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Departamento de Ciencias Fisiológicas, Pontificia Universidad Católica de Chile, Santiago, Chile; and Department of Pediatrics, University of Chicago, Chicago, Illinois
ABSTRACT I. GENERAL COMMENTS II. BACKGROUND A. Intercellular Communication B. Gap Junction Structure III. MOLECULAR COMPONENTS OF GAP JUNCTIONS A. Gap Junction Proteins B. The Family of Chordate Gap Junction Protein Subunits C. Connexin Genes D. Transcriptional Regulation and Gene Promoters E. Posttranscriptional Regulation of Connexin mRNA Levels F. Connexin Topology and Secondary Structure IV. BIOCHEMISTRY A. Phosphorylation of Connexins B. Other Protein Modifications C. Interaction and Colocalization of Connexins With Other Proteins V. FORMATION AND DEGRADATION OF GAP JUNCTIONS A. Connexin Biosynthesis and Gap Junction Assembly B. Gap Junction and Connexin Degradation VI. HEMICHANNELS A. Opening and Closing Hemichannels B. Hemichannel Functions VII. GAP JUNCTIONS IN CARDIOVASCULAR, DIGESTIVE, REPRODUCTIVE, AND IMMUNE SYSTEMS A. Cardiovascular System B. Digestive System 1. Gastrointestinal tract 2. Accessory glands C. Reproductive System 1. Gap junctions in the female reproductive system 2. Gap junctions in the male reproductive system D. Immune System VIII. GAP JUNCTION CHANNELS AND DISEASE A. Connexin Mutations Related to Human Peripheral Neuropathy B. Connexin Mutations Related to Deafness and Skin Disorders C. Connexin Mutations Related to Human Cataracts IX. PHYLOGENY X. PERSPECTIVES NOTE ADDED IN PROOF
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ABSTRACT |
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I. GENERAL COMMENTS |
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Due to limitations of space, some of the earlier investigations and some specific areas of gap junction biology that have shown significant advance in recent years (e.g., nervous system) have not been considered in detail in the current review. The reader is referred to previous reviews for more detailed information on earlier studies (35, 48, 66, 67, 190, 539, 551). Several reviews have been published recently on gap junction structure (534, 548, 665), trafficking of protein subunits (154, 314, 317, 666), channel gating (36, 59, 108), pharmacological regulation (45, 499, 550), and functional features of the channels (353, 443, 540, 614). Other reviews have covered the roles and regulation of gap junctions in development (338, 352) and in various mature tissues and organs, including vascular tissue (60, 91), urogenital smooth muscle (269), the heart (50) and cardiovascular system (128), pancreas (377), endocrine glands (395), the nervous system (65, 122, 123, 168, 546, 552), and the immune system (11, 502, 503). Other reports describe the regulation and role of gap junctions in tissue injury (120) and the involvement of gap junctional communication in the phenotypes observed in connexin knock-out animals and/or different diseases (134, 540, 647, 662).
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II. BACKGROUND |
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The first indications that a pathway for direct cell-to-cell communication might exist substantially preceded discovery of the gap junction structure. Weidmann (637), working with strips of myocardium, found that the space constant for the spread of current extends beyond the expected value for a single Purkinje fiber. He suggested that this phenomenon might be explained by the existence of a low-resistance intercellular pathway. Stronger evidence supporting the existence of such a pathway was provided by the discovery of electrical transmission at the giant crayfish motor synapses (170). Later, this type of pathway was demonstrated in other excitable tissues (22, 34, 162) and in nonexcitable cell types (184, 356, 432, 468, 482). Today, it is accepted that cells of most invertebrate and vertebrate tissues can communicate with their neighbors via a low-resistance intercellular pathway. In vertebrates, only a few cell types do not form gap junctions in their fully differentiated state, including red blood cells, spermatozoa, and skeletal muscle. Nevertheless, the progenitors of these cells do express gap junctions (98, 386, 469, 495).
Electron microscopy studies provided evidence for a structure responsible for intercellular electrical transmission. Robertson (491) demonstrated the structural units of the neuronal junction. In studies of excitable tissues, a close apposition between the plasma membranes of adjacent cells was observed when current transfer occurred, but it was absent when electrical transmission was not detectable (22, 440, 482). This intercellular junction has been given several names, including nexus, macula communicans, and gap junction. The designation "gap junction" from the work of Revel and Karnovsky (481) has prevailed despite the contradiction between the function and the morphological characteristics that it denotes.
The structure of gap junctions and their constituent channels have been significantly elucidated. In transmission electron micrographs of ultrathin tissue sections, gap junctions appear as regions where the plasma membranes of adjacent cells closely approach each other, but appear to be separated by a small gap of 2-3 nm (33, 481, 491) (Fig. 1A). Electron micrographs of freeze-fracture replicas of vertebrate junctions show 8.5- to 9.5-nm particles on the P face either free or in plaque-like arrays with complementary pits on the E face (Fig. 1C). In many cases, these particles (termed connexons) are regularly ordered in a hexagonal pattern that has allowed detailed structural studies by other techniques. Studies of gap junctions using atomic force microscopy (AFM) (226, 318) show a dense packing of particles with a center-to-center distance of 9-10 nm and a similar long-range hexagonal packing. The particles contain a porelike structure with a diameter of 2 nm, a depth of 1 nm, and a width of 3.8 nm based on freeze-fracture and AFM analyses (226, 318, 481). X-ray diffraction examination of isolated gap junction membranes (78, 362) and Fourier analysis of low-dose electron micrographs (595) show that each particle or connexon within a gap junction forms a cylinder with an apparent aqueous pore in the center. The wall of the connexon is formed of six protein subunits (362). Other studies suggest that these subunits may move with respect to each other and control channel opening (594) (Fig. 3, inset 1).
