localization of CaM and gap junctions, and binding of CaM to peptides mimicking connexin domains identified as CaM targets. Our gating model envisions Ca2+-CaM to directly gate the channels by acting as a plug (Cork gating model), and probably by also affecting connexin conformation.
... GJ facilitates the coordination of electrical activity across the cells [12]. Since channels and hemichannels formed by Cxs also allow for the exchange of ions and molecules between the inside and outside of the cell, they are also seen as mediators of tissue homeostasis [13][14][15][16]. It is widely documented that GJ channels, through the passage of multiple signaling molecules, can modulate cell growth and apoptotic cell death, depending on the cellular context [17,18]. ...
Glucotoxicity may exert its deleterious effects on pancreatic β-cell function via a myriad of mechanisms, leading to impaired insulin secretion and, eventually, type 2 diabetes. β-cell communication requires gap junction channels to be present among these cells. Gap junctions are constituted by transmembrane proteins of the connexins (Cxs) family. Two Cx genes have been identified in β cells, Cx36 and Cx30.2. We have found evidence that the glucose concentration on its own is sufficient to regulate Cx30.2 gene expression in mouse islets. In this work, we examine the involvement of the Cx30.2 protein in the survival of β cells (RIN-m5F). Methods: RIN-m5F cells were cultured in 5 mM D-glucose (normal) or 30 mM D-glucose (high glucose) for 24 h. Cx30.2 siRNAs was used to downregulate Cx30.2 expression. Apoptosis was measured by means of TUNEL, an annexin V staining method, and the cleaved form of the caspase-3 protein was determined using Western blot. Results: High glucose did not induce apoptosis in RIN-m5F β cells after 24 h; interestingly, high glucose increased the Cx30.2 total protein levels. Moreover, this work found that the downregulation of Cx30.2 expression in high glucose promoted apoptosis in RIN-m5F cells. Conclusion: The data suggest that the upregulation of Cx30.2 protects β cells from hyperglycemia-induced apoptosis. Furthermore, Cx30.2 may be a promising avenue of therapeutic investigation for the treatment of glucose metabolic disorders.
... The copyright holder for this preprint (which this version posted April 16, 2024. ; https://doi.org/10.1101/2024.04.14.589464 doi: bioRxiv preprint likely arises from the stability of gap junctions facilitated by interaction between juxtaposed hemichannels (Sosinsky and Nicholson, 2005). It is noteworthy, however, that the expression of compatible gap junction subunits does not guarantee the assembly of gap junction connections between adjacent neurons in vivo (Bhattacharya et al., 2019;Fukuda, 2017;Greb et al., 2017;White et al., 1992;Yao et al., 2016). ...
Electrical coupling is vital to neural communication, facilitating synchronized activity among neurons. Despite its significance, the precise mechanisms governing the establishment of gap junction connections between specific neurons remain elusive. Here, we identified that the PVC interneuron in Caenorhabditis elegans forms gap junction connections with the PVR interneuron. The transcriptional regulator CFI-1/ARID3 is specifically expressed in the PVC but not PVR interneuron. Reducing cfi-1 expression in the PVC interneuron leads to enhanced gap junction formation in the PVR neuron, while ectopic expression of cfi-1 in the PVR neuron restores the proper level of gap junction connections in the PVC neuron, along with the normal touch response. These findings unveil the pivotal role of CFI-1/ARID3 in bidirectionally regulating the formation of gap junctions within a specific neuronal pair, shedding light on the intricate molecular mechanisms governing neuronal connectivity in vivo .
SUMMARY STATEMENT
Proper electrical coupling could be achieved by either of the partner cells; however distinctive influence on neuron circuits maybe achieved.
... Transmembrane domains (TM1-TM4) perform a variety of functions i.e., enzyme catalysis, transport across membranes, transducing signals as receptors of hormones and growth factors, and energy transfer in ATP synthesis. Cytoplasmic loop controls the activity and permeability properties of the channels [19]. ...
Background
To identify the underlying genetic defects in autosomal dominant (ADCC) and autosomal recessive (ARCC) congenital cataract families from North India.
Methods
Detailed family histories were collected, pedigrees drawn followed by slit-lamp examination and lens photography. Mutation screening was performed using Sanger sequencing in the known candidate genes for crystallins, connexins, and membrane proteins. The pathogenicity of identified variants was assessed bioinformatically.
Results
In two ADCC families (CC-281 and CC-3015) with posterior lenticonus cataract, a novel change c.263C > T (p.P88L) in GJA3 in CC-281 family and a previously reported substitution c.388C > T (p.R130C) in LIM2 in CC-3015 family was observed. In an ARCC family (CC-3005) having central pulverulent cataract, a novel frameshift deletion (c.764delT;p.L255R46fs) in GJA3 was detected. The observed variants segregated completely with phenotypes in the affected members and were neither present in unaffected family members nor in the ethnically matched 150 controls (tested for two novel variants), hence excluding these as polymorphisms.
Conclusions
Present study identified two novel mutations i.e., c.263C > T;p.P88L and c.764delT;p.L255R46fs in GJA3 in an ADCC and an ARCC family having posterior lenticonus and central pulverulent cataract, respectively. In another ADCC family with posterior lenticonus cataract, a previously reported mutation c.388C > T;p.R130C in LIM2 was observed. R130 may be a mutation hotspot as previously ADCC families from different ethnicities (UK/Czechia, China, Spain, Japan) also harbored the same substitution, however, with different phenotypes i.e., nuclear pulverulent, membranous, nuclear, lamellar, and sutural/lamellar. Findings in present study thus expand the mutation spectrum and phenotypic heterogeneity linked with GJA3 and LIM2.
... Transmembrane domains carry out a wide range of tasks, including energy transfer during ATP generation, membrane transport, signal transduction as hormone and growth factor receptors, and enzyme catalysis. The cytoplasmic loop regulates the channels permeability and activity (Saez et al. 2003;Sosinsky and Nicholson 2005). Cx46 is essential for the coupling of fiber cells, particularly mature fibers in the central core of the lens. ...
Congenital cataract an opacity of the eye lens is present at birth and results in visual impairment during early childhood. If left untreated, it can lead to permanent blindness. Its prevalence is ten times higher in developing countries like India. Thus, we aimed to investigate the underlying genetic defects in three autosomal dominant congenital cataract (ADCC) families from North India. Detailed family histories were collected, pedigrees drawn followed by slit-lamp examination and lens photography. Mutation screening was performed in the candidate genes for crystallins, connexins, and membrane proteins by Sanger sequencing. Pathogenicity of novel variant was assessed bioinformatically. In an ADCC (CC-3006) family with bilateral membranous cataract and microcornea, a novel change (c.1114C>T;p.P372S) in GJA3 has been detected. In other two ADCC families affected with subcapsular (CC-286) and shrunken membranous hypermature cataract (CC-3014), a nonsense mutation (c.463C>T;p.Q155X) in CRY βB2 and a frameshift deletion (c.590_591delAG;p.E197VfsX22) in CRY βA1/A3 respectively, are observed. These variants segregated completely with the phenotypes in respective families and were absent in their unaffected family members and unrelated controls (tested for novel variant in GJA3). Earlier p.Q155X (CRYβB2) and p.E197VfsX22 (CRYβA1/A3) are reported with entirely different phenotypes. Thus, findings in present study expand the mutation spectrum and phenotypic heterogeneity linked with GJA3, CRYβB2, and CRYβA1/A3 for congenital cataracts. Identifying underlying genetic defects is essential for disease management and appropriate genetic counseling.
... The remaining gap junctions can facilitate the exchange of protective and harmful metabolites between healthy and damaged cells, protecting damaged cells to some extent. Various studies have shown that the phosphorylation state of the astrocyte Cx43 C-terminus is an important mediator of the regulation of gap junction channels and hemichannels after ischemic stroke, thereby affecting astrocyte and neuronal function [76,77]. It has been reported that the C-terminus of Cx43 in astrocytes after ischemic stroke can be phosphorylated by a variety of protein kinases, including protein kinase C, mitogen-activated protein kinase (MAPK), pp60Src kinase, and casein kinase 1δ, inducing Cx43 internalization and further promoting the uncoupling process of astrocytes [18,[78][79][80][81][82]. ...
Connexin 43 (Cx43) is most widely distributed in mammals, especially in the cardiovascular and nervous systems. Its phosphorylation state has been found to be regulated by the action of more than ten kinases and phosphatases, including mitogen-activated protein kinase/extracellular signaling and regulating kinase signaling. In addition, the phosphorylation status of different phosphorylation sites affects its own synthesis and assembly and the function of the gap junctions (GJs) to varying degrees. The phosphorylation of Cx43 can affect the permeability, electrical conductivity, and gating properties of GJs, thereby having various effects on intercellular communication and affecting physiological or pathological processes in vitro and in vivo. Therefore, clarifying the relationship between Cx43 phosphorylation and specific disease processes will help us better understand the disease. Based on the above clinical and preclinical findings, we present in this review the functional significance of Cx43 phosphorylation in multiple diseases and discuss the potential of Cx43 as a drug target in Cx43-related disease pathophysiology, with an emphasis on the importance of connexin 43 as an emerging therapeutic target in cardiac and neuroprotection.
... **P < 0.01, significantly different from the group of analgesics used alone at 0 µg/ml (or ng/ml), ## P < 0.01, significantly different from the group of analgesics concurrent use with TMZ at 0 µg/ml (or ng/ml) phosphorylation ( Fig. 5D and E). Considering that connexin phosphorylation is closely related to the MAPK signal pathway [12,13], the expression of ERK, JNK, p38, and their phosphorylation were explored further, and the results are shown in Fig. 5F. The results revealed that Cx43 phosphorylation was facilitated through phosphorylation of the ERK pathway, with no involvement of the JNK and p38 pathways. ...
This study investigated the effect of frequently used analgesics in cancer pain management (flurbiprofen (FLU), tramadol (TRA), and morphine (MOR)) and a novel α2-adrenergic agonist (dexmedetomidine, DEX) on temozolomide (TMZ) sensitivity in glioma cells. Cell counting kit-8 and colony-formation assays were performed to analyze the viability of U87 and SHG-44 cell lines. A high and low cell density of colony method, pharmacological methods, and connexin43 mimetic peptide GAP27 were used to manipulate the function of gap junctions; “Parachute” dye coupling and western blot were employed to determine junctional channel transfer ability and connexin expression. The results showed that DEX (in the concentration range of 0.1 to 5.0 ng/ml) and TRA (in the concentration range of 1.0 to 10.0 µg/ml) reduced the TMZ cytotoxicity in a concentration-dependent manner but was only observed with high cell density (having formed gap junction). The cell viability percentage was 71.3 to 86.8% when DEX was applied at 5.0 ng/ml, while tramadol showed 69.6 to 83.7% viability at 5.0 μg/ml in U87 cells. Similarly, 5.0 ng/ml of DEX resulted in 62.6 to 80.5%, and 5.0 μg/ml TRA showed 63.5 to 77.3% viability in SHG-44 cells. Further investigating the impact of analgesics on gap junctions, only DEX and TRA were found to decrease channel dye transfer through connexin phosphorylation and ERK pathway, while no such effect was observed for FLU and MOR. Analgesics that can affect junctional communication may compromise the effectiveness of TMZ when used simultaneously.
... Specifically, cadherins are attached to the actin cytoskeleton via catenins (Viggiano et al. 2015). Gap junctions and desmosomes contribute to intracellular interaction and cell-cell binding, respectively (Garrod and Chidgey 2008;Sosinsky and Nicholson 2005). These complicated multiprotein structures strengthen the resistance of the gut epithelial barrier to potentially harmful molecules and pathogens in the lumen (Allam-Ndoul, Castonguay-Paradis, and Veilleux 2020). ...
Human milk oligosaccharides (hMOs) in mothers' milk play a crucial role in guiding the colonization of microbiota and gut-immune barrier development in infants. Non-digestible carbohydrates (NDCs) such as synthetic single hMOs, galacto-oligosaccharides (GOS), inulin-type fructans and pectin oligomers have been added to infant formula to substitute some hMOs' functions. HMOs and NDCs can modulate the gut-immune barrier, which is a multiple-layered functional unit consisting of microbiota, a mucus layer, gut epithelium, and the immune system. There is increasing evidence that the structures of the complex polysaccharides may influence their efficacy in modulating the gut-immune barrier. This review focuses on the role of different structures of individual hMOs and commonly applied NDCs in infant formulas in (i) direct regulation of the gut-immune barrier in a microbiota-independent manner and in (ii) modulation of microbiota composition and microbial metabolites of these polysaccharides in a microbiota-dependent manner. Both have been shown to be essential for guiding the development of an adequate immune barrier, but the effects are very dependent on the structural features of hMO or NDC. This knowledge might lead to tailored infant formulas for specific target groups.
... In connexin 46, approximately 20 mutations linked with different cataract phenotypes are detected in the EL1 and EL2 loops, which are reported to be essential for docking interactions between hemichannels (26,27). In another ADCC (PC-12) family with nuclear cataract in the present study, we have detected p.Trp45Ser substitution in connexin 46. ...
Aims
The study aims to detect the underlying genetic defect in two autosomal dominant congenital cataract (ADCC) families.
