SMART MODE:

NORMAL GENOMIC

• Cason N, White TW, Cheng S, Goodenough DA, Valdimarsson G
• Molecular cloning, expression analysis, and functional characterization of connexin44.1: a zebrafish lens gap junction protein.
• Dev Dyn. 2001; 221: 238-47
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• The connexin family of genes codes for proteins that oligomerize into a connexon of six subunits to form one half of the gap junction channel. Gap junctions are plasma membrane structures that mediate intercellular communication by joining the cytoplasm of two cells, allowing the passage of small molecules and metabolites, and contributing significantly to the maintenance of tissue homeostasis. The signaling mediated by these junctions appears to be necessary for the correct timing of key developmental events. This communication is especially important in the avascular lens where the intercellular passage of metabolites, second messengers, and ions is necessary to maintain the correct ionic balance in the lens fibre cells, and prevent cataract formation. To characterize the role that the connexin genes play in development, a novel connexin was cloned from zebrafish. A genomic clone was isolated that contained a 1,173 base open reading frame. The nucleotide sequence in this open reading frame shows extensive sequence similarity to mouse connexin50 (Cx50), chicken Cx45.6, sheep Cx49, and human Cx50. The protein encoded by this open reading frame contains 391 amino acids, with a predicted molecular weight of 44.1 kDa and a typical connexin transmembrane topology. By using the LN54 radiation hybrid panel, the Cx44.1 gene was mapped to linkage group 1. Whole-mount in situ hybridization and Northern blot analyses were performed on zebrafish embryos at various developmental stages to characterize the developmental expression of the Cx44.1 message. The ocular lens was the only tissue in which Cx44.1 transcripts were detected. The transcripts were first detected in the lens around 24 hr post fertilization and remained detectable until 120 hr post fertilization. Electrophysiological analysis of Cx44.1 channels revealed gating properties that were virtually identical to the mouse and chicken orthologues of Cx44.1. Copyright 2001 Wiley-Liss, Inc.

• Ahmad S, Martin PE, Evans WH
• Assembly of gap junction channels: mechanism, effects of calmodulin antagonists and identification of connexin oligomerization determinants.
• Eur J Biochem. 2001; 268: 4544-52
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• The assembly of connexins (Cxs) into gap junction intercellular communication channels was studied. An in vitro cell-free synthesis system showed that formation of the hexameric connexon hemichannels involved dimeric and tetrameric connexin intermediates. Cx32 contains two putative cytoplasmic calmodulin-binding sites, and their role in gap junction channel assembly was investigated. The oligomerization of Cx32 into connexons was reversibly inhibited by a calmodulin-binding synthetic peptide, and by W7, a naphthalene sulfonamide calmodulin antagonist. Removing the calmodulin-binding site located at the carboxyl tail of Cx32 limited connexon formation and resulted in an accumulation of intermediate connexin oligomers. This truncation mutant, Cx32Delta215, when transiently expressed in COS-7 cells, accumulated intracellularly and had failed to target to gap junctions. Immunoprecipitation studies suggested that a C-terminal sequence of Cx32 incorporating the calmodulin-binding site was required for the formation of hetero-oligomers of Cx26 and Cx32 but not for Cx32 homomeric association. A chimera, Cx32TM3CFTR, in which the third transmembrane and proposed channel lining sequence of Cx32 was substituted by a transmembrane sequence of the cystic fibrosis transmembrane conductance regulator, did not oligomerize in vitro and it accumulated intracellularly when expressed in COS-7 cells. The results indicate that amino-acid sequences in the third transmembrane domain and a calmodulin-binding domain in the cytoplasmic tail of Cx32 are likely candidates for regulating connexin oligomerization.

• Saffitz JE, Yamada KA
• Closing the gap in understanding the regulation of intercellular communication.
• Cardiovasc Res. 2000; 45: 807-9

• Trosko JE, Chang CC, Wilson MR, Upham B, Hayashi T, Wade M
• Gap junctions and the regulation of cellular functions of stem cells during development and differentiation.
• Methods. 2000; 20: 245-64
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• In multicellular organisms, the role of gap junction intercellular communication (GJIC) in the regulation of cell proliferation, cell differentiation, and apoptosis is becoming increasingly recognized as one of the major cellular functions from the start of the fertilized egg, through normal development of the embryo and fetus, to the sexual maturation of the adult and ultimately to the maintenance of health of the aging adult. Given that the function of this membrane-associated protein channel is to synchronize electrotonic or metabolic functions, differential regulation of function at the transcriptional, translational, and posttranslational levels of a family of highly evolutionarily conserved genes (connexins) needs to be considered. Both inherited mutations and environmental modulation of GJIC could, in principle, affect the function of gap junctions to control cell proliferation, cell differentiation, and apoptosis, thereby leading to a wide variety of pathologies. We review a few techniques used to characterize the ability of stem and progenitor cells to perform GJIC.

• Meda P
• Probing the function of connexin channels in primary tissues.
• Methods. 2000; 20: 232-44
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• Connexin channels provide for a widespread mechanism of cell-to-cell cross-talk within primary tissues, which is mediated by intercellular exchanges of cytoplasmic ions and molecules. Experimental and clinical studies have recently provided evidence that these exchanges are most likely to play multiple roles, which are critical for the proper development and function of primary tissues. There is also increasing evidence that major clinical disorders may result when the formation and function of connexin channels are altered. Still, the physiological functions that the cell-to-cell communication mediated by connexin channels subserve in most primary tissues are still uncertain. Here, I review two approaches that may aid in identifying these specific functions.