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The structure of the gap junction channel has been refined in recent years (Fig. 1D). Using gap junctions isolated from cells expressing a recombinant truncated gap junction protein, Unger et al. (592) demonstrated the dodecameric structure of the channel at a resolution of 7 Å in the membrane plane and 21 Å in the vertical direction; each hemichannel contains 24 
-helices corresponding to the four transmembrane domains of the six protein subunits (592, 593). These structures were also suggested by circular dichroism (77) and X-ray patterns obtained from oriented gap junctions (577). The outer diameter of the hemichannel is 
70 Å at the intracellular region and 50 Å at the extracellular regions giving the complete channel an hour glass-like appearance. The channel pore narrows from 40 Å diameter at the cytoplasmic side to 15 Å at the extracellular side of the bilayer and then widens to 25 Å in the extracellular region (451, 592, 593). The connexon interfaces within a channel must be tightly bound to form a tight seal. Thus the docking domain provides a high resistance to the leakage of current carrying ions between the channel lumen and the extracellular space. Recently, conformational changes of the cytoplasmic and extracellular surfaces in gap junction plaques containing channels made of native connexin26, a protein subunit, have been imaged using AFM (394). The carboxy terminus reversibly collapses when increased forces are applied to the AFM stylus. The presence of Ca2+ in the buffer solution reduces the diameter of the extracellular channel entrance from 1.5 to 0.6 nm, a conformational change fully reversible and specific among the divalent cations. Calcium induces the formation of microdomains, and the plaque height increases in 0.6 nm (from 17.4 to 18 nm), indicating that Ca2+ induces conformational changes affecting the structure of connexin26 membrane channels (394).
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III. MOLECULAR COMPONENTS OF GAP JUNCTIONS |
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Gap junction-enriched preparations, similar to those utilized for structural studies, were used for biochemical identification of protein components; initially, it was thought that all gap junctions contained the same protein. Evidence for differences in gap junction proteins came from the differing electrophoretic mobilities (21-70 kDa) of bands detected by SDS-PAGE in gap junction-enriched preparations from different tissues (211, 219, 285, 366). In support of this, analysis of nonproteolized heart gap junctions by electron microscopy showed a fuzzy interface that was absent in isolated liver gap junctions or proteolyzed heart gap junctions (365, 535). Comparison of heart and liver gap junctional proteins by two-dimensional peptide mapping of proteolytic fragments followed by high-pressure liquid chromatography revealed differences between these proteins (200). Furthermore, microsequencing of the amino-terminal regions of the hepatic and cardiac proteins revealed both similarities and dissimilarities (414). Subsequently, sequencing of a lens gap junction protein showed that it differed from both the liver and heart proteins (284).
B. The Family of Chordate Gap Junction Protein Subunits
The production of antibodies to the main protein in isolated liver gap junction preparations, and the synthesis of oligonucleotide probes based on the amino-terminal sequence information, allowed the isolation of rat and human cDNA clones encoding a liver gap junction protein of 32 kDa (300, 442). Proof that this protein produces gap junction channels came from its expression in Xenopus oocytes (138, 566, 639). It became evident that there was a family of subunit gap junction proteins when Beyer et al. (52) isolated cDNA clones encoding a related, but clearly different, polypeptide of 43 kDa corresponding to a rat heart gap junction protein, which they named connexin43 (Cx43). While connexins were originally denoted according to the tissue of origin or the apparent size of a polypeptide as determined by SDS-PAGE, it soon became clear that such designations were inappropriate, because many of these proteins are not uniquely expressed in a single tissue (52), and their mobilities on SDS-PAGE may vary with electrophoresis conditions (196). Therefore, to distinguish members of this family, a standard nomenclature was needed. The most widely used system uses the word connexin (often abbreviated Cx) followed by a suffix indicating the predicted molecular mass of the polypeptide connexin (in kilodaltons) to distinguish different protein subunits (52, 53). In some cases, a prefix is added to indicate the species of origin. The finding that two (or more) connexins may have similar molecular mass has led to the use of a decimal point to distinguish them (e.g., mCx30.3 or mCx31.1). Some confusion has also been created when orthologous and functionally homologous connexins have different molecular masses (e.g., rat Cx46 vs. bovine Cx44 vs. chicken Cx56). An alternative (but also problematic and less widely used) nomenclature in which connexins are classified in subclasses according to amino acid sequence homology has also been proposed (423, 486).
Subsequently, a number of cloning strategies including hybridization screening of genomic and cDNA libraries at reduced stringency and use of the polymerase chain reaction with degenerate/consensus primers have been successfully applied to identify additional members of the connexin family (for recent review, see Ref. 653).
Recently, the screening of the mouse and human genomic databases has revealed 19 and 20 connexin genes in the mouse and human genome, respectively (55, 359, 417, 653). A dendrogram for all identified human connexins is shown in Figure 2A. The genes for various connexins have been localized to human (32, 92, 161, 214, 233, 271, 417, 654) and mouse (9, 93, 207, 215, 233, 521) chromosomes. Connexin genes are found in many different chromosomes, but there is a cluster of connexins in a region of mouse chromosome 4 (214) which is syntenic to a region in human chromosome 1. The genes for many connexins have been cloned, and the gene structure of other connexins has been partially assessed by DNA blotting and by the polymerase chain reaction (for a review, see Ref. 653). The available information indicates that many connexin genes have a similar organization, and virtually all have only single copies in the haploid genome (653). The sequence similarities between connexins and their similar gene structures suggest that they have arisen by gene duplication. In humans, a Cx43 pseudogene has been identified (163).
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Most connexin genes have a first exon containing only 5'-untranslated (UTR) sequences and a large second exon containing the complete coding region as well as all remaining untranslated sequences (Fig. 2B). Exceptions to this gene structure are the Cx32, Cx36, and Cx45 genes. The human (and rodent) Cx32 gene contains alternative first exons (containing only 5'-UTR) whose use is tissue specific (411, 545, 547); some bovine Cx32 transcripts have three exons, two of which contain only 5'-UTR (135). The Cx36 (and the Morone americana Cx34.7) gene contains two exons, both of which contain untranslated and translated sequences. The coding region is interrupted by an intron (32, 97, 422, 423, 544). The Cx45 gene contains three exons; exons 1 and 2 contain only 5'-UTR and exon 3 contains the full coding region and 3'-UTR (19, 242); all Cx45 transcripts examined contain exons 2 and 3, while some also contain exon 1 (242).