Methods
A detailed family history was collected, pedigrees were drawn, and slit-lamp examination and lens photography were performed. Mutation screening was carried out in the genes for crystallins and connexins by PCR and Sanger sequencing. Ethnically matched controls were tested for the identified variants. Different bioinformatics tools were used to assess the pathogenicity of the observed variants.
Results
In an ADCC family with total cataract, a novel change (c.166A > G) (p.Thr56Ala) in GJA8 was identified. In another ADCC family with nuclear cataract, c.134G > C (p.Trp45Ser) in GJA3 has been detected. These variants co-segregated completely in patients in their respective families and were neither observed in unaffected family members nor in ethnically matched 100 controls, excluding them as polymorphisms.
Conclusions
The present study identifies a novel variant c.166A > G (p.Thr56Ala) in GJA8 in an ADCC family having total cataract and a previously known mutation c.134G > C (p.Trp45Ser) in GJA3 in another ADCC family. Thr56 in GJA8 seems to be a mutation hotspot, as previously an ADCC Mauritanian family harbored a different substitution (p.Thr56Pro) at the same codon, although for a different phenotype (nuclear cataract). Similarly, Trp45 in GJA3 appears as a mutation hotspot, as p.Trp45Ser has previously been reported for nuclear cataract in a Chinese ADCC family. p.Thr56 (GJA8) and p.Trp45 (GJA3 ) are in the extracellular loop 1 (EL1) in their respective connexin proteins, which, along with EL2, are essential for gap junction formation, hemichannel docking, and regulating the voltage gating of the channels. Hence, residues in these regions seem crucial for maintaining eye lens transparency.
... Transmembrane domains perform a variety of functions such as enzyme catalysis, transport across membranes, transducing signals as receptors of hormones and growth factors, and energy transfer in ATP synthesis. Cytoplasmic loop controls the activity and permeability properties of the channels (29,30). ...
Background
To investigate the underlying genetic defects in two autosomal dominant (ADCC) and an autosomal recessive (ARCC) congenital cataract families from North India.
Methods
Detailed family history was collected, and pedigrees drawn followed by slit-lamp examination and lens photography. Mutation screening performed using Sanger sequencing in the known candidate genes for crystallins, connexins, and membrane proteins. Various bioinformatics tools were used to assess the pathogenicity of the identified variants.
Results
In two ADCC families with posterior lenticonus cataract, a novel change (c.263C > T; p.P88L) in GJA3 (CC-281 family) and a previously reported substitution (c.388C > T; p.R130C) in LIM2 in second ADCC family (CC-3015) was observed. In an ARCC family (CC-3005) having bilateral central pulverulent cataract, a novel frameshift deletion c.764delT; p.L255R46fs) in GJA3 was detected. The observed variants segregated completely with the phenotypes in the affected members and were neither present in unaffected family members nor in the ethnically matched 150 controls, hence excluding these as polymorphisms.
Conclusions
Present study identified two novel mutations i.e., c.263C > T (p.P88L) and c.764delT (p.L255R46fs) in GJA3 in an ADCC and an ARCC family having posterior lenticonus and central pulverulent cataract, respectively. In second ADCC family with posterior lenticonus cataract, a previously reported mutation c.388C > T (p.R130C) in LIM2 was observed. This indicates R130 a mutation hotspot as previously two ADCC families one each of European and Chinese descent also harbored the same substitution, however, with different phenotypes i.e., pulverulent and nuclear cataracts. Findings in the present study thus expand the mutation spectrum and phenotypic heterogeneity linked with GJA3 and LIM2.
... The role of hemichannels is to exchange ions and small molecules between the cytosol and the extracellular space, while GJs are responsible for the direct diffusion of these molecules between adjacent cells. Channels composed of different Cx isoforms show specific and characteristic molecular permeability [1][2][3]. Therefore, these channels are involved in various physiological functions, including glandular secretion [4][5][6][7][8]. In most glands, Cx26, Cx32, and Cx43 are the ones mainly expressed. ...
Connexins (Cxs) are transmembrane proteins involved in the formation of hemichannels and gap junctions (GJs). GJs are involved in various physiological functions, including secretion in glandular tissue. It has been demonstrated that Cx26, Cx32, and Cx43 are mainly expressed in glands, but no data are available in human salivary glands to date. The aim of our study was to investigate the presence and the localization of Cxs in human minor labial salivary glands. Immunofluorescence and immunoelectron microscopy were employed to evaluate the Cx26, Cx32, and Cx43 protein in human labial salivary gland biopsies (hLSGBs). RT-PCR was also used to detect their mRNA expression. Cx expression was found at both the mRNA and protein levels in all hLSGBs analysed. Cxs were observed at the level of the duct and acinar cells, as well as in myoepithelial cells. The localization of the three Cx types was very similar, suggesting colocalization of these Cxs in the same connexons. These results demonstrated the presence of Cxs in human salivary glands for the first time. Moreover, the few samples with primary Sjögren’s Syndrome analysed only by immunofluorescence showed an alteration of the Cx expression, indicating that these proteins could be involved in salivary gland dysfunctions.
... Biological encodings of classical information have lower limits of 1-2 nm in radius, e.g. the size of a typical protein active site (Liang et al. 1998) or a gap-junction channel (Sosinsky and Nicholson 2005), and about 200 fs in time, e.g. the response time of rhodopsin to photons (Wang et al. 1994). Cellular response times, even for bioelectric responses, are orders of magnitude larger and involve much larger areas. ...
Theories of consciousness and cognition that assume a neural substrate automatically regard phylogenetically basal, nonneural systems as nonconscious and noncognitive. Here, we advance a scale-free characterization of consciousness and cognition that regards basal systems, including synthetic constructs, as not only informative about the structure and function of experience in more complex systems but also as offering distinct advantages for experimental manipulation. Our "minimal physicalist" approach makes no assumptions beyond those of quantum information theory, and hence is applicable from the molecular scale upwards. We show that standard concepts including integrated information, state broadcasting via small-world networks, and hierarchical Bayesian inference emerge naturally in this setting, and that common phenomena including stigmergic memory, perceptual coarse-graining, and attention switching follow directly from the thermodynamic requirements of classical computation. We show that the self-representation that lies at the heart of human autonoetic awareness can be traced as far back as, and serves the same basic functions as, the stress response in bacteria and other basal systems.
... Gap junctions (GJs) are a class of membrane channels that provide a direct passageway between neighbouring cells and facilitate the exchange of ions and small molecules (Saez et al. 2003;Goodenough & Paul, 2009). This type of cell-to-cell communication is facilitated by a unique channel architecture, whereby a continuous water-filled pore ∼1.5 nm in diameter is formed between two opposing cell membranes, effectively coupling the cytoplasms of adjoined cells (Sosinsky & Nicholson, 2005). These direct passageways are essential for enabling fast transmission of electrical signals in the brain and heart and for facilitating long-range metabolic coupling in most tissues. ...
Key points
Gap junctions formed by different connexins are expressed throughout the body and harbour unique channel properties that have not been fully defined mechanistically.
Recent structural studies by cryo‐electron microscopy have produced high‐resolution models of the related but functionally distinct lens connexins (Cx50 and Cx46) captured in a stable open state, opening the door for structure–function comparison.
Here, we conducted comparative molecular dynamics simulation and electrophysiology studies to dissect the isoform‐specific differences in Cx46 and Cx50 intercellular channel function.
We show that key determinants Cx46 and Cx50 gap junction channel open stability and unitary conductance are shaped by structural and dynamic features of their N‐terminal domains, in particular the residue at the 9th position and differences in hydrophobic anchoring sites.
The results of this study establish the open state Cx46/50 structural models as archetypes for structure–function studies targeted at elucidating the mechanism of gap junction channels and the molecular basis of disease‐causing variants.
Abstract
Connexins form intercellular communication channels, known as gap junctions (GJs), that facilitate diverse physiological roles, from long‐range electrical and chemical coupling to coordinating development and nutrient exchange. GJs formed by different connexin isoforms harbour unique channel properties that have not been fully defined mechanistically. Recent structural studies on Cx46 and Cx50 defined a novel and stable open state and implicated the amino‐terminal (NT) domain as a major contributor for isoform‐specific functional differences between these closely related lens connexins. To better understand these differences, we constructed models corresponding to wildtype Cx50 and Cx46 GJs, NT domain swapped chimeras, and point variants at the 9th residue for comparative molecular dynamics (MD) simulation and electrophysiology studies. All constructs formed functional GJ channels, except the chimeric Cx46‐50NT variant, which correlated with an introduced steric clash and increased dynamical behaviour (instability) of the NT domain observed by MD simulation. Single channel conductance correlated well with free‐energy landscapes predicted by MD, but resulted in a surprisingly greater degree of effect. Additionally, we observed significant effects on transjunctional voltage‐dependent gating (Vj gating) and/or open state dwell times induced by the designed NT domain variants. Together, these studies indicate intra‐ and inter‐subunit interactions involving both hydrophobic and charged residues within the NT domains of Cx46 and Cx50 play important roles in defining GJ open state stability and single channel conductance, and establish the open state Cx46/50 structural models as archetypes for structure–function studies targeted at elucidating GJ channel mechanisms and the molecular basis of cataract‐linked connexin variants.
... There are 21 known connexin isoforms in the human genome, which are named according to their molecular weight (in kD). Most cells and tissues express several connexin isoforms [65,66]. ...
Gap junctions are molecular structures that allow communication between neighboring cells. It has been shown that gap junctional intercellular communication (GJIC) is notoriously reduced in cancer cells compared to their normal counterparts. Ouabain, a plant derived substance, widely known for its therapeutic properties on the heart, has been shown to play a role in several types of cancer, although its mechanism of action is not yet fully understood. Since we have previously shown that ouabain enhances GJIC in epithelial cells (MDCK), here we probed whether ouabain affects GJIC in a variety of cancer cell lines, including cervico-uterine (CasKi, SiHa and Hela), breast (MDA-MB-321 and MCF7), lung (A549), colon (SW480) and pancreas (HPAF-II). For this purpose, we conducted dye transfer assays to measure and compare GJIC in monolayers of cells with and without treatment with ouabain (0.1, 1, 10, 50 and 500 nM). We found that ouabain induces a statistically significant enhancement of GJIC in all of these cancer cell lines, albeit with distinct sensitivity. Additionally, we show that synthesis of new nucleotides or protein subunits is not required, and that Csrc, ErK1/2 and ROCK-Rho mediate the signaling mechanisms. These results may contribute to explaining how ouabain influences cancer.
... Connexins (Cx) are gap junction proteins involved in cell-to-cell communication. Connexins assemble in the plasma membrane to form gap junctions by assembling six connexin subunits to form a connexon [1,2]. Connexons bridge two neighboring cells by creating a channel between the two cells that mediate the transport of ions, nutrients, signaling molecules, as well as miRNAs between the two cells [3][4][5][6]. ...
The term lung disease describes a broad category of disorders that impair lung function. More than 35 million Americans have a preventable chronic lung disease with high mortality rates due to limited treatment efficacy. The recent increase in patients with lung disease highlights the need to increase our understanding of mechanisms driving lung inflammation. Connexins, gap junction proteins, and more specifically connexin 43 (Cx43), are abundantly expressed in the lung and are known to play a role in lung diseases. This review focuses on the role of Cx43 in pathology associated with acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD) and asthma. Additionally, we discuss the role of Cx43 in preventing disease through the transfer of mitochondria between cells. We aim to highlight the need to better understand what cell types are expressing Cx43 and how this expression influences lung disease.
... We have previously demonstrated that gap junctions expand the area of cell death affected by NTP [16]. Gap junctions promote intercellular communication through the transfer of charged and neutral species of up to 1 kilodalton (kDa) including ROS, RNS, and other free radicals [23]. Gap junctions enable the transport of ROS molecules between cells via the bystander effect as demonstrated by radiation treatment [24]. ...
Metastatic melanoma cells overexpressing gap junctions were assayed for their ability to propagate cell death by a novel combination therapy that generates reactive oxygen species (ROS) by both 1) non-thermal plasma (NTP) and 2) tirapazamine (TPZ) under hypoxic conditions. Results demonstrate additive-to-synergistic effects of combination therapy compared to each agent individually. NTP induces highly localized cell death in target areas whereas TPZ partially reduces viability over the total surface area. However, when high gap junction expression was induced in melanoma cells, effects of combination NTP+TPZ therapy was augmented, spreading cell death across the entire plate. Similarly, in vivo studies of human metastatic melanoma in a mouse tumor model demonstrate that the combined effect of NTP+TPZ causes a 90% reduction in tumor volume, specifically in the model expressing gap junctions. Treatment with NTP+TPZ increases gene expression in the apoptotic pathway and oxidative stress while decreasing genes related to cell migration. Immune response was also elicited through differential regulation of cytokines and chemokines, suggesting potential for this therapy to induce a cytotoxic immune response with fewer side effects than current therapies. Interestingly, the gap junction protein, Cx26 was upregulated following treatment with NTP+TPZ and these gap junctions were shown to maintain functionality during the onset of treatment. Therefore, we propose that gap junctions both increase the efficacy of NTP+TPZ and perpetuate a positive feedback mechanism of gap junction expression and tumoricidal activity. Our unique approach to ROS induction in tumor cells with NTP+TPZ shows potential as a novel cancer treatment.