• Berthoud VM, Tadros PN, Beyer EC
• Connexin and gap junction degradation.
• Methods. 2000; 20: 180-7
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• Many of the subunit proteins (connexins) that form gap junctions are rather dynamic, with half-lives of only a few hours. Thus, alterations in connexin turnover and degradation may represent significant mechanisms for the regulation of intercellular communication. We describe a pharmacological approach to determining pathways of connexin degradation. Cell cultures are left untreated or treated with inhibitors of lysosomal or proteasomal proteolysis. Changes in connexin levels, localization, or decay curves (derived from pulse-chase experiments) are assessed by immunoblotting, immunofluorescence, and immunoprecipitation, respectively. Such experiments have provided evidence that connexin43 degradation involves both the lysosome and the proteasome.

• Berthoud VM, Beyer EC, Seul KH
• Peptide inhibitors of intercellular communication.
• Am J Physiol Lung Cell Mol Physiol. 2000; 279: 61922-61922

• Musil LS, Le AC, VanSlyke JK, Roberts LM
• Regulation of connexin degradation as a mechanism to increase gap junction assembly and function.
• J Biol Chem. 2000; 275: 25207-15
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• Connexins, the integral membrane protein constituents of gap junctions, are degraded at a rate (t(12) = 1.5-5 h) much faster than most other cell surface proteins. Although the turnover of connexins has been shown to be sensitive to inhibitors of either the lysosome or of the proteasome, how connexins are targeted for degradation and whether this process can be regulated to affect intercellular communication is unknown. We show here that reducing connexin degradation with inhibitors of the proteasome (but not with lysosomal blockers) is associated with a striking increase in gap junction assembly and intercellular dye transfer in cells inefficient in both processes under basal conditions. The effect of proteasome inhibitors on wild-type connexin stability, assembly, and function was mimicked by treatment of assembly-inefficient cells with inhibitors of protein synthesis such as cycloheximide. Sensitivity of connexin degradation to cycloheximide, but not to proteasome inhibitors, was abolished when connexins were rendered structurally abnormal by perturbation of essential disulfide bonds or by mutation. Our findings provide the first evidence that intercellular communication can be up-regulated at the level of connexin turnover and that a short-lived protein may be required for conformationally mature connexins to become substrates of proteasomal degradation.

• Falk MM
• Cell-free synthesis for analyzing the membrane integration, oligomerization, and assembly characteristics of gap junction connexins.
• Methods. 2000; 20: 165-79
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• For gap junction channels to function, their subunit proteins, referred to as connexins, have to be synthesized and inserted into the cell membrane in their native configuration. Like other transmembrane proteins, connexins are synthesized and inserted cotranslationally into the endoplasmic reticulum membrane. Membrane insertion is followed by their assembly and transport to the plasma membrane. Finally, the end-to-end pairing of two half-channels, referred to as connexons, each provided by one of two neighboring cells, and clustering of the channels into larger plaques complete the gap junction channel formation. Gap junction channel formation is further complicated by the potential assembly of homo- as well as heterooligomeric connexons, and the pairing of identical or different connexons into homo- and heterotypic gap junction channels. In this article, I describe the cell-free synthesis approach that we have used to study the biosynthesis of connexins and gap junction channels. Special emphasis is placed on the synthesis of full-length, membrane-integrated connexins, assembly into gap junction connexons, homo- as well as heterooligomerization, and characterization of connexin-specific assembly signals.

• Lampe PD, Lau AF
• Regulation of gap junctions by phosphorylation of connexins.
• Arch Biochem Biophys. 2000; 384: 205-15
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• Gap junctions are a unique type of intercellular junction found in most animal cell types. Gap junctions permit the intercellular passage of small molecules and have been implicated in diverse biological processes, such as development, cellular metabolism, and cellular growth control. In vertebrates, gap junctions are composed of proteins from the "connexin" gene family. The majority of connexins are modified posttranslationally by phosphorylation, primarily on serine amino acids; however, phosphotyrosine has also been detected in connexin from cells coexpressing nonreceptor tyrosine protein kinases. Connexins are targeted by numerous protein kinases, of which some have been identified: protein kinase C, mitogen-activated protein kinase, and the v-Src tyrosine protein kinase. Phosphorylation has been implicated in the regulation of a broad variety of connexin processes, such as the trafficking, assembly/disassembly, degradation, as well as the gating of gap junction channels. This review examines the consequences of connexin phosphorylation for the regulation of gap junctional communication.

• Werner R, Hudder A
• Gap junctions.
• Methods. 2000; 20: 127-8

• Falk MM
• Biosynthesis and structural composition of gap junction intercellular membrane channels.
• Eur J Cell Biol. 2000; 79: 564-74
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• Gap junction channels assemble as dodecameric complexes, in which a hexameric connexon (hemichannel) in one plasma membrane docks end-to-end with a connexon in the membrane of a closely apposed cell to provide direct cell-to-cell communication. Synthesis, assembly, and trafficking of the gap junction channel subunit proteins referred to as connexins, largely appear to follow the general secretory pathway for membrane proteins. The connexin subunits can assemble into homo-, as well as distinct hetero-oligomeric connexons. Assembly appears to be based on specific signals located within the connexin polypeptides. Plaque formation by the clustering of gap junction channels in the plane of the membrane, as well as channel degradation are poorly understood processes that are topics of current research. Recently, we tagged connexins with the autofluorescent reporter green fluorescent protein (GFP), and its cyan (CFP), and yellow (YFP) color variants and combined this reporter technology with single, and dual-color, high resolution deconvolution microscopy, computational volume rendering, and time-lapse microscopy to examine the detailed organization, structural composition, and dynamics of gap junctions in live cells. This technology provided for the first time a realistic, three-dimensional impression of gap junctions as they appear in the plasma membranes of adjoining cells, and revealed an excitingly detailed structural organization of gap junctions never seen before in live cells. Here, I summarize recent progress in areas encompassing the synthesis, assembly and structural composition of gap junctions with a special emphasis on the recent results we obtained using cell-free translation/ membrane-protein translocation, and autofluorescent reporters in combination with live-cell deconvolution microscopy.