D. Transcriptional Regulation and Gene Promoters
In various cell types, different treatments are known to increase the levels of connexin mRNA due to enhanced transcription. For example, cAMP induces elevation of the Cx43 transcription rate in Morris hepatoma cells (380). Moreover, the transcription rates of Cx32 and Cx26, but not Cx43, are upregulated by glucocorticoids in liver-derived cells (479), and the phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] induces transcriptional up-regulation of Cx26 in human immortalized MCF-10 mammary epithelial cells (340). Also, the large increase of Cx43 in myometrium before parturition is at least in part due to increased transcription (433, 486). Thus knowledge of connexin gene structure and identification of regulatory elements will allow us to understand the transcriptional regulation of connexin expression.
The basal promoter of the rat Cx32 gene was first localized to a region -179 to 0 or -134 to 0, immediately upstream of the first exon (17). The mouse Cx32 gene contains two putative binding sites for the transcription factor HNF-1 and consensus motifs for NF-1 as well as NF
B within the 680 bp upstream of the main transcription start site (215). Cx32 cDNA clones from rat or mouse liver, sciatic nerve, and embryonic stem cells differ (545). They correspond to different splicing variants with a different 5'-UTR or exon 1, each with its own promoter region (545).
The human Cx32 gene can be transcribed from two tissue-specific promoters, P1 and P2, leading to the production of two mRNAs with different 5'-UTRs through the use of alternative exons (411). The P1 promoter is functional in liver and pancreas and is located more than 8 kb upstream of the translation start codon, while the P2 promoter is functional in nerve cells and is located 497 bp upstream from the translation start codon (411). Cx32 expression in vitro is strongly activated by direct binding of SOX10, a common transcription factor for other myelin genes (56). Methylation of the Cx32 (and Cx43) promoter at Msp I/Hpa II restriction sites correlates well with cell type-dependent decreased transcriptional activity (459).
Sequence analysis of mouse Cx37 gene has revealed that the regions flanking exon I contain a consensus "TATA box" 43 bp 5' from the transcription start site preceded by several putative transcription factor binding sites and a 282-bp truncated L1Md transposon (529).
Determination of the structure and sequence of the Cx43 gene and analysis of trans-activation have elucidated some of the hormonal and pharmacological regulation of Cx43 expression. Sullivan et al. (565) and Yu et al. (674) cloned the mouse and rat Cx43 genes, identified the transcriptional start site, and showed that there were a number of putative transcription factor binding sites within the 5'-flanking region including a TATA box, AP-1, AP-2, Sp1, CRE sites, and half-palindromic estrogen response elements. The basal promoter is contained within 110 bp 5' to the start site (88). DNase I footprinting assays have confirmed the use of AP-1 (-44 to -36) and Sp1 (-77 to -69 and -59 to -48) consensus sequences; moreover, mutation of this AP-1 site abolishes phorbol ester-induced transactivation of Cx43 gene transcription in uterine smooth muscle cells (178). Transfection studies using Cx43 constructs linked to luciferase have shown the responsiveness of Cx43 transcription to estrogen, parathyroid hormone, and prostaglandin (94, 674). cAMP, which can also increase Cx43 expression (380), may serve as the mediator of the parathyroid hormone and prostaglandin effects (519) through the Cx43 promoter cAMP response elements (18). The mechanism of the estrogen-induced response is not so clear; Piersanti and Lye (460) have suggested that it occurs indirectly through production of c-fos, which interacts with the AP-1 sites. Electrophoretic mobility shift assays have demonstrated c-jun and Sp1 binding at the AP1 and Sp1 sites, respectively (141). In positions -480 and -464 of the rat gene, there is a thyroid hormone binding sequence that is able to activate the promoter (561).
Rat Cx43 and Cx30 transcription may be transactivated by the Wnt-1 signaling pathway, probably through TCF/LEF binding elements (376, 600). These are HMG box transcription factors that bind to 
-catenin in response to activation of the Wnt-1 signaling pathway, and not through a cAMP-induced pathway (600). Functional analysis demonstrated that mouse Cx43 promoter constructs could be activated in zebrafish embryonic neural crest cells (87), suggesting that the sequence motifs and transcriptional regulation involved in targeting Cx43 expression to neural crest cells are evolutionarily conserved in zebrafish and mouse embryos.
The Cx40 mRNA sequence is identical in all tissues studied (198). Thus transcriptional regulation may account for the differences in levels of Cx40 transcripts detected during development and among different tissues. Transient transfection studies have demonstrated that the basal promoter activity of the Cx40 gene lies within 300 bp upstream of the transcription start site and that a strong negative regulatory element is present in the intron in the region from +100 to +297 bp (530). Moreover, potential transcription binding sites include AP-1, AP-2, Sp1, TRE, p53, and a TATA box located near exon 1 (530). Transcription of Cx40 is transactivated by Tbx5 (T-box transcription factor 5) and the homeodomain transcription factor Nkx2-5 (62). Heterozygous and homozygous Tbx5-deficient mice have similar features to the human Holt-Oram syndrome (62), and it has been suggested that decreased Cx40 transcription may contribute to the cardiac conduction defects observed in this syndrome.
The mouse Cx26 promoter contains at least two transcription start sites and is located in a very GC-rich region without a TATA box, reminiscent of promoters of housekeeping genes (215). Consensus sequences for a metal response element, NFB, and six GC boxes were found within 600 bp upstream of the Cx26 transcription start sites in mouse and human DNA (215, 276). Moreover, the human sequence contains a YY1-like binding site and a consensus mammary gland factor binding site (276). The region -140 to -113 of the Cx26 promoter includes an Sp binding site overlapping with an AP-2 binding site which are responsible for the upregulation of Cx26 in the myometrium during pregnancy and in the mammary gland during lactation (590).
The Cx31 gene promoter region contains five putative binding sites for the GATA transcription factor, a NFB element, a CAAT box, and E-box/E-box related sequences (172). Interestingly, if part of the intron sequence is deleted, the promoter activity is lost (172).
E. Posttranscriptional Regulation of Connexin mRNA Levels
The abundance of mRNA can be affected by conditions that increase or reduce its stability. The 5'-UTR of the Cx43 mRNA contains a strong internal ribosome entry site that confers increased transcription levels (518; for a review, see Ref. 638).