... The C-terminal of astrocytic Cx43 has critical roles in regulating astrocytic functions. Various studies have indicated that the phosphorylation status of the astrocytic Cx43 C-terminal is an important mediator modulating the gap junction channels and hemichannels after ischemic stroke, thus influencing the functions of astrocytes and neurons (Figure 2) (95). It has been reported that the C-terminal of astrocytic Cx43 could be phosphorylated after ischemic stroke via several protein kinases including protein kinase C (96), mitogen-activated protein kinase (MAPK) (97), pp60Src kinase (98), and casein kinase 1δ (99), inducing Cx43 internalization, further contributing to the uncoupling process of astrocytes (100,101). ...
Ischemic stroke is a multi-factorial cerebrovascular disease with high worldwide morbidity and mortality. In the past few years, multiple studies have revealed the underlying mechanism of ischemia/reperfusion injury, including calcium overload, amino acid toxicity, oxidative stress, and inflammation. Connexin 43 (Cx43), the predominant connexin protein in astrocytes, has been recently proven to display non-substitutable roles in the pathology of ischemic stroke development and progression through forming gap junctions and hemichannels. Under normal conditions, astrocytic Cx43 could be found in hemichannels or in the coupling with other hemichannels on astrocytes, neurons, or oligodendrocytes to form the neuro–glial syncytium, which is involved in metabolites exchange between communicated cells, thus maintaining the homeostasis of the CNS environment. In ischemic stroke, the phosphorylation of Cx43 might cause the degradation of gap junctions and the opening of hemichannels, contributing to the release of inflammatory mediators. However, the remaining gap junctions could facilitate the exchange of protective and harmful metabolites between healthy and injured cells, protecting the injured cells to some extent or damaging the healthy cells depending on the balance of the exchange of protective and harmful metabolites. In this study, we review the changes in astrocytic Cx43 expression and distribution as well as the influence of these changes on the function of astrocytes and other cells in the CNS, providing new insight into the pathology of ischemic stroke injury; we also discuss the potential of astrocytic Cx43 as a target for the treatment of ischemic stroke.
... Connexin 43 (Cx43) is the most ubiquitous connexin isoform, and is widely distributed in most tissues, including cardiac cells [1]. In the heart, connexins form aggregations of intercellular channels known as gap junctions, which are mainly located at cardiomyocyte poles [2]. Gap junctions constitute low resistance pathways that are essential to allow electrical current flow between connected cells [3]. ...
Previous studies demonstrated a reduction in myocardial scar size in heterozygous Cx43+/- mice subjected to permanent coronary occlusion. However, patients presenting with ST segment elevation myocardial infarction often undergo rapid coronary revascularization leading to prompt restoration of coronary flow. Therefore, we aimed to assess changes in scar size and left ventricular remodeling following transient myocardial ischemia (45 min) followed by 14 days of reperfusion using Cx43fl/fl (controls) and Cx43Cre-ER(T)/fl inducible knock-out (Cx43 content: 50%) mice treated with vehicle or 4-hydroxytamoxifen (4-OHT) to induce a Cre-ER(T)-mediated global deletion of the Cx43 floxed allele. The scar area (picrosirius red), measured 14 days after transient coronary occlusion, was similarly reduced in both vehicle and 4-OHT-treated Cx43Cre-ER(T)/fl mice, compared to Cx43fl/fl animals, having normal Cx43 levels (15.78% ± 3.42% and 16.54% ± 2.31% vs. 25.40% ± 3.14% and 22.43% ± 3.88% in vehicle and 4-OHT-treated mice, respectively, p = 0.027). Left ventricular dilatation was significantly attenuated in both Cx43-deficient groups (p = 0.037 for left ventricular end-diastolic diameter). These protective effects were correlated with an attenuated enhancement in pro-transforming growth factor beta 1 (TGFβ1) expression after reperfusion. In conclusion, our data demonstrate that Cx43 deficiency induces a protective effect on scar formation after transient coronary occlusion in mice, an effect associated with reduced left ventricular remodeling and attenuated enhancement in pro-TGFβ1 expression.
... Cxs assemble to form hexameric connexons or hemichannels, which form dodecameric gap junction channels (GJCs) by end-to-end docking of the hemichannels from adjacent cells. The E1 and E2 loops form the extracellular domain that mediates docking between hemichannels (Abascal and Zardoya, 2013;Bao et al., 2004;Foote et al., 1998;Sosinsky and Nicholson, 2005;Yeager, 2009;Yeager and Harris, 2007;Yeager and Nicholson, 1996). ...
Gap junction channels (GJCs) mediate intercellular communication and are gated by numerous conditions such as pH. The electron cryomicroscopy (cryo-EM) structure of Cx26 GJC at physiological pH recapitulates previous GJC structures in lipid bilayers. At pH 6.4, we identify two conformational states, one resembling the open physiological-pH structure and a closed conformation that displays six threads of density, that join to form a pore-occluding density. Crosslinking and hydrogen-deuterium exchange mass spectrometry reveal closer association between the N-terminal (NT) domains and the cytoplasmic loops (CL) at acidic pH. Previous electrophysiologic studies suggest an association between NT residue N14 and H100 near M2, which may trigger the observed movement of M2 toward M1 in our cryo-EM maps, thereby accounting for additional NT-CL crosslinks at acidic pH. We propose that these pH-induced interactions and conformational changes result in extension, ordering, and association of the acetylated NT domains to form a hexameric “ball-and-chain” gating particle.
... Physiology and Сell Biology элект рическую передачу сигнала, широко распространены между клетками как возбудимых, так и невозбудимых тканей [13; 14]. Из них наибольший интерес представляют так называемые щелевые соединения (gap junctions), использующие трансмембранные белки коннексины (позвоночные) или иннексины (беспозвоночные) в качестве структурной составляющей контактов подобного рода -коннексонов и иннексонов [15][16][17]. Другие группы синапсов, опосредующих прямую передачу электрического сигнала от клетки к клетке, фактически являют собой контакты с химическим способом передачи -тормозные синапсы на маутнеровских нейронах рыб [18] или синапс цилиарного ганглия цыпленка [19; 20]. Их структурные особенности, связанные с большой площадью контакта пре-и постсинаптических мембран и окружением области синаптической щели клетками глии с развитой миелиновой оболочкой, позволяют избежать потерь в пространстве интерстиция, обеспечив прямую передачу электрического импульса от пресинаптической клетки к постсинаптической. ...
The article deals with the structure functional configuration of gap junctions and control of their permeability. Based on the reactions of identified electrical synapse between V. D. 1 and R. Pa. D. 2 neurons within central nervous system of mollusc Lymnaea stagnalis the responses of mentioned above connections to temperature changes, acid-base balance (pH) shifts and increase of reactive oxygen species (hydrogen peroxide) level were analyzed. Time-course of effects observed (minutes), give the evidence of fast dynamic modulation of electrotonical coupling. The fluctuations in it conductance seems to be due to the changes in conductivity of exist gap junctions channels (innexons), but not to the alterations in their number in the junction area. It is assumed that the action of these agents realize by reversible conformational transition of gap junction structural proteins (innexins).
... Connexins are a family of membrane proteins with a characteristic structure consisting of four transmembrane domains, cytoplasmic amino-and carboxi-terminal domains, and an extracellular loop [1]. Hexameric connexin assemblies, known as connexons or hemichannels, dock with connexons from adjacent cells, to form intercellular channels, that put into contact the cytoplasms of neighboring cells. ...
Connexin 43 (Cx43) is essential for cardiac electrical coupling, but its effects on myocardial fibrosis is controversial. Here, we analyzed the role of Cx43 in myocardial fibrosis caused by angiotensin II (AngII) using Cx43fl/fl and Cx43Cre-ER(T)/fl inducible knock-out (Cx43 content: 50%) mice treated with vehicle or 4-hydroxytamoxifen (4-OHT) to induce a Cre-ER(T)-mediated global deletion of the Cx43 floxed allele. Myocardial collagen content was enhanced by AngII in all groups (n = 8–10/group, p < 0.05). However, animals with partial Cx43 deficiency (vehicle-treated Cx43Cre-ER(T)/fl) had a significantly higher AngII-induced collagen accumulation that reverted when treated with 4-OHT, which abolished Cx43 expression. The exaggerated fibrotic response to AngII in partially deficient Cx43Cre-ER(T)/fl mice was associated with enhanced p38 MAPK activation and was not evident in Cx43 heterozygous (Cx43+/-) mice. In contrast, normalization of interstitial collagen in 4-OHT-treated Cx43Cre-ER(T)/fl animals correlated with enhanced MMP-9 activity, IL-6 and NOX2 mRNA expression, and macrophage content, and with reduced -SMA and SM22 in isolated fibroblasts. In conclusion, our data demonstrates an exaggerated, p38 MAPK-dependent, fibrotic response to AngII in partially deficient Cx43Cre-ER(T)/fl mice, and a paradoxical normalization of collagen deposition in animals with an almost complete Cx43 ablation, an effect associated with increased MMP-9 activity and inflammatory response and reduced fibroblasts differentiation.
... Gap junctions (GJs) directly connect the cytoplasm of adjacent cells, mediating the intercellular transmission of signaling molecules. Six transmembrane connexin (Cx) monomers are arranged in a circle to form a hemichannel, and then two hemichannels from neighboring plasma membranes are docked to form the GJ (9,10). Cx expression is distinct in a variety of tissues, and Cx32 is the major GJ protein in hepatocytes (11,12). ...
Gap junctions (GJs), which are important plasma membrane channels for the transfer of signaling molecules between adjacent cells, have been implicated in drug-induced liver injury. However, the influence and the underlying mechanisms of GJs in propylthiouracil (PTU)-induced hepatotoxicity are unclear. In the present study, distinct manipulations were performed to regulate GJ function in the BRL-3A rat liver cell line. The results indicated that the toxic effect of PTU in BRL-3A cells was mediated by GJ intercellular communication, as cell death was significantly attenuated in the absence of functional GJ channels. Furthermore, the specific knockdown of connexin-32 (Cx32; a major GJ component protein in hepatocytes) using small interfering RNA was observed to decrease necrosis, intracellular PTU content and the level of reactive oxygen species (ROS) following PTU exposure. These observations demonstrated that suppressing GJ Cx32 could confer protection against PTU-induced cytotoxicity through decreasing the accumulation of PTU and ROS. To the best of our knowledge, the present study is the first to demonstrate the role and possible underlying mechanisms of GJs in the regulation of PTU-induced toxicity in BRL-3A rat liver cells.
... While other explanations of this property are possible, an impairment of adhesive function (Weber et al., 2002) seems the most likely. However, the fact that RS1 is a soluble molecule is potentially problematic because known cell-cell adhesion systems such as cadherins (Al-Amoudi et al., 2011), connexins (Sosinsky and Nicholson, 2005), and claudins (Haseloff et al., 2015) tend to have obvious membrane-embedded moieties. On the other hand, all these junctions are built from paired back-to-back oligomers, a property shared by RS1. ...
Mutations in the retinal protein retinoschisin (RS1) cause progressive loss of vision in young males, a form of macular degeneration called X-linked retinoschisis (XLRS). We previously solved the structure of RS1, a 16-mer composed of paired back-to-back octameric rings. Here, we show by cryo–electron microscopy that RS1 16-mers can assemble into extensive branched networks. We classified the different configurations, finding four types of interaction between the RS1 molecules. The predominant configuration is a linear strand with a wavy appearance. Three less frequent types constitute the branch points of the network. In all cases, the “spikes” around the periphery of the double rings are involved in these interactions. In the linear strand, a loop (usually referred to as spike 1) occurs on both sides of the interface between neighboring molecules. Mutations in this loop suppress secretion, indicating the possibility of intracellular higher-order assembly. These observations suggest that branched networks of RS1 may play a stabilizing role in maintaining the integrity of the retina.
... At the structural level, the basic topology of a connexin protein consists of four transmembrane segments (TM1-4), two extracellular loops (E1 and E2), and one cytoplasmic loop (CL), with both the N-and C-terminal domains facing the cytosol. The assembly of six connexin proteins forms a hemichannel that is trafficked to the plasma membrane, where it can dock with another hemichannel of an adjacent cell to form a gap junction channel (GJC; Goodenough et al., 1996;Gaietta et al., 2002;Sosinsky and Nicholson, 2005). Both unpaired he-michannels and GJCs are permeable to atomic ions and small molecules (Harris, 2001). ...
A group of human mutations within the N-terminal (NT) domain of connexin 26 (Cx26) hemichannels produce aberrant channel activity, which gives rise to deafness and skin disorders, including keratitis-ichthyosis-deafness (KID) syndrome. Structural and functional studies indicate that the NT of connexin hemichannels is folded into the pore, where it plays important roles in permeability and gating. In this study, we explore the molecular basis by which N14K, an NT KID mutant, promotes gain of function. In macroscopic and single-channel recordings, we find that the N14K mutant favors the open conformation of hemichannels, shifts calcium and voltage sensitivity, and slows deactivation kinetics. Multiple copies of MD simulations of WT and N14K hemichannels, followed by the Kolmogorov–Smirnov significance test (KS test) of the distributions of interaction energies, reveal that the N14K mutation significantly disrupts pairwise interactions that occur in WT hemichannels between residue K15 of one subunit and residue E101 of the adjacent subunit (E101 being located at the transition between transmembrane segment 2 [TM2] and the cytoplasmic loop [CL]). Double mutant cycle analysis supports coupling between the NT and the TM2/CL transition in WT hemichannels, which is disrupted in N14K mutant hemichannels. KS tests of the α carbon correlation coefficients calculated over MD trajectories suggest that the effects of the N14K mutation are not confined to the K15–E101 pairs but extend to essentially all pairwise residue correlations between the NT and TM2/CL interface. Together, our data indicate that the N14K mutation increases hemichannel open probability by disrupting interactions between the NT and the TM2/CL region of the adjacent connexin subunit. This suggests that NT–TM2/CL interactions facilitate Cx26 hemichannel closure.