• Richard G
• Connexins: a connection with the skin.
• Exp Dermatol. 2000; 9: 77-96
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• The intercellular signaling system mediated by connexin channels is crucial for maintaining tissue homeostasis, growth control, development, and synchronized response of cells to stimuli. This review summarizes the structure, assembly, and properties of the components of the complex and diverse connexin system, and their biological functions in skin. The importance of gap junctional intercellular communication for normal development and differentiation of human epidermis as well as the hearing function of the inner ear is illustrated by the examples of erythrokeratodermia variabilis and palmoplantar keratoderma associated with hearing loss. These autosomal dominant inherited disorders are caused by germline mutations in the connexin genes GJB3 (encoding connexin-31) and GJB2 (encoding connexin-26), respectively. Recent functional studies of individual connexin mutations suggest that they may exert a dominant inhibitory effect on normal connexin channel function and perturb gap junctional intercellular communication, resulting in phenotypic manifestation in patients with these disorders.

• Martin PE, Mambetisaeva ET, Archer DA, George CH, Evans WH
• Analysis of gap junction assembly using mutated connexins detected in Charcot-Marie-Tooth X-linked disease.
• J Neurochem. 2000; 74: 711-20
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• The assembly of gap junction intercellular communication channels was studied by analysis of the molecular basis of the dysfunction of connexin 32 mutations associated with the X-linked form of Charcot-Marie-Tooth disease in which peripheral nervous transmission is impaired. A cell-free translation system showed that six recombinant connexin 32 mutated proteins-four point mutations at the cytoplasmic amino terminus, one at the membrane aspect of the cytoplasmic carboxyl terminus, and a deletion in the intracellular loop-were inserted into microsomal membranes and oligomerised into connexon hemichannels with varying efficiencies. The functionality of the connexons was determined by the ability of HeLa cells expressing the respective connexin cDNAs to transfer Lucifer yellow. The intracellular trafficking properties of the mutated connexins were determined by immunocytochemistry. The results show a relationship between intracellular interruption of connexin trafficking, the efficiency of intercellular communication, and the severity of the disease phenotype. Intracellular retention was explained either by deficiencies in the ability of connexins to oligomerise or by mutational changes at two targeting motifs. The results point to dominance of two specific targeting motifs: one at the amino terminus and one at the membrane aspect of the cytoplasmically located carboxyl tail. An intracellular loop deletion of six amino acids, associated with a mild phenotype, showed partial oligomerisation and low intercellular dye transfer compared with wild-type connexin 32. The results show that modifications in trafficking and assembly of gap junction channels emerge as a major feature of Charcot-Marie-Tooth X-linked disease.

• Nagy JI, Rash JE
• Connexins and gap junctions of astrocytes and oligodendrocytes in the CNS.
• Brain Res Brain Res Rev. 2000; 32: 29-44
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• This review article summarizes early and recent literature on the structure, distribution and composition of gap junctions between astrocytes and oligodendrocytes, and the differential expression of glial connexins in adult and developing mammalian CNS. In addition to an overview of the topic, discussion is focused on the organization of homologous gap junctional interactions between astrocytes and between oligodendrocytes as well as on heterologous junctional coupling between astrocytes and oligodendrocytes. The homotypic and heterotypic nature of these gap junctions is related to the connexins known to be produced by glial cells in the intact brain and spinal cord. Emphasis is placed on the ultrastructural level of analysis required to attribute gap junction and connexin deployment to particular cell types and subcellular locations. Our aim is to provide a firm basis for consideration of anticipated rapid advances in understanding of structural relationships of gap junctions and connexins within the glial gap junctional syncytium. Conclusions to date suggest that the glial syncytium is more complex than previously appreciated and that glial pathways of junctional communication may not only be determined by the presence of gap junctions, but also by the connexin composition and conductance regulation of junctional channels.

• White TW, Bruzzone R
• Intercellular communication in the eye: clarifying the need for connexin diversity.
• Brain Res Brain Res Rev. 2000; 32: 130-7
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• In the vertebrate eye, virtually every cell type is directly coupled to its neighbors by intercellular channels present in gap junctions. Although these structures share the common property of allowing adjacent cells to directly exchange ions, second messengers and small metabolites, intercellular channels in the eye also play a specific role in distinct functions such as neuronal transmission at electrotonic synapses in the retina, and the maintenance of homeostasis in the avascular lens. The structural proteins comprising these channels, the connexins (Cx), are a multigene family of which many members are expressed in the eye, even in the same cell type. This molecular heterogeneity poses the crucial question of whether and how a diversity in gap junctional structural proteins influences intercellular communication in ocular tissues. This review will focus on two recent advances in the understanding of connexin diversity in regard to the eye. First, connexin knockouts have demonstrated that postnatal development and homeostasis in the lens requires multiple connexin proteins. Secondly, functional characterization of new connexins that are abundantly expressed in the retina has revealed biophysical properties that mimic those recorded from retinal neurons.