Elevated cAMP concentrations increase the levels of Cx32 mRNA in primary cultures of rat hepatocytes without detectable changes in the transcriptional rate, suggesting an enhanced Cx32 mRNA stability (505). Moreover, during liver regeneration, the reduction in Cx26 and Cx32 mRNA occurs without changes in transcriptional rate, suggesting a posttranscriptional mechanism (293). In this system, the administration of cycloheximide, a protein synthesis inhibitor, completely inhibits the disappearance of Cx26 mRNA, but not of Cx32 mRNA, suggesting the participation of protein factors in destabilization of the Cx26 mRNA (293). The reduction in liver Cx32 steady-state mRNA levels induced by bacterial lipopolysaccharide appears to occur at the posttranscriptional level, since the rate of degradation of this message is higher than the rate of transcription of the gene (185).
F. Connexin Topology and Secondary Structure
The amino acid sequences derived from each cloned cDNA have been used to predict the structure of connexins. Hydropathy plots of several connexins predict the presence of four hydrophobic domains, with a carboxy-terminal hydrophilic tail. In addition, three hydrophilic domains separate the hydrophobic regions (52, 220, 442) (Fig. 3, inset 2). The possible connexin membrane topologies derived from these data were tested by examining the protease sensitivity of isolated liver and heart gap junctions. Antisera raised against synthetic oligopeptides representing various segments of Cx26, Cx32, and Cx43 were used to map the connexin topology. The amino terminus, the carboxy terminus, and a loop in the middle of the protein are all located on the cytoplasmic side of the junctional membrane, and the predicted extracellular connexin domains can be detected on the extracellular surfaces of the junctional membranes (218, 382, 471, 663, 679, 682) (Fig. 3, inset 2). The four transmembrane spanning regions and the extracellular loops contain many identical residues or conservative substitutions among the different connexins. In contrast, the cytoplasmic domains are unique to each connexin; the cytoplasmic loop and the carboxy terminus vary extensively in length and amino acid composition.
Sequence analysis and expression of wild-type or mutant connexins are approaches that are actively being used to determine residues within these sequences that may be responsible for various channel properties. The transmembrane regions must be responsible for spanning the plasma membrane and forming the aqueous pore. Three transmembrane regions M1, M2, and M4 contain mainly hydrophobic amino acid residues, while M3 also contains a number of charged (positive and negative) amino acid residues. Therefore, M3 has been modeled as an amphipathic -helix, and it has been suggested that its hydrophilic face might line the pore of the gap junction channel (35). Structural studies (593) indicate that regions of two -helices line the inner wall of the channel. Scanning cysteine mutagenesis of Cx46 hemichannels in which amino acid residues from M1 and M3 were replaced by cysteines indicated that both transmembrane regions contribute to the lining of the pore (681). Scanning cysteine mutagenesis of Cx32 channels suggests that M2 also contributes to the lining of the pore (543).
The extracellular regions must be responsible for the "docking" of connexons which occurs by collision of two connexons, each provided by each of two adjacent cells. This process apparently occurs through noncovalent forces (180). White and co-workers (643, 648) demonstrated the importance of the second extracellular loop (E2) in determining which connexins can interact to form heterotypic channels. Synthetic peptides homologous to the extracellular loop 2 of Cx40 or Cx43 selectively block the function of the corresponding connexin (304). Werner et al. (640) showed that site-directed mutagenesis of amino acid residues within the extracellular loops that differ among connexins alter but do not prevent gap junctional communication. Biochemical studies have shown that cysteines in the extracellular loops participate in forming intramolecular but not intermolecular disulfide bonds (252, 475). Mutation of any of these cysteines to serines prevents gap junction channel formation (109, 110), emphasizing the importance of the secondary and tertiary structures imposed on the connexins by the disulfide bonds. Alteration of the position of the cysteines in Cx32 can seriously impair the ability to form functional channels (165); these data have been used to model this region as adopting a -sheet conformation. The cysteine residues involved in formation of the intramolecular disulfide bonds in Cx43 have been studied using site-directed mutagenesis (580); however, these differ from those proposed for Cx32 (165). In expression systems, different connexins form channels with differing properties including unitary conductance, selectivity/permeability, and gating/regulation (23, 73, 142, 144, 162, 292, 367, 389, 391, 617-620). Some of the unique properties must be conferred by the unique portions of the connexin sequences (many of which are located within or near the cytoplasm). The main differences in the primary sequence between two closely related connexins, Cx26 and Cx30, are located in the cytoplasmic loop and carboxy-terminal domains. Exchange of one or both of these domains has demonstrated that the cytoplasmic domains interfere directly or indirectly with the permeability, conductance, and voltage gating of the channels in HeLa cells transfected with Cx26/30 chimeras (367).
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IV. BIOCHEMISTRY |
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All studied connexins with the exception of Cx26 (508, 585) are phosphoproteins (for previous reviews, see Refs. 100, 322, 506). Connexin phosphorylation has been implicated in the regulation of intercellular communication through a number of mechanisms, including connexin biosynthesis, trafficking, assembly, membrane insertion, channel gating, internalization, and degradation (16, 42, 44, 146, 217, 229, 320, 330, 390, 401, 426, 470, 567, 586, 670, 669). It is likely that some connexin phosphorylation occurs before insertion into the plasma membrane, but some must occur at the plasma membrane to affect gating. Nevertheless, the specific phosphorylation sites and functional role for most of them have not been elucidated.
The most extensively studied connexin in terms of modification by protein kinases and phosphatases is Cx43. This protein contains several consensus sites for phosphorylation in the carboxy terminus, several of which have been identified as target sites for specific protein kinases. Fusion proteins containing the cytoplasmic tail of Cx43 or synthetic peptides corresponding to the deduced sequences in this region are phosphorylated by protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and cdc2 kinase (323, 507, 575, 634). The identified phosphorylation sites are Ser-368 and Ser-372 phosphorylated by PKC (507) and Ser-255, Ser-279, and Ser-282 phosphorylated by MAPK (633, 634). Although phosphorylated Cx43 can be found at the plasma membrane (400), phosphorylation of Cx43 could also happen in intracellular compartments (103, 315).