... Gap junctions (GJ s) are bicellular structures that are formed by the conglomeration of several cell-cell channels at cell-to-cell contact sites (Loewenstein, 1981;Sosinsky and Nicholson, 2005). The direct exchange of small molecules (≤1500 Da) between the cytoplasmic interiors of contiguous cells through these channels maintains cytoplasmic continuity through buffering of nutrients and sharing of second messengers (Goodenough and Paul, 2009;Loewenstein, 1981). ...
Defects in assembly of gap junction-forming proteins, called connexins (Cxs), are observed in a variety of cancers. Connexin32 (Cx32; also known as GJB1) is expressed by the polarized cells in epithelia. We discovered two dileucine-based motifs, which govern the intracellular sorting and endocytosis of transmembrane proteins, in the C-terminal tail of Cx32 and explored their role in regulating its endocytosis and gap junction-forming abilities in pancreatic and prostate cancer cells. One motif, designated as LI, was located near the juxtamembrane domain, whereas the other, designated as LL, was located distally. We also discovered a non-canonical motif, designated as LR, in the C-terminal tail. Our results showed that rendering these motifs non-functional had no effect on the intracellular sorting of Cx32. However, rendering the LL or LR motif nonfunctional enhanced the formation of gap junctions by inhibiting Cx32 endocytosis by the clathrin-mediated pathway. Rendering the LI motif nonfunctional inhibited gap junction formation by augmenting the endocytosis of Cx32 via the LL and LR motifs. Our studies have defined distinct roles of these motifs in regulating the endocytosis of Cx32 and its gap junction-forming ability.
This article has an associated First Person interview with the first author of the paper.
... The connexins represent a family of integral membrane proteins that are able to form intercellular channels (Sosinsky and Nicholson, 2005). These channels assemble into distinct plasma membrane domains known as gap junctions, which can consist of up to several thousand connexin protein subunits. ...
Intercellular communication via gap junctions has an important role in controlling cell growth and in maintaining tissue homeostasis. Connexin43 is the most abundantly expressed gap junction channel protein in humans and acts as a tumor suppressor in multiple tissue types. Connexin43 is often dysregulated at the post-translational level during cancer development, resulting in loss of gap junctions. However, the molecular basis underlying the aberrant regulation of connexin43 in cancer cells has remained elusive. Here, we demonstrate that the oncogenic E3 ubiquitin ligase NEDD4 regulates the connexin43 protein level in HeLa cells, both under basal conditions and in response to protein kinase C activation. Furthermore, overexpression of NEDD4, but not a catalytically inactive form of NEDD4, was found to result in nearly complete loss of gap junctions and increased lysosomal degradation of connexin43 in both HeLa and C33A cervical carcinoma cells. Collectively, the data provide new insights into the molecular basis underlying the regulation of gap junction size and represent the first evidence that an oncogenic E3 ubiquitin ligase promotes loss of gap junctions and connexin43 degradation in human carcinoma cells.
... Each hemichannel is composed of six protein subunits termed connexins (CXs). About 20 CXs have been identified in mammalian tissues, and they usually form three types of gap junctions: 1) homotypic channels with two identical connexons composed of one type of CX subunit; 2) heterotypic channels with homomeric connexons, each containing a different type of CX; and 3) heterotypic channels with heteromeric connexons [3][4] . In lens, gap junctions are composed of three types of CXs: CX43 (or Gja1) is present in lens epithelial cells; CX46 (or Gja3) mainly expresses in the fiber cells; and CX50 (or Gja8) is present in epithelial and fiber cells [5][6][7] . ...
Aim:
To investigate the genetic mutations that are associated the hereditary autosomal dominant cataract in a Chinese family.
Methods:
A Chinese family consisting of 20 cataract patients (including 9 male and 11 female) and 2 unaffected individuals from 5 generations were diagnosed to be a typical autosomal dominant cataract pedigree. Genomic DNA samples were extracted from the peripheral blood cells of the participants in this pedigree. Exon sequence was used for genetic mutation screening. In silico analysis was used to study the structure characteristics of connexin 46 (CX46) mutant. Immunoblotting was conduceted for testing the expression of CX46.
Results:
To determine the involved genetic mutations, 11 well-known cataract-associated genes (cryaa, cryab, crybb1, crybb2, crygc, crygd, Gja3, Gja8, Hsf4, Mip and Pitx3) were chosen for genetic mutation test by using exon sequencing. A novel cytosine insertion at position 1195 of CX46 cDNA (c.1194_1195ins C) was found in the samples of 5 tested cataract patients but not in the unaffected 2 individuals nor in normal controls, which resulted in 30 amino acids more extension in CX46C-terminus (cx46fs400) compared with the wild-type CX46. In silico protein structure analysis indicated that the mutant showed distinctive hydrophobicity and protein secondary structure compared with the wild-type CX46. The immunoblot results revealed that CX46 protein, which expressed in the aging cataract lens tissues, was absence in the proband lens. In contrast, CX50, alpha A-crystallin and alphaB-crystallin expressed equally in both proband and aging cataract tissues. Those results revealed that the cx46fs400 mutation could impair CX46 protein expression.
Conclusion:
The insertion of cytosine at position 1195 of CX46 cDNA is a novel mutation site that is associated with the autosomal dominant cataracts in this Chinese family. The C-terminal frameshift mutation is involved in regulating CX46 protein expression.
... Gap junction channel gating is tightly regulated by numerous stimuli, such as changes in voltage, pH, and calcium concentration, as previously reviewed by Nielsen et al. [90] and Sosinsky and Nicholson [91]. In addition, the gating of Cx43 gap junction channels is controlled by phosphorylation at serine or tyrosine residues in the Cx43 CT by multiple kinases, as described in Section 3.1. ...
... Gap junctions consist of arrays of channels composed of proteins known as connexins [165]. Gap junction channels serve to interconnect adjacent cells by creating gated high-conductance pores that enable the passive diffusion of small molecules, water, and ions from the cytoplasm of one cell to another [166]. ...
Lung epithelial cells are oriented in order to properly function. Top-bottom orientation, called the apical–basolateral polarity axis, is determined by several cues in the cell microenvironment. Particularly critical are contacts between cells that establish a demarcation zone that defines the border between the apical and basolateral elements of the plasma membrane. This contact zone, called the apical junctional complex (AJC), is an area where the polarity complex proteins interlink with tight junctions, adherens junctions, and gap junctions. Each of these junctions has unique composition and function, moreover, their organization and function is coordinated. Junctions also serve as signaling platforms that regulate the traffic of transcription factors between the plasma membrane and nucleus. This chapter focuses on AJC components that have been best characterized for lung epithelial cells: adherens junctions, that serve as initiators of junction formation and sites that coordinate the actin cytoskeleton, tight junctions that regulate paracellular permeability of small solutes and ions through cell contact sites and gap junctions, communicating junctions that enable cytosolic molecules and ions to transfer from one cell to another.
... Gap junctions consist of arrays of channels composed of proteins known as connexins [165]. Gap junction channels serve to interconnect adjacent cells by creating gated high-conductance pores that enable the passive diffusion of small molecules, water, and ions from the cytoplasm of one cell to another [166]. ...
Background and aim:
A murine model mimicking osmotic demyelination syndrome (ODS) revealed with histology in the relay posterolateral (VPL) and ventral posteromedial (VPM) thalamic nuclei adjoined nerve cell bodies in chronic hyponatremia, amongst the damaged 12 h and 48 h after reinstatement of osmolality. This report aims to verify and complement with ultrastructure other neurophysiology, immunohistochemistry, and molecular biochemistry data to assess the connexin-36 protein, as part of those hinted close contacts.This ODS investigation included four groups of mice: Sham (NN; n = 13), hyponatremic (HN; n = 11), those sacrificed 12 h after a fast restoration of normal natremia (ODS12h; n = 6) and mice sacrificed 48 h afterward, or ODS48 h (n = 9). Out of these, thalamic zones samples included NN (n = 2), HN (n = 2), ODS12h (n = 3) and ODS48h (n = 3).
Results:
Ultrastructure illustrated junctions between nerve cell bodies that were immunolabeled with connexin36 (Cx36) with light microscopy and Western blots. These cell's junctions were reminiscent of low resistance junctions characterized in other regions of the CNS with electrophysiology. Contiguous neurons showed neurolemma contacts in intact and damaged tissues according to their location in the ODS zones, at 12 h and 48 h post correction along with other demyelinating alterations. Neurons and ephaptic contact measurements indicated the highest alterations, including nerve cell necrosis in the ODS epicenter and damages decreased toward the outskirts of the demyelinated zone.
Conclusion:
Ephapses contained C × 36between intact or ODS injured neurons in the thalamus appeared to be resilient beyond the core degraded tissue injuries. These could maintain intercellular ionic and metabolite exchanges between these lesser injured regions and, thus, would partake to some brain plasticity repairs.
Gap junctions are specialized regions of the plasma membrane containing clusters of channels that provide for the diffusion of ions and small molecules between adjacent cells. A fundamental role of gap junctions is to coordinate the functions of cells in tissues. Cancer pathogenesis is usually associated with loss of intercellular communication mediated by gap junctions, which may affect tumor growth and the response to radio- and chemotherapy. Gap junction channels consist of integral membrane proteins termed connexins. In addition to their canonical roles in cell-cell communication, connexins modulate a range of signal transduction pathways via interactions with proteins such as β-catenin, c-Src, and PTEN. Consequently, connexins can regulate cellular processes such as cell growth, migration, and differentiation through both channel-dependent and independent mechanisms. Gap junctions are dynamic plasma membrane entities, and by modulating the rate at which connexins undergo endocytosis and sorting to lysosomes for degradation, cells rapidly adjust the level of gap junctions in response to alterations in the intracellular or extracellular milieu. Current experimental evidence indicates that aberrant trafficking of connexins in the endocytic system is intrinsically involved in mediating the loss of gap junctions during carcinogenesis. This review highlights the role played by the endocytic system in controlling connexin degradation, and consequently gap junction levels, and discusses how dysregulation of these processes contributes to the loss of gap junctions during cancer development. We also discuss the therapeutic implications of aberrant endocytic trafficking of connexins in cancer cells.
Exosomes are nanosized vesicles that carry biologically diverse molecules for intercellular communication. Researchers have been trying to engineer exosomes for therapeutic purposes by using different approaches to deliver biologically active molecules to the various target cells efficiently. Recent technological advances may allow the biodistribution and pharmacokinetics of exosomes to be modified to meet scientific needs with respect to specific diseases. However, it is essential to determine an exosome's optimal dosage and potential side effects before its clinical use. Significant breakthroughs have been made in recent decades concerning exosome labelling and imaging techniques. These tools provide in situ monitoring of exosome biodistribution and pharmacokinetics and pinpoint targetability. However, because exosomes are nanometres in size and vary significantly in contents, a deeper understanding is required to ensure accurate monitoring before they can be applied in clinical settings. Different research groups have established different approaches to elucidate the roles of exosomes and visualize their spatial properties. This review covers current and emerging strategies for in vivo and in vitro exosome imaging and tracking for potential studies. image
Heart failure (HF) is the end stage of various cardiovascular diseases, with high morbidity and mortality, and is associated with a poor prognosis. One of the primary causes of HF is aortic valve disease, manifested by progressive aortic valve stenosis (AVS), resulting in increased left ventricular load, ventricular hypertrophy, ultimately ventricular dysfunction, and HF. Early assessment of the degree of cardiomyopathy and timely intervention is expected to improve patients’ cardiac function and delay or even avoid the occurrence of HF. The Wnt signaling pathway is mainly involved in regulating myocardial insufficiency after valve stenosis. Connexin 43 protein (Cx43) is an essential target of Wnt signaling pathway that forms gap junction (GJ) structures and is widely distributed in various organs and tissues, especially in the heart. The distribution and transformation of Cx43 among cardiac cells are crucial for the development of HF. To specifically label Cx43 in vivo, we established a new Cx43-BFP-GFP mouse model with two loxp sites on both sides of the tag BFP-polyA box, which can be removed by Cre recombination. This double-reporter line endowed us with a powerful genetic tool for determining the area, spatial distribution, and functional status of Cx43. It also indicated changes in electrical conduction between cells in a steady or diseased state.
Electrical synapses are established between specific neurons and within distinct subcellular compartments, but the mechanisms that direct gap junction assembly in the nervous system are largely unknown. Here, we show that a developmental program tunes cAMP signaling to direct the neuron-specific assembly and placement of electrical synapses in the C. elegans motor circuit. We use live-cell imaging to visualize electrical synapses in vivo and an optogenetic assay to confirm that they are functional. In ventral A class (VA) motor neurons, the UNC-4 transcription factor blocks expression of cAMP antagonists that promote gap junction miswiring. In unc-4 mutants, VA electrical synapses are established with an alternative synaptic partner and are repositioned from the VA axon to soma. cAMP counters these effects by driving gap junction trafficking into the VA axon for electrical synapse assembly. Thus, our experiments establish that cAMP regulates gap junction trafficking for the biogenesis of functional electrical synapses.