• Saez JC et al.
• Gap junctions in cells of the immune system: structure, regulation and possible functional roles.
• Braz J Med Biol Res. 2000; 33: 447-55
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• Gap junction channels are sites of cytoplasmic communication between contacting cells. In vertebrates, they consist of protein subunits denoted connexins (Cxs) which are encoded by a gene family. According to their Cx composition, gap junction channels show different gating and permeability properties that define which ions and small molecules permeate them. Differences in Cx primary sequences suggest that channels composed of different Cxs are regulated differentially by intracellular pathways under specific physiological conditions. Functional roles of gap junction channels could be defined by the relative importance of permeant substances, resulting in coordination of electrical and/or metabolic cellular responses. Cells of the native and specific immune systems establish transient homo- and heterocellular contacts at various steps of the immune response. Morphological and functional studies reported during the last three decades have revealed that many intercellular contacts between cells in the immune response present gap junctions or "gap junction-like" structures. Partial characterization of the molecular composition of some of these plasma membrane structures and regulatory mechanisms that control them have been published recently. Studies designed to elucidate their physiological roles suggest that they might permit coordination of cellular events which favor the effective and timely response of the immune system.

• Saffitz JE
• Regulation of intercellular coupling in acute and chronic heart disease.
• Braz J Med Biol Res. 2000; 33: 407-13
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• Effective pump function of the heart depends on the precise control of spatial and temporal patterns of electrical activation. Accordingly, the distribution and function of gap junction channels are important determinants of the conduction properties of myocardium and undoubtedly play other roles in intercellular communication crucial to normal cardiac function. Recent advances have begun to elucidate mechanisms by which the heart regulates intercellular electrical coupling at gap junctions in response to stress or injury. Although responses to increased load or injury are generally adaptive in nature, remodeling of intercellular junctions under conditions of severe stress creates anatomic substrates conducive to the development of lethal ventricular arrhythmias. Potential mechanisms controlling the level of intercellular communication in the heart include regulation of connexin turnover dynamics and phosphorylation.

• Beyer EC, Gemel J, Seul KH, Larson DM, Banach K, Brink PR
• Modulation of intercellular communication by differential regulation and heteromeric mixing of co-expressed connexins.
• Braz J Med Biol Res. 2000; 33: 391-7
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• Intercellular communication may be regulated by the differential expression of subunit gap junction proteins (connexins) which form channels with differing gating and permeability properties. Endothelial cells express three different connexins (connexin37, connexin40, and connexin43) in vivo. To study the differential regulation of expression and synthesis of connexin37 and connexin43, we used cultured bovine aortic endothelial cells which contain these two connexins in vitro. RNA blots demonstrated discordant expression of these two connexins during growth to confluency. RNA blots and immunoblots showed that levels of these connexins were modulated by treatment of cultures with transforming growth factor-ss1. To examine the potential ability of these connexins to form heteromeric channels (containing different connexins within the same hemi-channel), we stably transfected connexin43-containing normal rat kidney (NRK) cells with connexin37 or connexin40. In the transfected cells, both connexin proteins were abundantly produced and localized in identical distributions as detected by immunofluorescence. Double whole-cell patch-clamp studies showed that co-expressing cells exhibited unitary channel conductances and gating characteristics that could not be explained by hemi-channels formed of either connexin alone. These observations suggest that these connexins can readily mix with connexin43 to form heteromeric channels and that the intercellular communication between cells is determined not only by the properties of individual connexins, but also by the interactions of those connexins to form heteromeric channels with novel properties. Furthermore, modulation of levels of the co-expressed connexins during cell proliferation or by cytokines may alter the relative abundance of different heteromeric combinations.

• Nicholson BJ, Weber PA, Cao F, Chang H, Lampe P, Goldberg G
• The molecular basis of selective permeability of connexins is complex and includes both size and charge.
• Braz J Med Biol Res. 2000; 33: 369-78
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• Although gap junction channels are still widely viewed as large, non-specific pores connecting cells, the diversity in the connexin family has led more attention to be focused on their permeability characteristics. We summarize here the current status of these investigations, both published and on-going, that reveal both charge and size selectivity between gap junction channels composed of different connexins. In particular, this review will focus on quantitative approaches that monitor the expression level of the connexins, so that it is clear that differences that are seen can be attributed to channel properties. The degree of selectivity that is observed is modest compared to other channels, but is likely to be significant for biological molecules that are labile within the cell. Of particular relevance to the in vivo function of gap junctions, recent studies are summarized that demonstrate that the connexin phenotype can control the nature of the endogenous traffic between cells, with consequent effects on biological effects of gap junctions such as tumor suppression.

• Rozental R, Carvalho AC, Spray DC
• Gap junctions in the cardiovascular and immune systems.
• Braz J Med Biol Res. 2000; 33: 365-8
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• Gap junctions are clusters of intercellular channels directly connecting the cytoplasm of adjacent cells. These channels are formed by proteins named connexins and are present in all metazoan organisms where they serve diverse functions ranging from control of cell growth and differentiation to electric conduction in excitable tissues. In this overview we describe the presence of connexins in the cardiovascular and lympho-hematopoietic systems giving the reader a summary of the topics to be covered throughout this edition and a historical perspective of the discovery of gap junctions in the immune system.