Epidermal growth factor (EGF)-, vascular endothelial growth factor (VEGF)-, fibroblast growth factor 2 (FGF-2)-, and platelet-derived growth factor (PDGF)-induced Cx43 phosphorylation can be mediated by intracellular kinase pathways that activate MAPK also termed ERK1/2 (132, 229, 230, 263, 564, 664) (Fig. 4). In some systems, this effect appears to occur in a PKC-independent manner (449), but in other systems it can be prevented by PKC inhibition, but not by inhibition of the ERK1/2 pathway (133). In T51B cells, this effect requires the activation of both PKC and ERK1/2 (229). MAPK activation may also mediate the induction of Cx43 phosphorylation by lysophosphatidic acid (LPA) (221). The observations are consistent with the variations in crosstalk between different protein kinase-dependent pathways in different cell types (e.g., differences in the relative activity of each pathway and different subcellular compartmentalization of different protein kinases). It has been confirmed that Cx43 is a MAPK substrate in vivo and that phosphorylation on Ser-255, Ser-279, and/or Ser-282 is sufficient to disrupt gap junctional intercellular communication (634). Accordingly, 3T3 A31 fibroblasts transfected with a carboxy-terminal truncated Cx43 remain highly coupled after treatment with PDGF (388). Nevertheless, other studies suggest that other unknown factors besides phosphorylation are required for disruption of Cx43-mediated cell-to-cell communication (231, 232). The consensus sites for MAPK and cdc-2 kinase present in Cx43 are apparently shared, since the two-dimensional tryptic maps of the carboxy terminus phosphorylated by MAPK or cdc-2 kinase appear identical (507). Yet, comparison of two-dimensional phosphotryptic peptide analysis of the p34(cdc2)/cyclin B kinase-phosphorylated carboxy terminus and of the P3 phosphoform of Cx43 immunoprecipitated from mitotic cells reveals several differences. This suggests that in vivo Cx43 phosphorylation would involve the participation of other protein kinase(s), which may be activated by the p34(cdc2)/cyclin B kinase (321).
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Numerous reports have described changes in the state of phosphorylation of Cx43 through PKC-dependent pathways. The pattern of Cx43 phosphorylation and the functional consequences are cell type dependent (for reviews, see Refs. 322, 506). Ser-368 in Cx43 appears to be an important site that may underlie the cellular uncoupling that follows TPA treatment, since mutation of Ser-368 partially prevents this effect (323, 349).
The products of various oncogenes are tyrosine kinases that disrupt gap junctional communication by phosphorylation of tyrosine residues in Cx43. Oncogene products with tyrosine kinase activity phosphorylate Cx43 in vivo (102, 160, 302) and in vitro (357). Similarly, c-Src seems to be involved in loss of gap junctional communication induced when ligands bind to c-Src-associated receptors (466). The interaction with v-Src is mediated by a proline-rich motif of Cx43, Pro-274-Pro-284 (264). Moreover, in vitro studies indicate that Cx43 is a substrate for v-Src tyrosine kinase and that phosphorylation of Cx43 occurs in Tyr-247 and Tyr-265 (345). Expression in Cx43-null cells of Cx43 constructs in which Tyr-247 and/or Tyr-265 were replaced with phenylalanines suggests that phosphorylation of both tyrosine residues is required for the complete v-Src-induced disruption of gap junction channel communication (345).
In rat (but not human) Cx43, Ser-257 is flanked by a proline and a lysine, making it a possible site for phosphorylation by cGMP-dependent protein kinase. Accordingly, the extent of phosphorylation of rat Cx43, but not of human Cx43, expressed in SkHep1 cells is increased by 8-bromo-cGMP (306).
Some studies have shown that activation of cAMP-dependent protein kinase (cAMP-dPK) leads to a rapid augmentation in Cx43 phosphorylation as detected by immunoblotting (193) and increased intercellular communication (69, 186). However, direct phosphorylation of Cx43 by cAMP-dPK has not been documented. The polypeptides corresponding to the carboxy terminus of Cx43 are not phosphorylated by cAMP-dPK (509, 575). Moreover, activation of cAMP-dPK does not change the 32P incorporation into Cx43 transfected into fibroblasts derived from Cx43-null mice (575).
Cx32 is phosphorylated by cAMP-dPK, PKC, Ca2+/calmodulin-dependent kinase II, and EGF receptor (130, 508, 511, 569, 570, 586). In rat hepatocytes, the effect of cAMP-dPK is temporally correlated with increased junctional conductance (511). Similarly, in the human colonic cell line T84, a rapid (<20 min) increase in intercellular communication mediated by Cx32 gap junctions is induced by an increase in intracellular cAMP concentration (84). Functional correlates of Cx32 phosphorylation by EGF receptor or Ca2+/calmodulin-dependent protein kinase have not been reported.
The stoichiometry of Cx32 phosphorylation in vitro by PKC or cAMP-dPK is between 0.1 and 1 mol phosphate/mol Cx32 (508, 511, 570), suggesting that not every channel or not every subunit within a channel is phosphorylated. The stoichiometry of in vivo phosphorylation and identification of the cell compartment(s) in which Cx32 phosphorylation occurs have not been reported. Cx32 is phosphorylated in Ser-233 by both cAMP-dPK and PKC (508). It is likely that PKC phosphorylates other amino acid residues that are not phosphorylated by cAMP-dPK (570). Increased incorporation of 32P into Cx32 is observed when isolated liver gap junctions (previously phosphorylated by cAMP-dPK) are incubated with PKC (570); moreover, two-dimensional tryptic phosphopeptide maps of Cx32 immunoprecipitated from hepatocytes treated with PKC activators show a higher number of 32P-labeled peptides than the maps of Cx32 obtained from cells treated with cAMP-dPK activators (508). Phosphorylation of Ser-233 is not required for formation of functional channels (640), but its possible role in other functions has not been studied. The tyrosine residues phosphorylated after activation of EGF receptor (130) have not been identified.