In humans, protoplasmic astrocytes are found with high density in the cortex and hippocampus (both parts responsible for memory and cognition) and each astrocyte is in contact with two million of synapses. Astrocytes are responsible for the synaptogenesis and the removal of synapses, which is the basis of learning and memory. The synaptogenesis increases sevenfold with the addition of astrocytes to the neuronal culture media. Astrocytes divide the brain and spinal cord into separate domains, including neurons, synaptic terminals and blood vessels and are integrated by protoplasmic astrocytic appendages. Astrocytes are also responsible for the formation of a single, wide lattice called the syncytium, which can store and process a high volume of information and transmit voluminous messages through intracellular gap junctions. It is relatively slow and is in turn a reason for thinking and gradual use of the information stored in memory. Astrocytes are mainly involved in the intercellular diffusion of calcium signals and the tripartite synapses of neurons and astrocytes are more in the gray matter; in most cases, the astrocyte membrane completely covers the pre-synaptic and postsynaptic ends. Similar to neurons, astrocytes also exhibit cellular memory and connect and integrate with neurons both homocellularly and heterocellularly. New circuits are formed far from the damaged site with the degeneration of brain tissues, the brain adaptation process and replacement of astrocytes and long-term memory is preserved with the cooperation of astrocytes close to the lesion site. Cognitive decline is evident in aging and research shows that there is no obvious neuronal death while the death of astrocytes is evident in aging. The volume of astroglia cells decreases in schizophrenia, which is accompanied by impaired limen and cognition and radiotherapy of glioma causes disorders and reduces cognition and memory. In Alzheimer's, the hippocampus is destroyed together with thinning the site of contact with the anterior cortex of the forehead (brain scans).
Gap junctions consist of arrays of intercellular channels that enable adjacent cells to communicate both electrically and metabolically. Gap junctions have a wide diversity of physiological functions, playing critical roles in both excitable and non-excitable tissues. Gap junction channels are formed by integral membrane proteins called connexins. Inherited or acquired alterations in connexins are associated with numerous diseases, including heart failure, neuropathologies, deafness, skin disorders, cataracts and cancer. Gap junctions are highly dynamic structures and by modulating the turnover rate of connexins, cells can rapidly alter the number of gap junction channels at the plasma membrane in response to extracellular or intracellular cues. Increasing evidence suggests that ubiquitination has important roles in the regulation of endoplasmic reticulum-associated degradation of connexins as well as in the modulation of gap junction endocytosis and post-endocytic sorting of connexins to lysosomes. In recent years, researchers have also started to provide insights into the physiological roles of connexin ubiquitination in specific tissue types. This review provides an overview of the advances made in understanding the roles of connexin ubiquitination in the regulation of gap junction intercellular communication and discusses the emerging physiological and pathophysiological implications of these processes.
The use of antibiotics in diets has been restricted in several countries as a precautionary measure to avoid development of antibiotic resistance among pathogenic microorganisms. This regulation promoted the exploration of natural plant bioactive compounds (PBCs) as feed additives to improve productivity, welfare and health of livestock and poultry. Along with several beneficial attributes of PBCs, including antimicrobial, antioxidant and various pharmacological effects, they also improve barrier function and nutrient transport in the gastrointestinal (GI) tract. This comprehensive review discusses the effects of different PBCs on the integrity, nutrient transport and permeability of GI epithelia and their mechanism of actions. Dietary PBCs influence the maintenance and enhancement of GI integrity via a number of mechanisms including altered signaling pathways and expression of several tight junction proteins (claudins, occludin, and zonula occludens proteins), altered expression of various cytokines, chemokines, complement components and their transcription factors, goblet cell abundance and mucin gene expression, and the modulation of the cellular immune system. They also affect nutrient transporter gene expression and active absorption of nutrients, minerals and ammonia. One intriguing perspective is to select an effective dose at which a specific PBC could improve GI barrier function and nutrient absorption. The effective doses and clear-cut molecular mechanisms for PBCs are yet to be elucidated to understand discrepant observations among different studies and to improve the targeted biotechnological and pharmaceutical uses of PBCs in farm animals. The latter will also enable a more successful use of such PBCs in humans.
Connexins are ubiquitous channel forming proteins that assemble as plasma membrane hemichannels and as intercellular gap junction channels that directly connect cells. In the heart, gap junction channels electrically connect myocytes and specialized conductive tissues to coordinate the atrial and ventricular contraction/relaxation cycles and pump function. In blood vessels, these channels facilitate long-distance endothelial cell communication, synchronize smooth muscle cell contraction, and support endothelial-smooth muscle cell communication. In the central nervous system they form cellular syncytia and coordinate neural function. Gap junction channels are normally open and hemichannels are normally closed, but pathologic conditions may restrict gap junction communication and promote hemichannel opening, thereby disturbing a delicate cellular communication balance. Until recently, most connexin-targeting agents exhibited little specificity and several off-target effects. Recent work with peptide-based approaches has demonstrated improved specificity and opened avenues for a more rational approach toward independently modulating the function of gap junctions and hemichannels. We here review the role of connexins and their channels in cardiovascular and neurovascular health and disease, focusing on crucial regulatory aspects and identification of potential targets to modify their function. We conclude that peptide-based investigations have raised several new opportunities for interfering with connexins and their channels that may soon allow preservation of gap junction communication, inhibition of hemichannel opening, and mitigation of inflammatory signaling. © 2017 by The American Society for Pharmacology and Experimental Therapeutics.
Neuroendocrine cells are a unique type of neuron that release hormones, often peptides, into the blood. Because these hormones typically govern significant phenomena, such as metabolism, reproduction, or survival, their secretion needs to be well timed. Consequently, the cells of most neuroendocrine systems are linked by electrical coupling or electrical synapses. These connections are made by gap junction proteins (vertebrate connexins or invertebrate innexins), which form intercellular channels that pass small ions (ionic current) and larger solutes between cells. Electrically coupled neuroendocrine cells are usually united by nonrectifying, voltage-independent junctions that promote the synchronization of both action potential firing and hormone release. By comparing and contrasting different organisms and tissues, this chapter attempts to demonstrate that electrical synapses are a common means of ensuring proper neuroendocrine connectivity and function.
We have proposed that the gap junctions between amphibian blastomeres are comprised of voltage-sensitive channels. The kinetic properties of the junctional conductance are here studied under voltage damp. When the transjunctional voltage is stepped to a new voltage of the same polarity, the junctional conductance changes as a single exponential to a steady-state level. The time constant of the conductance change is determined by the existing transjunctional voltage and is independent of the previous voltage. For each voltage polarity, the relations between voltage, time constant, and steady-state conductance are well modeled by a reversible two-state reaction scheme in which the calculated rate constants for the transitions between the states are exponential functions of voltage. The calculated rate constant for the transition to the low-conductance state is approximately twice as voltage dependent as that for the transition to the high-conductance state. When the transjunctional voltage polarity is reversed, the junctional conductance undergoes a transient recovery. The polarity reversal data are well modeled by a reaction scheme in which the junctional channel has two gates, each with opposite voltage sensitiv- ity, and in which an open gate may close only if the gate in series with it is open. A simple explanation for this contingent gating is a mechanism in which each gate senses only the local voltage drop within the channel.
Micrographs of isolated gap junction specimens, negatively stained with one molybdate, three tungstate and three uranyl stains, were recorded at low and high irradiation. Fourier-averaged images of the negatively stained gap junctions have been self-consistently scaled to identify conserved and variable features. Intrinsic features in the hexagonally averaged images have been distinguished from residual noise by statistical comparisons among similarly prepared specimens. The cationic uranyl stains can penetrate the axial connexon channel, whereas the anionic stains are largely excluded; these observations indicate that the channel is negatively charged. Variability in the extent of the axial stain penetration, and enhancement of this staining by radiation damage and heating may be accounted for by a leaky, labile channel gate. The peripheral stain concentrations marking the perimeter of the skewed, six-lobed connexon image and the stain-excluding region at the 3-fold axis of the lattice, which are seen only under conditions of low irradiation with both anionic and cationic stains, are identified as intrinsic features of the isolated gap junction structure. The stain concentrations located ~30 Å from the connexon center appear to be symmetrically related on opposite sides of the junction by non-crystallographic 2-fold axes oriented ~8 ° to the lattice axes at the plane of the gap. The radiation-sensitive hexagonal features seen in the negatively stained images may correspond to substructure on the cytoplasmic surfaces of the paired gap junction membranes.
The unitary conductances and permeability sequences of the rat connexin40 (rCx40) gap junction channels to seven monovalent cations and anions were studied in rCx40-transfected neuroblastoma 2A (N2A) cell pairs using the dual whole cell recording technique. Chloride salt cation substitutions (115 mM principal salt) resulted in the following junctional maximal single channel current-voltage relationship slope conductances (γj in pS): CsCl (153), RbCl (148), KCl (142), NaCl (115), LiCl (86), TMACl (71), TEACl (63). Reversible block of the rCx40 channel was observed with TBA. Potassium anion salt γj are: Kglutamate (160), Kacetate (160), Kaspartate (158), KNO3 (157), KF (148), KCl (142), and KBr (132). Ion selectivity was verified by measuring reversal potentials for current in rCx40 gap junction channels with asymmetric salt solutions in the two electrodes and using the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The permeabilities relative to Li+ are: Cs+ (1.38), Rb+ (1.32), K+ (1.31), Na+ (1.16), TMA+ (0.53), TEA+ (0.45), TBA+ (0.03), Cl− (0.19), glutamate− (0.04), and NO3− (0.14), assuming that the monovalent anions permeate the channel by forming ion pairs with permeant monovalent cations within the pore thereby causing proportionate decreases in the channel conductance. This hypothesis can account for why the predicted increasing conductances with increasing ion mobilities in an essentially aqueous channel were not observed for anions in the rCx40 channel. The rCx40 effective channel radius is estimated to be 6.6 Å from a theoretical fit of the relationship of relative permeability and cation radius.
The scanning tunneling microscope is proposed as a method to measure forces as small as 10-18 N. As one application for this concept, we introduce a new type of microscope capable of investigating surfaces of insulators on an atomic scale. The atomic force microscope is a combination of the principles of the scanning tunneling microscope and the stylus profilometer. It incorporates a probe that does not damage the surface. Our preliminary results in air demonstrate a lateral resolution of 30 ÅA and a vertical resolution less than 1 Å.
Suppression of gap-junctional communica- tion by various protein kinases, growth factors, and on- cogenes frequently correlates with enhanced mitogene- sis. The oncogene v -src appears to cause acute closure of gap junction channels. Tyr265 in the COOH-terminal tail of connexin 43 (Cx43) has been implicated as a po- tential target of v -src , although v -src action has also been associated with changes in serine phosphoryla- tion. We have investigated the mechanism of this acute regulation through mutagenesis of Cx43 expressed in Xenopus laevis oocyte pairs. Truncations of the COOH- terminal domain led to an almost complete loss of response of Cx43 to v -src , but this was restored by coex- pression of the independent COOH-terminal polypep- tide. This suggests a ball and chain gating mechanism, similar to the mechanism proposed for pH gating of Cx43, and K 1 channel inactivation. Surprisingly, we found that v -src mediated gating of Cx43 did not re- quire the tyrosine site, but did seem to depend on the presence of two potential SH3 binding domains and the mitogen-activated protein (MAP) kinase phosphoryla- tion sites within them. Further point mutagenesis and pharmacological studies in normal rat kidney (NRK) cells implicated MAP kinase in the gating response to v -src , while the stable binding of v -src to Cx43 (in part mediated by SH3 domains) did not correlate with its ability to mediate channel closure. This suggests a common link between closure of gap junctions by v -src and other mitogens, such as EGF and lysophosphatidic acid (LPA).
Gap junctions are formed by a family of homologous proteins termed
connexins. Their channels are dodecamers, and homomeric forms differ in
their properties with respect to control by voltage and other gating
stimuli. We report here the properties of coupling from expression of
connexin complementary RNAs (cRNAs; sense to mRNA, antisense to cDNA) in
Xenopus oocyte pairs in which endogenous coupling was blocked by
injection of DNA oligonucleotides antisense to the mRNA of Cx38, the
principal endogenous connexin. We found that a connexin recently
sequenced from rat liver, Cx26, formed functional gap junctions whose
conductance exhibited voltage dependence with unusual characteristics
suggestive of two gating mechanisms. Junctional conductance (g_j) was
increased to a small degree by depolarization and decreased by
hyperpolarization of either cell in a coupled pair, indicating
dependence on the potential between the inside and outside of the cells
(Vi-o). These changes were fast compared with the resolution
of their measurement (ca. 10 ms). On a slower timescale, large
transjunctional potentials (V_j) of either sign caused a more
substantial decrease in conductance similar to that previously reported
for several other gap junctions. Homotypic junctions formed of another
connexin, Cx32, exhibited a similar slow dependence on V_j but no
dependence on Vi-o. In contrast, heterotypic junctions
between an oocyte expressing Cx26 and one expressing Cx32 were
electrically asymmetric; they exhibited a greater fast change in g_j,
which depended, however, on V_j, such that g_j increased with relative
positivity on the Cx26 side and decreased with relative negativity on
the Cx26 side. There was also a large slow decrease in g_j in response
to V_j for relative positivity on the Cx26 side but not for V_j of the
opposite sign. These data indicate that properties of the hemichannels
contributed by the two connexins in the heterotypic case were changed
from their properties in homotypic junctions. The fast change in g_j may
involve a mechanism analogous to that at fast rectifying electrical
synapses. Experiments in which oocytes expressing Cx32 were paired with
oocytes expressing both Cx26 and Cx32 demonstrated that asymmetric
junctions would form between oocytes expressing both connexins, thereby
confirming their potential relevance in vivo, where the same coupled
cells are known to express both proteins.