• Jongsma HJ
• Diversity of gap junctional proteins: does it play a role in cardiac excitation?
• J Cardiovasc Electrophysiol. 2000; 11: 228-30

• Delmar M
• Gap junctions as active signaling molecules for synchronous cardiac function.
• J Cardiovasc Electrophysiol. 2000; 11: 118-20

• White TW, Bruzzone R
• Gap junctions: fates worse than death?
• Curr Biol. 2000; 10: 6858-6858
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• The reasons for the molecular heterogeneity of connexin channels in vivo remain unclear. Functional replacement of one connexin gene with another has now revealed unexpected phenotypes and shows that cellular homeostasis depends not simply on cell-cell communication but also on the correct types of connexin.

• Diez JA, Ahmad S, Evans WH
• Assembly of heteromeric connexons in guinea-pig liver en route to the Golgi apparatus, plasma membrane and gap junctions.
• Eur J Biochem. 1999; 262: 142-8
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• Guinea-pig liver gap junctions are constructed from approximately equal amounts of connexins 26 and 32. The assembly of these connexins into connexon hemichannels and gap junctions was studied using antibodies specific to each connexin. Intracellular membranes were shown to contain low amounts of connexin 26 relative to connexin 32 in contrast to the equal connexin ratios detected in lateral plasma membranes and gap junctions. Assembly of gap junctions requires oligomerization of connexins into connexons that may be homomeric or heteromeric. Immunoprecipitation using antibodies to connexins 26 and 32 showed that liver gap junctions were heteromeric. A chemical cross-linking procedure showed that connexons solubilized from guinea-pig liver gap junctions were constructed of hexameric assemblies of connexin subunits. The intracellular site of oligomerization of connexins was investigated by velocity sedimentation in sucrose-detergent gradients. Oligomers of connexins 26 and 32 were extensively present in Golgi membranes and oligomeric intermediates, especially of connexin 26, were detected in the endoplasmic reticulum-Golgi intermediate subcellular fraction. Two intracellular trafficking pathways that may account for the delivery of connexin 26 to the plasma membrane and explain the heteromeric nature of liver gap junctions are discussed.

• Simon AM
• Gap junctions: more roles and new structural data.
• Trends Cell Biol. 1999; 9: 169-70

• Suchyna TM, Nitsche JM, Chilton M, Harris AL, Veenstra RD, Nicholson BJ
• Different ionic selectivities for connexins 26 and 32 produce rectifying gap junction channels.
• Biophys J. 1999; 77: 2968-87
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• The functional diversity of gap junction intercellular channels arising from the large number of connexin isoforms is significantly increased by heterotypic interactions between members of this family. This is particularly evident in the rectifying behavior of Cx26/Cx32 heterotypic channels (. Proc. Natl. Acad. Sci. USA. 88:8410-8414). The channel properties responsible for producing the rectifying current observed for Cx26/Cx32 heterotypic gap junction channels were determined in transfected mouse neuroblastoma 2A (N2A) cells. Transfectants revealed maximum unitary conductances (gamma(j)) of 135 pS for Cx26 and 53 pS for Cx32 homotypic channels in 120 mM KCl. Anionic substitution of glutamate for Cl indicated that Cx26 channels favored cations by 2.6:1, whereas Cx32 channels were relatively nonselective with respect to charge. In Cx26/Cx32 heterotypic cell pairs, the macroscopic fast rectification of the current-voltage relationship was fully explained at the single-channel level by a rectifying gamma(j) that increased by a factor of 2.9 as the transjunctional voltage (V(j)) changed from -100 to +100 mV with the Cx26 cell as the positive pole. A model of electrodiffusion of ions through the gap junction pore based on Nernst-Planck equations for ion concentrations and the Poisson equation for the electrical potential within the junction is developed. Selectivity characteristics are ascribed to each hemichannel based on either pore features (treated as uniform along the length of the hemichannel) or entrance effects unique to each connexin. Both analytical GHK approximations and full numerical solutions predict rectifying characteristics for Cx32/Cx26 heterotypic channels, although not to the full extent seen empirically. The model predicts that asymmetries in the conductance/permeability properties of the hemichannels (also cast as Donnan potentials) will produce either an accumulation or a depletion of ions within the channel, depending on voltage polarity, that will result in rectification.

• Bevans CG, Harris AL
• Regulation of connexin channels by pH. Direct action of the protonated form of taurine and other aminosulfonates.
• J Biol Chem. 1999; 274: 3711-9
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• Protonated aminosulfonate compounds directly inhibit connexin channel activity. This was demonstrated by pH-dependent connexin channel activity in Good's pH buffers (MES (4-morpholineethanesulfonic acid), HEPES, and TAPS (3-?[2-hydroxy-1, 1-bis(hydroxymethyl)ethyl]amino]-1-propanesulfonic acid)) that have an aminosulfonate moiety in common and by the absence of pH-dependent channel activity in pH buffers without an aminosulfonate moiety (maleate, Tris, and bicarbonate). The pH-activity relation was shifted according to the pKa of each aminosulfonate pH buffer. At constant pH, increased aminosulfonate concentration inhibited channel activity. Taurine, a ubiquitous cytoplasmic aminosulfonic acid, had the same effect at physiological concentrations. These data raise the possibility that effects on connexin channel activity previously attributed to protonation of connexin may be mediated instead by protonation of cytoplasmic regulators, such as taurine. Modulation by aminosulfonates is specific for heteromeric connexin channels containing connexin-26; it does not occur significantly for homomeric connexin-32 channels. The identification of taurine as a cytoplasmic compound that directly interacts with and modulates connexin channel activity is likely to facilitate understanding of cellular modulation of connexin channels and lead to the development of reagents for use in structure-function studies of connexin protein.