Lens connexins are phosphoproteins, and changes in their state of phosphorylation correlate with changes in cell-cell communication (39, 248, 514, 670). In ovine lens, Cx49 is phosphorylated by casein kinase I (89), and inhibition of casein kinase I increases intercellular communication between cultured lens cells (90). Moreover, Cx45.6 is phosphorylated at Ser-363 by casein kinase II; this phosphorylation inhibits the cleavage of the connexin carboxy terminus mediated by caspase-3-like protease (669). Cx46 is phosphorylated in serine and threonine by PKC in the lens cortex (514). Two phosphorylation sites have been identified in Cx56, Ser-118 and Ser-493. These sites are phosphorylated under basal conditions in lentoid-containing cultures. Upon activation of PKC, phosphorylation is enhanced in Ser-118 (39). Phosphorylation of Ser-118 correlates with a reduction in intercellular communication (39). The Cx56 phosphorylation and reduced intercellular communication induced by a tumor promoter phorbol ester appear to be mediated by PKC-
(44).
Phosphorylation of other connexins (e.g., Cx31, Cx37, Cx40, and Cx45) in transfected cells has been demonstrated directly by incorporation of 32P into the protein or shift in their electrophoretic mobilities after treatments that increase protein kinase activity (313, 328, 583, 584, 610, 613). Mouse and human Cx37 expressed in cell lines are phosphorylated mainly in serine residues (328, 584). Mouse Cx45 is detected as a doublet that is reduced to a single 46-kDa band after treatment with alkaline phosphatase (613). After mutation or deletion of nine serine residues in the carboxy terminus of Cx45, phosphorylation is decreased by 90%, indicating that those serine residues represent main phosphorylation sites in the protein (217). Activation of cAMP-dPK leads to an electrophoretic mobility shift of human Cx40 that correlates with increased cell-to-cell communication (610). However, the identification of amino acid residues with functional significance and dissection of protein kinases responsible for their phosphorylation await further studies.
Finally, treatment of some cells with phosphatase inhibitors leads to increased Cx43 phosphorylation, suggesting the involvement of phosphoprotein phosphatases 2A and 2B (42, 104, 342). The phosphatases that mediate dephosphorylation of other connexins have not been reported.
B. Other Protein Modifications
The amino termini of several connexins contain potential consensus N-glycosylation sites, but they are not utilized (474, 630), presumably because they are located in the cytoplasm and therefore are not accessible to the glycosylating enzymes. Fatty acylation of connexins has been suggested by metabolic labeling studies (585), but in-depth studies have not been published.
Modification of Cx43 by ubiquitination has been demonstrated (309, 312, 501) (Fig. 3). The residue to which ubiquitin molecules are added has not been identified, but it is likely to be a lysine.
Cleavage of the carboxy terminus of lens fiber-specific connexins has been proposed to occur naturally during maturation of lens fiber cells (283). Studies of the ovine Cx50 have identified calpain as the enzyme that removes a 32-kDa portion of the carboxy tail of Cx50 (344, 669, 670). However, a similar phenomenon may not occur in chicken lenses, since cleavage of Cx56 was not observed when lens cortex and lens nucleus homogenates were prepared using EDTA (40).
C. Interaction and Colocalization of Connexins With Other Proteins
Connexins may interact with other proteins within the cell. Because gap junction isolation methods involve the use of relatively harsh detergents, interacting proteins do not copurify with connexins. However, other strategies have suggested some possible associated proteins. Blotting experiments have suggested Cx32 could bind calmodulin (604, 682). In addition, Cx43 contains SH2 domains that may be important for its interaction with tyrosine kinases such as pp60v-src (264, 358). Recent studies have suggested an association of Cx43, Cx45, Cx46, and Cx31.9 with the peripheral membrane protein ZO-1 (181, 310, 416, 417, 581) (Fig. 3), which was originally identified as a component of tight junctions. Binding of Cx32, but not Cx26, to another tight junction protein, occludin, has also been demonstrated by coimmunoprecipitation experiments (288, 289). The expression of Cx32, but not Cx32 truncated at position 220, wild-type Cx26, or Cx43, in Cx32-null hepatocytes enhances the expression of the tight junction proteins claudin, occludin, and ZO-1, which colocalize with Cx32, suggesting that the Cx32-mediated intercellular communication participates in the formation of tight junctions (290). With the use of a yeast two-hybrid protein interaction screen, the interaction of Cx43 through its carboxy terminus with the second PDZ domain of the ZO-1 protein has been demonstrated (181). The v-Src-induced disruption of gap junction plaques may occur through interaction of v-Src with ZO-1 (579).
Connexins appear to interact with caveolin-1, a structural protein of lipid raft domains (112, 520); this interaction seems to be cell type specific, and its functional significance is unknown. Colocalization of connexins with other membrane proteins, including FGF receptors (268), aquaporins (476), or cytoplasmic proteins such as calmodulin (549) and cytoskeletal proteins (182) has been demonstrated.
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V. FORMATION AND DEGRADATION OF GAP JUNCTIONS |
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The development of specific anti-connexin antibodies has made possible in vitro studies of connexin biosynthesis by metabolic labeling and immunoprecipitation (reviewed in Refs. 314, 317). The biosynthetic studies have been most extensive for Cx43. Pulse-chase studies show that Cx43 is initially synthesized as a 40- to 42-kDa polypeptide that is subsequently posttranslationally modified to forms with slightly slower mobilities on SDS-PAGE due to phosphorylation of serine residues (102, 160, 399, 400). Multiple phosphates are added to Cx43, and at least some of this phosphorylation occurs in a serine-rich sequence near the carboxy terminus. Phosphorylation of Cx43 to yield a phosphoform (P1) occurs soon after translation. Inhibition of protein trafficking with monensin or brefeldin A reveals that partial phosphorylation of Cx43 occurs before its exit from the Golgi apparatus (315, 470).