Direct intercellular communication occurs through specialized channels, which are formed by the interaction of two half-channels, or connexons, contributed by each of the two participating cells. The ability to establish intercellular communication is specified, in part, by the expression of different structural proteins, termed connexins. Connexins can control the establishment of intercellular communication by selectively pairing with some but not other family members. To characterize the protein domains that allow connexins to recognize and discriminate between alternative partners, we have created chimeras composed of selected regions of rat connexin43, which forms channels with Xenopus connexin38, and rat connexin32, which cannot. Pairs of Xenopus oocytes were used to test the ability of the chimeras to form homotypic channels with themselves, and heterotypic channels with the parent connexins or with endogenous Xenopus connexin38. While all hybrid molecules tested were efficiently expressed by oocytes, most were devoid of functional activity. A chimera consisting of connexin32 from the N terminus to the second transmembrane domain, fused to connexin43 from the middle cytoplasmic loop to the C terminus, designated as 3243H4, was able to pair functionally with Xenopus connexin38 and one of its parent connexins, connexin43. Voltage-dependent closure of heterotypic channels containing 3243H4 was asymmetric, exhibited novel characteristics that were not predicted by the behavior of the parent connexins and was dependent on the type of connexin with which 3243H4 was paired. In contrast, 3243H4 was unable to form functional channels with either itself or the other parent, connexin32. Together, these results suggest that these connexins are not composed of functionally exchangeable regions and that multiple domains, namely the middle cytoplasmic portion and the second extracellular domain, can influence the interactions between connexins present in adjacent cells. Furthermore, they indicate that voltage gating is not strictly intrinsic behavior for a given connexin, but can be modulated by the partner connexins to which they are paired. Finally, the finding that 3243H4 is functional only in heterotypic configurations, and cannot form homotypic channels, suggests the existence of a novel form of selectivity: self-discrimination. The latter property may represent another mechanism that operates to control the extent of communication between cells.
In Differentiated Service (DiffServ) networks, the routing algorithms used by the premium class traffic, due to the high priority afforded to that traffic, may have a significant impact not only on the premium class traffic itself, but on all other classes of traffic as well. The shortest hop-count routing scheme, used in current Internet, turns out to be no longer sufficient in DiffServ networks. This paper studies the problem of finding optimal routes for the premium-class traffic in a DiffServ domain, such that (1) no forwarding loop exists in the entire network in the context of hop-by-hop routing; and (2) the residual bandwidth on bottleneck links is maximized. This problem is called the Optimal Premium-class Routing (OPR) problem. We prove in this paper that the OPR problem is NP-hard.To handle the OPR problem, first, we analyze the strength and weaknesses of two existing algorithms (Widest-Shortest-Path algorithm and Bandwidth-inversion Shortest-Path algorithm). Second, we propose a novel heuristic algorithm, called the Enhanced Bandwidth-inversion Shortest-Path (EBSP) algorithm. We prove theoretically the correctness of the EBSP algorithm, i.e., we show that it is consistent and loop-free. Our extensive simulations in different network environments show clearly that the EBSP algorithm performs better when routing the premium traffic in complex, heterogeneous networks.
The potassium channel from Streptomyces lividans is an integral membrane protein with sequence similarity to all known K+ channels, particularly in the pore region. X-ray analysis with data to 3.2 angstroms reveals that four identical subunits
create an inverted teepee, or cone, cradling the selectivity filter of the pore in its outer end. The narrow selectivity filter
is only 12 angstroms long, whereas the remainder of the pore is wider and lined with hydrophobic amino acids. A large water-filled
cavity and helix dipoles are positioned so as to overcome electrostatic destabilization of an ion in the pore at the center
of the bilayer. Main chain carbonyl oxygen atoms from the K+ channel signature sequence line the selectivity filter, which is held open by structural constraints to coordinate K+ ions but not smaller Na+ ions. The selectivity filter contains two K+ ions about 7.5 angstroms apart. This configuration promotes ion conduction by exploiting electrostatic repulsive forces to
overcome attractive forces between K+ ions and the selectivity filter. The architecture of the pore establishes the physical principles underlying selective K+ conduction.
X-ray crystallographic methods and electron microscope image analysis have been used to correlate the structure and the chemical composition of gap junction plaques isolated intact from mouse liver. The requirement that the interpretations of X-ray, electron microscope, and chemical measurements be consistent reduces the uncertainties inherent in the separate observations and leads to a unified picture of the gap junction structures. Gap junctions are built up of units called connexons that are hexagonally arrayed in the pair of connected cell membranes. X-ray diffraction and electron microscope measurements show that the lattice constant of this array varies from about 80 to 90 A. Analysis of electron micrographs of negatively stained gap junctions shows that there is significant short range disorder in the junction lattice. even though the long range order of the array is remarkably regular. Analysis of the disorder provides information about the nature of the intermolecular forces that hold the array together.
Models for the spatial distribution of protein, lipid and water in gap junction structures have been constructed from the results of the analysis of X-ray diffraction data described here and the electron microscope and chemical data presented in the preceding paper (Caspar, D. L. D., D. A. Goodenough, L. Makowski, and W.C. Phillips. 1977. 74:605-628). The continuous intensity distribution on the meridian of the X-ray diffraction pattern was measured, and corrected for the effects of the partially ordered stacking and partial orientation of the junctions in the X-ray specimens. The electron density distribution in the direction perpendicular to the plane of the junction was calculated from the meridional intensity data. Determination of the interference function for the stacking of the junctions improved the accuracy of the electron density profile. The pair-correlation function, which provides information about the packing of junctions in the specimen, was calculated from the interference function. The intensities of the hexagonal lattice reflections on the equator of the X-ray pattern were used in coordination with the electron microscope data to calculate to the two-dimensional electron density projection onto the plane of the membrane. Differences in the structure of the connexons as seen in the meridional profile and equatorial projections were shown to be correlated to changes in lattice constant. The parts of the junction structure which are variable have been distinguished from the invariant parts by comparison of the X-ray data from different specimens. The combination of these results with electron microscope and chemical data provides low resolution three- dimensional representations of the structures of gap junctions.
The gap junction channel mediates an important form of intercellular communication, but its detailed study is hindered by inaccessibility in situ. We show here that connexin32, the major protein composing junctional channels in rat liver, forms ion channels in single bilayer membranes. The properties of these reconstituted connexin32 channels are characterized and compared with those of gap junction channels. The demonstration that connexin32 forms channels in single membranes has implications for assembly and regulation of junctional channels, and permits detailed study of the gating, permeability and modulation of this channel-forming protein.
Calcium signaling in C6 glioma cells in culture was examined with digital fluorescence video microscopy. C6 cells express low levels of the gap junction protein connexin43 and have correspondingly weak gap junctional communication as evidenced by dye coupling (Naus, C. C. G., J. F. Bechberger, S. Caveney, and J. X. Wilson. 1991. Neurosci. Lett. 126:33-36). Transfection of C6 cells with the cDNA encoding connexin43 resulted in clones with increased expression of connexin43 mRNA and protein and increased dye coupling, as well as markedly reduced rates of proliferation (Zhu, D., S. Caveney, G. M. Kidder, and C. C. Naus. 1991. Proc. Natl. Acad. Sci. USA. 88:1883-1887; Naus, C. C. G., D. Zhu, S. Todd, and G. M. Kidder. 1992. Cell Mol. Neurobiol. 12:163-175). Mechanical stimulation of a single cell in a culture of non-transfected C6 cells induced a wave of increased intracellular calcium concentration ([Ca2+]i) that showed little or no communication to adjacent cells. By contrast, mechanical stimulation of a single cell in cultures of C6 clones expressing transfected connexin43 cDNA induced a Ca2+ wave that was communicated to multiple surrounding cells, and the extent of communication was proportional to the level of expression of the connexin43 cDNA. These results provide direct evidence that intercellular Ca2+ signaling occurs via gap junctions. Ca2+ signaling through gap junctions may provide a means for the coordinated regulation of cellular function, including cell growth and differentiation.
This paper reports methods we have developed to solubilize gap junction channels, or connexons, from isolated gap junctions and to purify them in milligram quantities. Two sources of material are used: rat liver gap junctions and gap junctions produced by infecting insect cells with a baculovirus containing the cDNA for human liver beta 1 protein (connexin 32). Complete solubilization is obtained with long chain detergents (lauryl dimethyl amineoxide, dodecyl maltoside) and requires high ionic strength and high pH as well as reducing conditions. The purification involves chromatography on hydroxylapatite and gel filtration on Superose 6. A homogeneous product is indicated by a single band on a silver-stained gel and a homogeneous population of doughnut-shaped particles under the electron microscope. These particles have hexameric symmetry. The purified connexons have a tendency to form aggregates: filaments and sheets. The filaments grow by end-to-end association of connexons and are nonpolar, suggesting that the connexons are paired as in the cell-to-cell channel. The sheets grow by lateral association of the filaments.
Paired intercellular transmembrane channels, termed connexons, comprised of hexameric assemblies of gap junction protein, were isolated and purified from rat liver by exploiting their resistance to either Sarkosyl detergent solubilization or alkali extraction. The secondary structures of the gap junction proteins prepared by these methods were compared by circular dichroism (CD) spectroscopy. Both the spectra and the calculated net secondary structures of the proteins obtained by the two isolation methods were different. The protein isolated by the Sarkosyl treatment was found to be approximately 50% alpha-helical, while protein isolated by alkali extraction had a lower helix content (approximately 40%). In both types of preparations, however, the helical content of the gap junction protein was sufficiently large to be consistent with an all-helical model for the membrane-spanning parts of the structure. CD spectroscopy was also used to examine the effects of proteolytic digestion of the cytoplasmic domain on the net secondary structure of the detergent-treated gap junction protein. The membrane-bound fragments had a slightly higher proportion of their residues that were alpha-helical in nature, suggesting that the transmembrane and/or intra-gap domains are indeed enriched in this type of secondary structure. This information constrains the range of models which can be realistically proposed for the channel structure.
A new member of the connexin gene family has been identified and designated rat connexin-31 (Cx31) based on its predicted molecular mass of 30,960 daltons. Cx31 is 270 amino acids long and is coded for by a single copy gene. It is expressed as a 1.7-kilobase mRNA that is detected in placenta, Harderian gland, skin, and eye. Cx31 is highly conserved and can be detected in species as distantly related to rat as Xenopus laevis. It exhibits extensive sequence similarity to the previously identified connexins, 58, 50, and 40% amino acid identity to Cx26, Cx32, and Cx43, respectively. When conservation of predicted phosphorylation sites is used to adjust the alignment of Cx31 to other connexins, a unique alignment of three predicted protein kinase C phosphorylation sites near the carboxyl terminus of Cx31 with three sites at the carboxyl terminus of Cx43 is revealed.
Gap junctions are formed by a family of homologous proteins termed connexins. Their channels are dodecamers, and homomeric forms differ in their properties with respect to control by voltage and other gating stimuli. We report here the properties of coupling from expression of connexin complementary RNAs (cRNAs; sense to mRNA, antisense to cDNA) in Xenopus oocyte pairs in which endogenous coupling was blocked by injection of DNA oligonucleotides antisense to the mRNA of Cx38, the principal endogenous connexin. We found that a connexin recently sequenced from rat liver, Cx26, formed functional gap junctions whose conductance exhibited voltage dependence with unusual characteristics suggestive of two gating mechanisms. Junctional conductance (gj) was increased to a small degree by depolarization and decreased by hyperpolarization of either cell in a coupled pair, indicating dependence on the potential between the inside and outside of the cells (Vi-o). These changes were fast compared with the resolution of their measurement (ca. 10 ms). On a slower timescale, large transjunctional potentials (Vj) of either sign caused a more substantial decrease in conductance similar to that previously reported for several other gap junctions. Homotypic junctions formed of another connexin, Cx32, exhibited a similar slow dependence on Vj but no dependence on Vi-o. In contrast, heterotypic junctions between an oocyte expressing Cx26 and one expressing Cx32 were electrically asymmetric; they exhibited a greater fast change in gj, which depended, however, on Vj, such that gj increased with relative positivity on the Cx26 side and decreased with relative negativity on the Cx26 side. There was also a large slow decrease in gj in response to Vj for relative positivity on the Cx26 side but not for Vj of the opposite sign. These data indicate that properties of the hemichannels contributed by the two connexins in the heterotypic case were changed from their properties in homotypic junctions. The fast change in gj may involve a mechanism analogous to that at fast rectifying electrical synapses. Experiments in which oocytes expressing Cx32 were paired with oocytes expressing both Cx26 and Cx32 demonstrated that asymmetric junctions would form between oocytes expressing both connexins, thereby confirming their potential relevance in vivo, where the same coupled cells are known to express both proteins.