• Bevans CG, Harris AL
• Direct high affinity modulation of connexin channel activity by cyclic nucleotides.
• J Biol Chem. 1999; 274: 3720-5
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• Connexin channels mediate molecular communication between cells. However, positive identification of biological ligands that directly and noncovalently modulate their activity has been elusive. This study demonstrates a high affinity inhibition of connexin channels by the purine cyclic monophosphates cAMP and cGMP. Purified homomeric connexin-32 and heteromeric connexin-32/connexin-26 channels were inhibited by exposure to nanomolar levels of the nucleotides prior to incorporation into membranes. Access to the site of action, or affinity for the nucleotides, was greatly reduced following incorporation of the connexin channels into membranes, where inhibition required millimolar concentrations of the nucleotides. The high affinity inhibition did not occur with similar concentrations of AMP, ADP, ATP, cTMP, or cCMP. This is the first report of a direct ligand effect on connexin channel function. The high affinity and specificity of the inhibition suggest a biological role in control of connexin channels and also may lead to the application of affinity reagents to study of connexin channel structure-function.

• Jalife J, Morley GE, Vaidya D
• Connexins and impulse propagation in the mouse heart.
• J Cardiovasc Electrophysiol. 1999; 10: 1649-63
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• Gap junction channels are essential for normal cardiac impulse propagation. Three gap junction proteins, known as connexins, are expressed in the heart: Cx40, Cx43, and Cx45. Each of these proteins forms channels with unique biophysical and electrophysiologic properties, as well as spatial distribution of expression throughout the heart. However, the specific functional role of the individual connexins in normal and abnormal propagation is unknown. The availability of genetically engineered mouse models, together with new developments in optical mapping technology, makes it possible to integrate knowledge about molecular mechanisms of intercellular communication and its regulation with our growing understanding of the microscopic and global dynamics of electrical impulse propagation during normal and abnormal cardiac rhythms. This article reviews knowledge on the mechanisms of cardiac impulse propagation, with particular focus on the role of cardiac connexins in electrical communication between cells. It summarizes results of recent studies on the electrophysiologic consequences of defects in the functional expression of specific gap junction channels in mice lacking either the Cx43 or Cx40 gene. It also reviews data obtained in a transgenic mouse model in which cell loss and remodeling of gap junction distribution leads to increased susceptibility to arrhythmias and sudden cardiac death. Overall, the results demonstrate that these are potentially powerful strategies for studying fundamental mechanisms of cardiac electrical activity and for testing the hypothesis that certain cardiac arrhythmias involve gap junction or other membrane channel dysfunction. These new approaches, which permit one to manipulate electrical wave propagation at the molecular level, should provide new insight into the detailed mechanisms of initiation, maintenance, and termination of cardiac arrhythmias, and may lead to more effective means to treat arrhythmias and prevent sudden cardiac death.

• Lo CW
• Genes, gene knockouts, and mutations in the analysis of gap junctions.
• Dev Genet. 1999; 24: 1-4
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• Gop junctions are cell junctions found between most cells and tissues. They contain membrane channels that mediate the cell-to-cell diffusion of ions, metabolites, and small cell signaling molecules. Cell-cell communication mediated by gap junctions has been proposed to have a variety of functions, including roles in regulating events in development, cell differentiation, and cell growth and proliferation. The analysis of these possibilities has been confounded by the fact that there are over a dozen connexin genes encoding polypeptides that make up vertebrate gap junctions. This complexity, coupled with the fact that most cells express multiple connexin isotypes, likely explains why recent studies using reverse genetic and genetic approaches to disrupt connexin gene function have yielded only limited insights into the physiological roles of gap junctions. Nevertheless, studies in vivo and in vitro together have provided evidence for gap junctions being involved in the regulation of cell metabolism, growth, and differentiation in restricted cell and tissue types. Surprisingly, studies in invertebrates suggest that their gap junctions are encoded not by connexins, but by a family of proteins referred to as innexins. Analysis of various Drosophila and C. elegans mutants suggest that innexins may be functional homologs to the connexins. However, whether innexins are the elusive invertebrate gap junction proteins or, rather, accessory proteins that facilitate gap junction formation remains an open question. Given the rapid progress being made in the cloning and functional analysis of gap junctions in many diverse species, confusion and difficulties with nomenclature are coming to a head in this rapidly expanding field. It may be timely to form a Nomenclature Committee to establish a uniform classification scheme for naming gap junction proteins.