The location at which connexins oligomerize into connexons is connexin-type dependent (116, 129, 370, 402, 474); as examples, it appears that Cx32 assembles in the endoplasmic reticulum (ER) (or ER/Golgi intermediate compartment) while Cx43 assembles in the trans-Golgi network (116, 129, 370, 402, 474). With the use of an in vitro cell-free transcription/translation system, it has been demonstrated that Cx26 hemichannels are integrated directly into plasma membranes in a posttranslational manner (5). In cells expressing two connexins, it is possible to find hemichannels formed by both connexins (heteromeric) (43, 246, 355, 372, 556) or hemichannels constituted by one or the other protein (homomeric). In studies performed in liver, oligomeric intermediates of Cx26 are detected in an ER/Golgi intermediate compartment while oligomers containing both Cx26 and Cx32 are preferentially detected in a Golgi membrane fraction (129). This might have implications for the formation of heteromeric connexons (see below). Newly synthesized connexins appear to be transported to the plasma membrane through two different pathways: one sensitive to brefeldin A (a drug that disrupts the Golgi compartment) and one sensitive to nocodazole (a drug that disassembles microtubules) (179, 370).
Connexons are inserted into the plasma membrane in a closed configuration (Fig. 3, inset 1) perhaps at regions of cadherin/catenin-mediated cell adhesion or near tight junctions (169). Formation of gap junctions requires appropriate cell adhesion, especially that mediated by Ca2+-dependent molecules (cadherins) (258, 400). Musil et al. (400) have demonstrated that Cx43 localizes intracellularly and not at gap junctional plaques in the noncommunicating cell lines L929 and S180. The Cx43 in these cells is incompletely phosphorylated. However, transfection of the S180 cells with the cell adhesion molecule LCAM (E-cadherin) restores gap junctional communication, phosphorylation of Cx43 (to a "P2" form), and presence of gap junction plaques. These findings suggest a relation between the ability of cells to more extensively phosphorylate Cx43 and the ability to form communicating junctions. They also suggest a hierarchy of events in the formation of intercellular junctions: a cell adhesion event is required before formation of gap junction plaques. Similar observations regarding the requirement of cadherin-mediated cell adhesion for development of gap junctions have been made in epidermal cells (258). Cell-to-cell contact sites mediated by the cadherin-catenin complex also seem to act as foci for gap junction formation as shown by colocalization of connexins with E-cadherin or -catenin during gap junction formation in regenerating liver (169). Moreover, antibodies to A-CAM (N-cadherin) or peptides representing extracellular domains of Cx43 inhibit gap junction formation in Novikoff hepatoma cells (381). However, the effects of increased cell adhesion seem to be cell type specific, since expression of exogenous cadherin decreased gap junctional communication between mouse L cells but increased it in Morris hepatoma cells (632). A role for Ca2+-independent cell adhesion molecules (e.g., N-CAM) has also been suggested (270).
Incorporation of connexons made of Cx43 into gap junction plaques correlates with resistance to solubilization in Triton X-100 and with additional phosphorylation (401). However, some studies suggest that phosphorylation may not be an absolute requirement for assembly or insertion of connexins into the plasma membrane. In exogenous expression systems, Cx26, a nonphosphorylated protein, is able to induce junctional currents (23). Moreover, truncated Cx43 or Cx32 constructs devoid of some phosphorylatable serine residues from their carboxy termini can still induce intercellular coupling (162, 351, 640). In addition, phosphorylation of Cx43 can occur in Madin-Darby canine kidney cells in the absence of Ca2+-dependent cell adhesion molecule activity and does not correlate with existence of intercellular coupling (42).
Formation of gap junction plaques requires clustering of gap junction channels. Increased levels of cAMP induce clustering of Cx43 gap junction channels and enhanced gap junction plaque growth through a mechanism that seems to be cell type dependent. In some cell types, this process depends on polymerized actin while in others it depends on intact microtubules and trafficking through intracellular membrane compartments (158, 256, 445, 631). It has recently been suggested that the carboxy terminus of Cx43 (including Ser-364) plays a role in the cAMP-induced clustering of gap junction channels (575). The differences observed between cell types might reflect differences in the source from which additional connexons come (i.e., a pool at or near the plasma membrane or a pool at earlier locations within the biosynthetic/exocytic pathway).
Connexins that are coexpressed in the same cell can localize to the same gap junction plaque (404, 413, 428, 585, 115, 228, 660, 668) or to distinct membrane domains (203, 553), suggesting the presence of a sequence motif in connexins that determines its topographic fate. Although differences in primary sequence between connexins might explain the delivery of hemichannels to specific plasma membrane domains, these sequences have not been identified for most connexins. It has been reported that the transmembrane domains, but none of the cytoplasmic domains, are required for the delivery of Cx32 to its specific destination in cultured epithelial cells (337). How gap junction channels aggregate and form small junctional plaques is not yet resolved. In ultrastructural studies, small particle aggregates have been found within particle poor, flattened regions of the plasma membrane as junctions are beginning to form between reaggregated cells (255). Growth of gap junctions occurs by incorporation of additional gap junction channels to the plasma membrane followed by their incorporation to the periphery of existing gap junction plaques (174, 331). Imaging of living cells transfected with fluorescent protein-tagged connexins suggests that connexins are delivered to the gap junction plaque in cytoplasmic vesicles (260) that move along microtubules (331). Large junctional plaques can form by coalescence of small junctional plaques (227, 282).
Some incompletely resolved issues are as follows: how connexins are brought together to assemble into connexons, how these connexons are incorporated into the plasma membrane, how gap junctional channels become part of a plaque, and which other proteins/factors participate in these processes. It is possible that no other factors are necessary for channel or plaque formation because purified connexons tend to aggregate in filaments and sheets (557).
B. Gap Junction and Connexin Degradation
Proteins in gap junctions are apparently rather shortlived. Based on in vivo labeling, Fallon and Goodenough (157) estimated that the half-life of a major polypeptide in liver gap junctions was in the range of 5 h. Similarly, pulse-chase experiments have shown that connexins have half-lives of 1-3 h in cultured cells (102, 115, 316, 586). A similar half-life has been measured for Cx43 in intact lens and heart (29, 399). However, at least some of the lens fiber connexins are more stable, with half-lives of 2-3 days or more (38, 247).