The connexins form a family of membrane spanning proteins that assemble into gap junction channels. The biophysical properties of these channels are dependent upon the constituent connexin isoform. To begin identifying the molecular basis for gap junction channel behavior in the human heart, a tissue that expresses connexin43, we used site-directed mutagenesis to generate mutant cDNAs of human connexin43 with shortened cytoplasmic tail domains. Premature stop codons were inserted, resulting in proteins corresponding in length to the mammalian isoforms connexin32 and connexin26, which are expressed primarily in liver. All constructs restore intercellular coupling when they are transfected into SKHep1 cells, a human hepatoma line that is communication deficient. Whereas wild-type connexin43 transfectants display two distinct unitary conductance values of about 60 and 100 pS, transfectants expressing the mutant proteins, from which 80 and 138 amino acids have been deleted, exhibit markedly different single-channel properties, with unitary conductance values of about 160 and 50 pS, respectively. Junctional conductance of channels composed of wild-type connexin43 is less voltage-sensitive compared with transfectants expressing wild-type connexin32. However, neither of the connexin43 truncation mutants alters this relative voltage insensitivity. These results suggest that the cytoplasmic tail domain is an important determinant of the unitary conductance event of gap junction channels but not their voltage dependence. Furthermore, since the mutant connexins are missing several consensus phosphorylation sites, modification of these particular sites may not be required for membrane insertion or assembly of human connexin43 into functional channels.
The membrane topology of connexin32, a principal polypeptide of gap junctions in diverse cell types, has been studied in rat and mouse hepatocyte gap junctions using site-specific antisera raised against synthetic oligopeptides corresponding to amino acid sequences deduced from cDNA clones. Based on published hydropathicity maps and identified protease-sensitive cleavage sites, oligopeptides were synthesized corresponding to two hydrophilic domains of connexin32, one predicted to face the cytoplasm, the other predicted to be directed extracellularly. Antisera were raised to keyhole limpet hemocyanin conjugates of the oligopeptides and used to map the distribution of their antigens using indirect immunocytochemistry on isolated gap junctions. The results directly demonstrated the cytoplasmic orientation of an antigen contained within amino acids 98-124 of the connexin32 sequence. The extracellular space in intact, isolated gap junctions is too small to permit binding of antibody molecules, necessitating the experimental separation of the junctional membranes to expose their extracellular surfaces using a urea/alkali procedure. While an antigen contained within amino acids 164-189 was visualized on the extracellular surfaces of some of the separated junctional membranes, variability in the observations and in the splitting procedure left ambiguities concerning the biological relevance of the observations after the denaturing conditions necessary to separate the junctional membranes. Using a different approach, however, the antigen could be exposed in intact liver using a hypertonic disaccharide junction-splitting procedure. The period of time of antigen exposure at the cell surface appears to peak at 30 s and disappear by 2-4 min. Taken together, these data demonstrate the extracellular orientation of an antigen contained within amino acids 164-189, which may be involved in cell-cell interaction within the gap junction.
The transformed or normal phenotype of cultured normal rat kidney cells infected with a temperature-sensitive mutant of avian sarcoma virus is conditional on the temperature at which the cells are grown. Using dye injection techniques, we show that junction-mediated dye transfer is also temperature-sensitive. The extent and rate of transfer between infected cells grown at the transformation-permissive temperature (35 degrees C) is significantly reduced when compared to infected cells grown at the nonpermissive temperature (40.5 degrees C) or uninfected cells grown at either temperature. Infected cells subjected to reciprocal temperature shifts express rapid and reversible alterations of dye transfer capacities, with responses evident by 15 min and completed by 60 min for temperature shifts in either direction. These results suggest that altered junctional capacities may be fundamental to the expression of the ASV-induced, transformed phenotype.
This paper reports methods we have developed to solubilize gap junction channels, or connexons, from isolated gap junctions and to purify them in milligram quantities. Two sources of material are used: rat liver gap junctions and gap junctions produced by infecting insect cells with a baculovirus containing the cDNA for human liver beta 1 protein (connexin 32). Complete solubilization is obtained with long chain detergents (lauryl dimethyl amineoxide, dodecyl maltoside) and requires high ionic strength and high pH as well as reducing conditions. The purification involves chromatography on hydroxylapatite and gel filtration on Superose 6. A homogeneous product is indicated by a single band on a silver-stained gel and a homogeneous population of doughnut-shaped particles under the electron microscope. These particles have hexameric symmetry. The purified connexons have a tendency to form aggregates: filaments and sheets. The filaments grow by end-to-end association of connexons and are nonpolar, suggesting that the connexons are paired as in the cell-to-cell channel. The sheets grow by lateral association of the filaments.
X-ray diffraction patterns have been recorded from partially oriented specimens of gap junctions isolated from mouse liver and suspended in sucrose solutions of different concentration and thus of different electron density. Analysis of these diffraction patterns has shown that sucrose is excluded from the 6-fold rotation axis of the junction lattice for a length of about 100 Å. This indicates that the aqueous channel of the junctions is in the closed, high resistance state in these preparations. Mapping of the sucrose-accessible space in the junction indicates that the cross-sectional area of the channel entrance on the cytoplasmic side of the membrane could be up to five times larger than the area of the transmembrane channel. Sucrose does not penetrate more than 20 Å into the membrane along the channel. Apparently the aqueous channel, 8 to 10 Å in radius for most of its length, is narrowed or blocked by a small feature about 50 Å from the center of the gap. Very close interactions exist between the gap junction protein and the lipid polar head groups on the cytoplasmic surface of the membrane. In this region, the protein intercalates between the polar head groups. These results suggest that the gap junction protein may have a functional two-domain structure. One domain, with a molecular weight of about 15,000, spans one bilayer and half of the gap and is contained largely within a radius of 25 Å from the 6-fold axis. The second domain is smaller and occupies the cytoplasmic surface of the gap junction membrane. Trypsin digestion removes about 4000 Mrmr from the cytoplasmic surface domain of the junction protein. Most of the material susceptible to trypsin digestion is located more than 28 å from the 6-fold axis.
Gap junction membrane channels mediate electrical and metabolic coupling between adjacent cells. The structure of a recombinant
cardiac gap junction channel was determined by electron crystallography at resolutions of 7.5 angstroms in the membrane plane
and 21 angstroms in the vertical direction. The dodecameric channel was formed by the end-to-end docking of two hexamers,
each of which displayed 24 rods of density in the membrane interior, which is consistent with an α-helical conformation for
the four transmembrane domains of each connexin subunit. The transmembrane α-helical rods contrasted with the double-layered
appearance of the extracellular domains. Although not indicative for a particular type of secondary structure, the protein
density that formed the extracellular vestibule provided a tight seal to exclude the exchange of substances with the extracellular
milieu.
Calcium signaling in C6 glioma cells in culture was examined with digital fluorescence video microscopy. C6 cells express low levels of the gap junction protein connexin43 and have correspondingly weak gap junctional communication as evidenced by dye coupling (Naus, C. C. G., J. F. Bechberger, S. Caveney, and J. X. Wilson. 1991. Neurosci. Lett. 126:33-36). Transfection of C6 cells with the cDNA encoding connexin43 resulted in clones with increased expression of connexin43 mRNA and protein and increased dye coupling, as well as markedly reduced rates of proliferation (Zhu, D., S. Caveney, G. M. Kidder, and C. C. Naus. 1991. Proc. Natl. Acad. Sci. USA. 88:1883-1887; Naus, C. C. G., D. Zhu, S. Todd, and G. M. Kidder. 1992. Cell Mol. Neurobiol. 12:163-175). Mechanical stimulation of a single cell in a culture of non-transfected C6 cells induced a wave of increased intracellular calcium concentration ([Ca2+]i) that showed little or no communication to adjacent cells. By contrast, mechanical stimulation of a single cell in cultures of C6 clones expressing transfected connexin43 cDNA induced a Ca2+ wave that was communicated to multiple surrounding cells, and the extent of communication was proportional to the level of expression of the connexin43 cDNA. These results provide direct evidence that intercellular Ca2+ signaling occurs via gap junctions. Ca2+ signaling through gap junctions may provide a means for the coordinated regulation of cellular function, including cell growth and differentiation.
This chapter discusses the structure and biochemistry of gap junction. The conduit for intercellular communication is formed by connexons in register among closely apposed cells. Gap junction channels can be viewed as having a modular design. Homology in the transmembrane sequences suggests that the bundle of 24 α helices that form each connexon is likely be a common architecture for the transmembrane channel. Similarly, sequence homology in the extracellular domains suggests similar mechanisms for connexon–connexon interactions. The identification of specific functional sites or domains is critical for understanding the regulation of gap junction channels. Instead of blindly accumulating site mutations, a better strategy may be the generation of chimeric connexins where a loop domain of one connexin can be substituted for another. Currently, there are numerous areas for continued exploration of the structural and functional properties of gap junction channels—delineation of the precise interactions between different regions in the channel; the precise folding of the extracellular loops that confers stability but also specificity in connexon–connexon pairing; the identification and orientation of the transmembrane α helices in the 3D map; and specific mechanisms for gating. The regulation of gap junction channels is particularly fascinating as they manifest properties of both ligand-gated channels.
It is the current view that gap junctions are not only passive conduits of electrical charge but also dynamic filters that modulate the passage of molecular messages within the cellular network; hence, not only the presence but also the regulation of connexins is important for synchronous tissue function. Research has focused on the regulation of connexins by two separate factors: intracellular pH and insulin (or IGF) exposure. Regulation of gap junction conductance by factors such as pHi and cytokines may be critical in the development of lethal cardiac arrhythmias during ischemia. Growth factor-mediated regulation of intercellular communication may also be important for normal cardiogenesis. Finally, changes in pH sensitivity after connexin processing may allow the preservation of cell–cell communication in the lens. Understanding the molecular events controlling connexin regulation and developing means to manipulate their occurrence allow learning more about the importance of intercellular communication both in health and disease.
Models for the spatial distribution of protein, lipid and water in gap junction structures have been constructed from the results of the analysis of X-ray diffraction data described here and the electron microscope and chemical data presented in the preceding paper (Caspar, D. L. D., D. A. Goodenough, L. Makowski, and W.C. Phillips. 1977. 74:605-628). The continuous intensity distribution on the meridian of the X-ray diffraction pattern was measured, and corrected for the effects of the partially ordered stacking and partial orientation of the junctions in the X-ray specimens. The electron density distribution in the direction perpendicular to the plane of the junction was calculated from the meridional intensity data. Determination of the interference function for the stacking of the junctions improved the accuracy of the electron density profile. The pair-correlation function, which provides information about the packing of junctions in the specimen, was calculated from the interference function. The intensities of the hexagonal lattice reflections on the equator of the X-ray pattern were used in coordination with the electron microscope data to calculate to the two-dimensional electron density projection onto the plane of the membrane. Differences in the structure of the connexons as seen in the meridional profile and equatorial projections were shown to be correlated to changes in lattice constant. The parts of the junction structure which are variable have been distinguished from the invariant parts by comparison of the X-ray data from different specimens. The combination of these results with electron microscope and chemical data provides low resolution three- dimensional representations of the structures of gap junctions.
of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.
α-Bungarotoxin (α-BTX) is a highly toxic snake neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. In the following we review multi-phase research of the design, synthesis and structure analysis of peptides that bind α-BTX and inhibit its binding to AChR. Structure-based design concomitant with biological information of the α-BTX/AChR system yielded 13-mer peptides that bind to α-BTX with high affinity and are potent inhibitors of α-BTX binding to AChR (IC50 of 2 nM). X-Ray and NMR spectroscopy reveal that the high-affinity peptides fold into an anti-parallel β-hairpin structure when bound to α-BTX. The structures of the bound peptides and the homologous loop of acetylcholine binding protein, a soluble analog of AChR, are remarkably similar. Their superposition indicates that the toxin wraps around the binding-site loop, and in addition, binds tightly at the interface of two of the receptor subunits and blocks access of acetylcholine to its binding site. The procedure described in this article may serve as a paradigm for obtaining high-affinity peptides in biochemical systems that contain a ligand and a receptor molecule.
In the heart, individual cardiac muscle cells are linked by gap junctions. These junctions form low resistance pathways along which the electrical impulse flows rapidly and repeatedly between all the cells of the myocardium, ensuring their synchronous contraction. To obtain probes for mapping the distribution of gap junctions in cardiac tissue, polyclonal antisera were raised to three synthetic peptides, each matching different cytoplasmically exposed portions of the sequence of connexin43, the major gap-junctional protein reported in the heart. The specificity of each antiserum for the peptide to which it was raised was established by dot blotting. New methods were developed for isolating enriched fractions of gap junctions from whole heart and from dissociated adult myocytes, in which detergent-treatment and raising the temperature (potentially damaging steps in previously described techniques) are avoided. Analysis of these fractions by SDS-polyacrylamide gel electrophoresis revealed major bands at 43 kDa (matching the molecular mass of connexin43) and at 70 kDa. Western blot experiments using our antisera indicated that both the 43-kDa and the 70-kDa bands represent cardiac gap-junctional proteins. Pre-embedding immunogold labelling of isolated gap junctions and post-embedding immunogold labelling of Lowicryl-embedded whole tissue demonstrated the specific binding of the antibodies to ultrastructurally defined gap junctions. One antiserum (raised to residues 131–142) was found to be particularly effective for cytochemical labelling. Using this antiserum for immunofluorescence labelling in combination with confocal scanning laser microscopy enabled highly sensitive detection and three-dimensional mapping of gap junctions through thick slices of cardiac tissue. By means of the serial optical sectioning ability of the confocal microscope, images of the entire gap junction population of complete en face-viewed disks were reconstructed. These reconstructions reveal the presence of large junctions arranged as a peripheral ring around the disk, with smaller junctions in an interior zone: an arrangement that may facilitate efficient intercellular transfer of current. By applying our immunolabelling techniques to tissue from hearts removed from transplant patients with advanced ischaemic heart disease, we have demonstrated that gap junction distribution between myocytes at the border zone of healed infarcts is markedly disordered. This abnormality may contribute to the genesis of reentrant arrhythmias in ischaemic heart disease.