• Francis D, Stergiopoulos K, Ek-Vitorin JF, Cao FL, Taffet SM, Delmar M
• Connexin diversity and gap junction regulation by pHi.
• Dev Genet. 1999; 24: 123-36
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• The molecular mechanisms controlling pH-sensitivity of gap junctions formed of two different connexins are yet to be determined. We used a proton-sensitive fluorophore and electrophysiological techniques to correlate changes in intracellular pH (pHi) with electrical coupling between connexin-expressing Xenopus oocytes. The pH sensitivities of alpha 3 (connexin46), alpha 2 (connexin38), and alpha 1 (connexin43) were studied when these proteins were expressed as: 1) nonjunctional hemichannels (for alpha 3 and alpha 2), 2) homotypic gap junctions, and 3) heterotypic gap junctions. We found that alpha 3 hemichannels are sensitive to changes in pHi within a physiological range (pKa = 7.13 +/- 0.03; Hill coefficient = 3.25 +/- 1.73; n = 8; mean +/- SEM); an even more alkaline pKa was obtained for alpha 2 hemichannels (pKa = 7.50 +/- 0.03; Hill coefficient = 3.22 +/- 0.66; n = 13). The pH sensitivity curves of alpha 2 and alpha 3 homotypic junctions were indistinguishable from those recorded from hemichannels of the same connexin. Based on a comparison of pKa values, both alpha 3 and alpha 2 gap junctions were more pHi-dependent than alpha 1. The pH sensitivity of alpha 2-containing heterotypic junctions could not be predicted from the behavior of the two connexons in the pair. When alpha 2 was paired with alpha 3, the pH sensitivity curve was similar to that obtained from alpha 2 homotypic pairs. Yet, pairing alpha 2 with alpha 1 shifted the curve similar to homotypic alpha 1 channels. Pairing alpha 2 with a less pH sensitive mutant of alpha 1 (M257) yielded the same curve as when alpha 1 was used. However, the pH sensitivity curve of alpha 3/alpha 1 channels was similar to alpha 3/alpha 3, while alpha 3/M257 was indistinguishable from alpha 3/alpha 1. Our results could not be consistently predicted by a probabilistic model of two independent gates in series. The data show that dissimilarities in the pH regulation of gap junctions are due to differences in the primary sequence of connexins. Moreover, we found that pH regulation is an intrinsic property of the hemichannels, but pH sensitivity is modified by the interactions between connexons. These interactions should provide a higher level of functional diversity to gap junctions that are formed by more than one connexin.

• Yamasaki H, Krutovskikh V, Mesnil M, Tanaka T, Zaidan-Dagli ML, Omori Y
• Role of connexin (gap junction) genes in cell growth control and carcinogenesis.
• C R Acad Sci III. 1999; 322: 151-9
• Display abstract

• Gap junctional intercellular communication (GJIC) is considered to play a key role in the maintenance of tissue independence and homeostasis in multicellular organisms by controlling the growth of GJIC-connected cells. Gap junction channels are composed of connexin molecules and, so far, more than a dozen different connexin genes have been shown to be expressed in mammals. Reflecting the importance of GJIC in various physiological functions, deletion of different connexin genes from mice results in various disorders, including cancers, heart malformation or conduction abnormality, cataract, etc. The possible involvement of aberrant GJIC in abnormal cell growth and carcinogenesis has long been postulated and recent studies in our own and other laboratories have confirmed that expression and function of connexin genes play an important role in cell growth control. Thus, almost all malignant cells show altered homologous and/or heterologous GJIC and are often associated with aberrant expression or localization of connexins. Aberrant localization of connexins in some tumour cells is associated with lack of function of cell adhesion molecules, suggesting the importance of cell-cell recognition for GJIC. Transfection of connexin genes into tumorigenic cells restores normal cell growth, supporting the idea that connexins form a family of tumour-suppressor genes. Some studies also show that specific connexins may be necessary to control growth of specific cell types. We have produced various dominant-negative mutants of Cx26, Cx32 and Cx43 and showed that some of them prevent the growth control exerted by the corresponding wild-type genes. However, we have found that connexins 32, 37 and 43 genes are rarely mutated in tumours. In some of these studies, we noted that connexin expression per se, rather than GJIC level, is more closely related to growth control, suggesting that connexins may have a GJIC-independent function. We have recently created a transgenic mouse strain in which a mutant Cx32 is specifically overexpressed in the liver. Studies with such mice indicate that Cx32 plays a key role in liver regeneration after partial hepatectomy. A decade ago, we proposed a method to enhance killing of cancer cells by diffusion of therapeutic agents through GJIC. Recently, we and others have shown that GJIC is responsible for the bystander effect seen in HSV-tk/ganciclovir gene therapy. Thus, connexin genes can exert dual effects in tumour control: tumour suppression and a bystander effect for cancer therapy.

• Yamasaki H et al.
• Connexins in tumour suppression and cancer therapy.
• Novartis Found Symp. 1999; 219: 241-54
• Display abstract

• Malignant cells usually show altered gap junctional intercellular communication and are often associated with aberrant expression or localization of connexins. Transfection of connexin genes into tumorigenic cells restores normal cell growth, suggesting that connexins form a family of tumour suppressor genes. Some studies have also shown that specific connexins may be necessary to control growth of specific cell types. Although we have found that genes encoding connexin32 (Cx32; beta 1), Cx37 (alpha 4) and Cx43 (alpha 1) are rarely mutated in tumours, our recent studies suggest that methylation of the connexin gene promoter may be a mechanism by which connexin gene expression is down-regulated in certain tumors. We have produced various dominant negative mutants of the genes encoding Cx26 (beta 2), Cx32 and Cx43, some of which prevent the growth control exerted by the corresponding wild-type genes. A decade ago, we proposed a method to enhance killing of cancer cells by diffusion of therapeutic agents through gap junctions. Recently, we and others have shown that gap junctional intercellular communication is responsible for the bystander effect seen in herpes simplex virus thymidine kinase/ganciclovir gene therapy. Thus, connexin genes can exert dual effects in tumour control: tumour suppression and a bystander effect for cancer therapy.

• Kumar NM
• Molecular biology of the interactions between connexins.
• Novartis Found Symp. 1999; 219: 6-16
• Display abstract

• The protein structural component of gap junctions is the connexin. Studies on the association properties of the connexins to form heteromeric connexons and heterotypic gap junctions are necessary for a complete understanding of the role of different connexins in gap junction function. The connexins are coded by a multigene family consisting of at least 16 members. Most cells express multiple types of connexin that can potentially associate to form gap junction channels containing more than one type of connexin. The permeability and gating characteristics of gap junction channels are dependent on the isoform and post-translational modifications present on the connexins and their association properties. Together with an observed selectivity in the association properties of the different connexins and the development of more specific perturbation approaches, these studies have provided insights into the significance of connexin diversity and the temporal expression patterns for connexins that have been determined in vivo in both developmental and differentiating systems.