Morphological and biochemical approaches have recently been applied to begin to elucidate the mechanisms of gap junction degradation (reviewed in Ref. 49). Electron microscopy studies have implied endosomal internalization of entire junctions forming "annular gap junctions" (Figs. 1B and 3) followed by degradation in lysosomal, multivesicular body, or autophagosomal compartments (260, 325, 326, 375, 397, 408, 447, 489, 533, 616). In vivo, these structures have been most frequently found following pathological insults such as ischemia or during tissue remodeling. Cell fractionation and immunoelectron microscopy studies have shown an association between connexins and lysosomes in some cultured cells (515, 616). Murray et al. (398) found that in SW-13 adrenal cortical tumor cells, many of the cytoplasmic annular Cx43 gap junction profiles are associated with myosin II, but not with tubulin, or vimentin-containing fibers. Disruption of microfilaments resulted in a decrease in the number and an increase in the size of these annular gap junctions, suggesting a role for myosin-containing cytoskeletal elements in annular gap junction turnover (327, 398). A recombinant Cx43 containing tetracysteine tags was recently used to demonstrate that older Cx43 is removed from the center of plaques into pleiomorphic vesicles of widely varying sizes (174).
Recently, investigators have used biochemical or immunochemical techniques to study proteolysis of the subunit gap junction proteins. Studies of Cx43 proteolysis in E36 Chinese hamster ovary cells have shown the major involvement of the ubiquitin-proteasome pathway in Cx43 degradation. In these cells, treatment with the protease inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN) induces accumulation of Cx43 and a significant prolongation of its half-life. Also, Cx43 degradation is abolished when ts20 Chinese hamster ovary cells, which contain a thermolabile ubiquitin activating enzyme, E1, are incubated at the restrictive temperature (309). Immunofluorescence studies in both primary cultures of cardiac myocytes and in established cell lines have shown that Cx43 gap junction plaques as well as the Cx43 polypeptide exhibit rapid turnover rates. The rapid disappearance of Cx43 staining can be blocked by inhibitors of either the proteasome or the lysosome, implicating both proteolytic systems in gap junction degradation (260, 311, 312). These studies have also shown that connexin degradation could be stimulated by cellular stress such as heat shock (311). It is possible that the proteasome is mainly involved in degradation of misfolded connexins (as occurs for many other proteins); this process is called ER-associated degradation and serves as a quality control check point. Misfolded connexins likely are dislocated from the ER and then degraded by the proteasome. The involvement of this pathway during connexin biosynthesis has been recently demonstrated for wild-type Cx43 (612). The participation of both proteasomal and lysosomal pathways in the degradation of Cx43 in the heart has also been demonstrated (29).
The published studies suggest that the preference of one degradative pathway over the other is cell type dependent and that within the same cell type the degradative pathway chosen might depend on the metabolic/pathological state of the cell. The experiments do not allow differentiation between the possibilities of a sequential (e.g., the carboxy tail of Cx43 is degraded in the proteasome while the core of the protein embedded in the membrane is degraded by the lysosome) or parallel (Cx43 can be degraded by the proteasome or the lysosome) pathway for degradation of connexins.
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VI. HEMICHANNELS |
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A. Opening and Closing Hemichannels
The opening of connexin hemichannels was first described based on the swelling and death of Xenopus oocytes injected with the RNA of Cx46 (444). Shortly thereafter, DeVries and Schwartz (125) reported that cultured solitary horizontal cells of the catfish retina express an endogenous current mediated by hemichannels that open upon reduction of the extracellular [Ca2+]. The current is reduced by external [Ca2+] higher than 1 mM, treatment with dopamine, or a weak acid and coincides with blockade of Lucifer yellow uptake. Subsequently, the electrophysiological characterization of macroscopic and single hemichannel currents have been reported for various connexins exogenously expressed in Xenopus oocytes or mammalian cell lines (137, 139, 205, 291, 341, 462, 471, 562, 589, 597, 599).
Opening of hemichannels formed by different connexin types is rather infrequent or absent under resting conditions but can be regulated by several factors. Low extracellular [Ca2+] enhances the opening of hemichannels in the Xenopus oocyte expression system (137, 139), in Novikoff cells (341), in some cardiac myocytes (291), in astrocytes (225, 562), and in transfected human osteo-blast-like cells (492). In addition, opening of Cx43 hemichannels has been induced by mechanical stimulation in cultured astrocytes (562). Agonists of hemichannel opening have also been described. The hemichannel-mediated currents expressed by retinal horizontal cells bathed in low extracellular [Ca2+] are enhanced by the antimalaric drugs quinidine or quinine (363). Quinine-induced dye uptake revealing opening of Cx43 hemichannels has also been observed in astrocytes (562), but not in Cx43 RNA-injected Xenopus oocytes (644). The quinine-induced hemichannel opening cannot be explained solely by the quinine-induced alkalinization because it is still observed using 80 mM buffered HEPES in the recording patch pipette (131). Alendronate, a drug used widely in the treatment of bone diseases, induces opening of hemichannels formed at least by Cx43 (462).
Lens connexins, including rat Cx46, bovine Cx44, and chicken Cx56, form functional hemichannels in Xenopus oocytes bathed in low or even normal extracellular [Ca2+] (137, 139, 205, 444). However, lens fibers have high input resistance, suggesting the absence of open hemichannels at the plasma membrane under physiological conditions. This apparent controversy might be explained by differences in the posttranscriptional modification of these connexins in oocytes with respect to that in lens fibers. These connexins are phosphoproteins and are detected as multiple bands in immunoblots of lens homogenates (40, 205, 444), but when expressed in Xenopus oocytes, they show the same electrophoretic mobilities as in vitro translated proteins (137, 205, 444). Sheep lens fibers containing Cx49 show an increase in dye coupling when treated with a casein kinase I inhibitor, suggesting that phosphorylation of the lens fiber connexin by the endogenous casein kinase I activity leads to closure of gap junction channels (89, 90). A similar mechanism might help in keeping lens fiber hemichannels closed to maintain the high input resistance of lens cells.
Opening of Cx43 hemichannels induced by low extracellular [Ca2+] can be blocked by activation of a PKC-dependent pathway (341, 350), sug