Gap junction channels are regulated by gates that close upon exposure to 100% CO2, probably via an increase in intracellular Ca2+ concentration, [Ca2+]i. For denning connexin (Cx) domain(s) involved in gating, we have studied chemical and voltage gating sensitivities of channels made of Cx38, Cx32 or chimeras of the above, expressed in Xenopus oocytes. Cx38 channels are more sensitive to CO2 and voltage than those of Cx32. A 3-min exposure to 100% CO2 reduces Cx38 junctional conductance (Gj) to 0% of initial values at a maximum rate of 25 %/min, whereas even a 15-min exposure to 100% CO2 reduces Cx32 Gj by approximately 50% at the slow rate of 9%/min. Of the various Cx32 mutants and Cx32/38 chimeras constructed, two chimeras (Cx32/38I and Cx32/38N) expressed functional channels. Upon exposure to CO2, channels made of Cx32/38I (Cx32 inner loop replaced with that of Cx38) reproduced precisely the uncoupling behavior of Cx38 channels in uncoupling magnitude and in both uncoupling and recoupling rates, whereas channels made of Cx32/38N (N-terminus replaced) behaved closer to Cx32 than to Cx38 channels. Cx38 channels were more voltage sensitive than those of Cx32, with V0, i.e., the transjunctional voltage at which voltage-sensitive conductance is half maximal = 35.3 and 59.5 mV, and n, i.e., equivalent gating charge =3.3 and 2.1, respectively. Of the two chimeras, Cx32/38I channels were similar to Cx38 channels, with V0 = 40.6 mV, Gj
min, i.e., the theoretical minimal normalized junctional conductance = 0.35 and n = 3.0, whereas Cx32/38 N channels displayed very low voltage sensitivity, with V0
= 84.8 mV, Gj
min = 0.5 and n = 1.1. The data suggest that the inner loop plays a major role in pH and voltage gating sensitivity, but whether other domains also participate in the gating mechanism cannot be excluded.
Gap junctions have been isolated from both rat heart and liver, tissues where junctions are typical in appearance and physiology. The purity of the fractions obtained was monitored by electron microscopy (thin-sectioning and negative staining) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The myocardial gap junctions are comprised of a single polypeptide of Mr 28,000, apparently derived from a protein of Mr 30,000. Hepatic gap junctions are also comprised of a single native protein of Mr 28,000 as previously reported. Exhaustive trypsin digestion of the isolated junctions cleaves both of these proteins similarly, while leaving their characteristic junctional lattice structures intact. However, comparison of heart and liver junctional proteins by two-dimensional peptide mapping of tryptic and α-chymotryptic fragments, followed by high pressure liquid chromatography, reveals no homology between these proteins.
The membrane topology and quaternary structure of rat cardiac gap junction ion channels containing α1, connexin (i.e. Cx43) have been examined using anti-peptide antibodies directed to seven different sites in the protein sequence, cleavage by an endogenous protease in heart tissue and electron microscopic image analysis of native and protease-cleaved two-dimensional membrane crystals of isolated cardiac gap junctions. Specificity of the peptide antibodies was established using dot immunoblotting, Western immunoblotting, immunofluorescence and immunoelectron microscopy. Based on the folding predicted by hydropathy analysis, five antibodies were directed to sites in cytoplasmic domains and two antibodies were directed to the two extracellular loop domains. Isolated gap junctions could not be labeled by the two extracellular loop antibodies using thin-section immunogold electron microscopy. This is consistent with the known narrowness of the extracellular gap region that presumably precludes penetration of antibody probes. However, cryo-sectioning rendered the extracellular domains accessible for immunolabeling. A cytoplasmic “loop” domain of at least Mr = 5100 (residues (101 to 142) is readily accessible to peptide antibody labeling. The native Mr = 43,000 protein can be protease-cleaved on the cytoplasmic side of the membrane, resulting in an Mr ≈ 30,000 membrane-bound fragment. Western immunoblots showed that protease cleavage occurs at the carboxy tail of the protein, and the cleavage site resides between amino acid residues 252–271. Immunoelectron microscopy demonstrated that the Mr ≈ 13,000 carboxy-terminal peptide(s) is released after protease cleavage and does not remain attached to the Mr ≈ 30,000 membrane-bound fragment via non-covalent interactions. Electron microscopic image analysis of two-dimensional membrane crystals of cardiac gap junctions revealed that the ion channels are formed by a hexagonal arrangement of protein subunits. This quaternary arrangement is not detectably altered by protease cleavage of the α1 polypeptide. Therefore, the Mr ≈ 13,000 carboxy-terminal domain is not involved in forming the transmembrane ion channel. The similar hexameric architecture of cardiac and liver gap junction connexons indicates conservation in the molecular design of the gap junction channels formed by α or β connexins.
A theory of dislocation-mediated melting in two dimensions is described
in detail, with an emphasis on results for triangular lattices on
both smooth and periodic substrates. The transition from solid to
liquid on a smooth substrate takes place in two steps with increasing
temperatures. Dissociation of dislocation pairs first drives a transition
out of a low-temperature solid phase, with algebraic decay of translational
order and long-range orientational order. This transition is into
a "'liquid-crystal"' phase characterized by exponential decay of
translational order, but power-law decay of sixfold orientational
order. Dissociation of disclination pairs at a higher temperature
then produces an isotropic fluid. The behavior of the specific heat,
structure factor, and various elastic constants near these transitions
is worked out. We also discuss the applicability of our results to
melting on a periodic substrate. Dislocation unbinding should describe
melting of a "'floating"' (and, in general, incommensurate) adsorbate
solid into a high-temperature fluid phase. The orientation bias imposed
by the substrate can alter or eliminate the disclination-unbinding
transition, however. Transitions from a floating solid into a low-temperature
registered or partially registered phase can also be mapped onto
the dislocation-unbinding transition, but only at certain special
values of the coverage. Substrate reciprocallattice vectors play
the role of Burger's vectors in this case.
Intercellular communication via gap junction membrane channels cannot occur until two apposing hemichannels (connexons) meet and dock to form a sealed cell-cell conduit. In particular, an important question is how does the structure at the extracellular surface influence the molecular recognition of the two connexons. In this study, cryoelectron microscopy and computer modeling provide evidence that the formation of the gap junction intercellular channel requires a 30 degree rotation between hemichannels for proper docking. With this amount of rotation, the peaks (protrusions) on one connexon fit into the valleys of the apposed connexon in the 3-D model, which would make for an ionically tight interface necessary for a functional cell-cell channel. Docking appears to be governed by a "lock and key" mechanism via a simple interdigitation of the six protrusions from each connexon. This interdigitation increases significantly the contact surface area and potential number of hydrogen bonds or hydrophobic interactions and/or other attractive interactions. Having a larger surface area than if the surfaces were flat would explain the biochemical requirements for conditions characterized previously for splitting of channels into hemichannels. The docked connexons were computationally fitted into two gap junction structures, which further confirmed the interdigitated manner of docking.
Gap junctional communication has been implicated in numerous cellular processes. However, the repertoire of specific transjunctional substances which mediate these processes remains relatively unexplored. A few selected secondary messengers have been identified, at least indirectly (e.g., cAMP and IP3) and phenotypic complementation experiments have indicated that gap junctions enable communicating cells to distribute nucleotide pools as a shared resource. The latter would include high energy compounds such as ADP and ATP, allowing cells to share energy resources. We have utilized a nonbiased process to directly capture, identify, and quantify transjunctional compounds from C6 glioma cells, the transformed phenotype of which has been ameliorated by transfection with connexin43 (Cx43). This technique involves the direct isolation, identification, and quantitation of radioactive transjunctional molecules that travel from metabolically labeled "donor" cells to "receiver" cells. This report demonstrates that ADP and/or ATP represents over 6% of the transjunctional material derived from glucose in Cx43-transfected C6 glioma cells. Furthermore, equilibration of these high energy metabolites among first order neighbors is shown to occur in less than 20 min of communication.
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Rat ovarian granulosa cells and mouse myocardial cells respond to cell-specific hormones by cyclic AMP-dependent mechanisms. In coculture, these heterologous cells communicate by means of gap junctions. Exposure of the cocultures to a hormone specific for one cell type causes the heterologous cells to respond through a cell contact-dependent mechanism. These studies suggest that this cross-stimulation results from the intercellular communication of a mediator that is common to both cell types. The communicated mediator may be cyclic AMP.
Gap junctions, containing regular hexagonal arrays of connexons, have been isolated from rat liver. The projected structures of these gap junctions have been studied to a resolution of 18 Å by electron microscopy of negatively stained samples. Two closely related forms of junction were produced that have different structures for the connexon, but the same hexagonal lattice constant. In one form the connexon is seen as a weakly contrasted annulus, which is broadest at the locations where other connexons come closest; in the other, the connexon is seen as a strongly contrasted annulus, which is broadest midway between the locations where other connexons come closest. The forms appear to reflect two configurations of the connexon subunits.
A new method for the isolation of gap junctions from mouse liver is described. Particular attention has been directed to minimising the effects of proteolysis during isolation. The purified membrane fragments retain the typical morphological features found in junctions of the intact liver.The junctions show two major polypeptides upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The apparent molecular weights are 26,000 for the more abundant species and 21,000 for the minor component. Preliminary protein chemical characterisation by fingerprint analysis suggests that the two polypeptides are structurally related. While an in vivo origin of the 21,000 molecular weight species cannot be excluded, the sensitivity of the junction proteins to proteolytic degradation in vitro suggests that the 21,000 molecular weight molecule may be a breakdown product of the major component.Image reconstruction methods applied to micrographs of negatively stained isolated junctions show that the membrane contains a close-packed hexagonal lattice of components having marked 6-fold symmetry. It is suggested that these represent hexamers of the 26,000 molecular weight protein.Lipid analysis performed on gap junctions isolated by different procedures shows that the lipid composition is strongly affected by the detergents employed during the isolation. A large amount of phopholipid, but not cholesterol, can be extracted from the structure without affecting its gross morphology. This result suggests that cholesterol is tightly bound to the junction protein and may play a role in determining the structure of the gap junction.
Many cell types growing in tissue culture form intercellular junctions which are permeable to nucleotides but not to RNA, DNA or protein.
Isolated gap junction plaques contain hexagonal crystalline arrays of membrane channels called connexons which are a suitable specimen for electron crystallography. Image analysis of gap junction lattices has shown that while there is sufficient lattice order for structural analysis to approximately 25 A, there is enough disorder in both the lattice and the connexon to create a family of related images. This review is focused on how these images can be interpreted in terms of what is known about both the connexon and its constituent protein, connexin.
when applied to cells of differenttissues (e.g., 3). These different effects may be attributable to the different complements of gap junctionproteins expressed by the different cell types, to tissuespecific as distinct from connexin-specificeffects, ormayrepresent effects on distinct synthetic or processingstages in the expression pathway (4). To evaluate theeffects ofphosphorylating and dephosphorylating treatments on gating properties (voltage dependence andunitary conductance) of gap junctions formed of aknown connexin, we transfected a communicationdeficient cell line (SKHep1) with cDNA encoding human connexin43 (hCx43;5). Furthermore, we comparedthe gating properties of the wild type channels withthose truncated so as to remove the phosphorylationsites (6). These studies indicate that unitary conductance and voltage dependent kinetics (but not equilibrium) properties ofthe human connexin43 gap junctionchannel are affected by phosphorylation, and that thepresence ofthe carboxyl terminal phosphorylation sitesis not required for functional expression of junctionalchannels.
Rat heart connexin-43 (RCx43) has been isolated using a modified procedure that is rapid and can be used with fresh or frozen hearts. When RCx43 is isolated in the presence of the alkylating reagent iodoacetamide, no intermolecular disulfide bonds are found. However, the alkylated RCx43 does have at least one intramolecular disulfide bond. By using site directed antibodies and proteolytic cleavage the location of the intramolecular disulfide bonding is shown between the two extracellular loops of RCx43.
The oocyte cell/cell-channel assay was used to identify amino acids involved in the process of cell/cell-channel formation. The expression of the rat liver gap-junction protein, connexin 32, in single oocytes, results in the accumulation of a pool of channel precursors. Upon pairing of such oocytes, cell/cell channels form rapidly from this pool. The rate of formation is affected by thiol-specific reagents and the pH. This suggests the involvement of extracellular cysteine residues in the channel formation process. Two connexin-32 mutants were generated by site-directed mutagenesis in which cysteine residues were replaced by serine. Both mutant connexins were unable to form cell/cell channels. Thus, the cysteine residues appear to play an important role in the channel formation process.
Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace “α” with “alpha”). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.