• Evans WH, Ahmad S, Diez J, George CH, Kendall JM, Martin PE
• Trafficking pathways leading to the formation of gap junctions.
• Novartis Found Symp. 1999; 219: 44-54
• Display abstract

• This chapter reports the mechanisms resulting in the assembly of gap junction intercellular communication channels. The connexin channel protein subunits are required to oligomerize into hexameric hemichannels (connexons) that may be homoor heteromeric in composition. Pairing of connexons in contacting cells leads to the formation of a gap junction unit. Subcellular fractionation studies using guinea-pig liver showed that oligomerization of connexins was complete on entry into Golgi, and that connexons showed heteromeric properties. The low ratio of connexin26 (Cx26; beta 2) relative to Cx32 (beta 1) in endomembranes compared to the approximately equal ratios found in plasma membranes and gap junctions suggest that Cx26 takes a non-classical route to the plasma membrane. Cultured cells, expressing connexin-aequorin chimeras, also provided evidence that Cx26 takes a more rapid non-classical route to the plasma membrane, because brefeldin A, a drug that disrupts the Golgi, had minimal effects on trafficking of Cx26 to the plasma membrane in contrast to its disruption of Cx32 trafficking. Finally, a cell-free approach for studying synthesis of connexons provided further evidence that Cx26 showed membrane insertion properties compatible with a more direct intracellular route to gap junctions. The presence of dual gap junction assembly pathways can explain many of the differential properties exhibited by connexins in cells.

• Sulkowski S, Sulkowska M, Skrzydlewska E
• Gap junctional intercellular communication and carcinogenesis.
• Pol J Pathol. 1999; 50: 227-33
• Display abstract

• Intercellular communications are indispensable for the maintenance of homeostasis in the multicellular organisms. Gap-type junctions are one of the most common and perhaps most interesting intercellular connections. Of various communication systems gap junctional intercellular communication is the only means for cells to directly exchange signals. Substantial progress has recently been made in the studies on the role of gap junctions both in experimental and human tumorigenesis. The present study is a review of the potential mechanisms associated with gap junctional cellular communication included in non-genotoxic tumorigenesis.

• White TW, Paul DL
• Genetic diseases and gene knockouts reveal diverse connexin functions.
• Annu Rev Physiol. 1999; 61: 283-310
• Display abstract

• Intercellular channels present in gap junctions allow cells to share small molecules and thus coordinate a wide range of behaviors. Remarkably, although junctions provide similar functions in all multicellular organisms, vertebrates and invertebrates use unrelated gene families to encode these channels. The recent identification of the invertebrate innexin family opens up powerful genetic systems to studies of intercellular communication. At the same time, new information on the physiological roles of vertebrate connexins has emerged from genetic studies. Mutations in connexin genes underlie a variety of human diseases, including deafness, demyelinating neuropathies, and lens cataracts. In addition, gene targeting of connexins in mice has provided new insights into connexin function and the significance of connexin diversity.

• Peracchia C, Wang XG, Peracchia LL
• Is the chemical gate of connexins voltage sensitive? Behavior of Cx32 wild-type and mutant channels.
• Am J Physiol. 1999; 276: 136173-136173
• Display abstract

• Connexin channels are gated by transjunctional voltage (Vj) or CO2 via distinct mechanisms. The cytoplasmic loop (CL) and arginines of a COOH-terminal domain (CT1) of connexin32 (Cx32) were shown to determine CO2 sensitivity, and a gating mechanism involving CL-CT1 association-dissociation was proposed. This study reports that Cx32 mutants, tandem, 5R/E, and 5R/N, designed to weaken CL-CT1 interactions, display atypical Vj and CO2 sensitivities when tested heterotypically with Cx32 wild-type channels in Xenopus oocytes. In tandems, two Cx32 monomers are linked NH2-to-COOH terminus. In 5R/E and 5R/N mutants, glutamates or asparagines replace CT1 arginines. On the basis of the intriguing sensitivity of the mutant-32 channel to Vj polarity, the existence of a "slow gate" distinct from the conventional Vj gate is proposed. To a lesser extent the slow gate manifests itself also in homotypic Cx32 channels. Mutant-32 channels are more CO2 sensitive than homotypic Cx32 channels, and CO2-induced chemical gating is reversed with relative depolarization of the mutant oocyte, suggesting Vj sensitivity of chemical gating. A hypothetical pore-plugging model involving an acidic cytosolic protein (possibly calmodulin) is discussed.

• He DS, Jiang JX, Taffet SM, Burt JM
• Formation of heteromeric gap junction channels by connexins 40 and 43 in vascular smooth muscle cells.
• Proc Natl Acad Sci U S A. 1999; 96: 6495-500
• Display abstract

• Connexin (Cx) 43 and Cx40 are coexpressed in several tissues, including cardiac atrial and ventricular myocytes and vascular smooth muscle. It has been shown that these Cxs form homomeric/homotypic channels with distinct permeability and gating properties but do not form functional homomeric/heterotypic channels. If these Cxs were to form heteromeric channels, they could display functional properties not well predicted by the homomeric forms. We assessed this possibility by using A7r5 cells, an embryonic rat aortic smooth muscle cell line that coexpresses Cxs 43 and 40. Connexons (hemichannels), which were isolated from these cells by density centrifugation and immunoprecipitated with antibody against Cx43, contained Cx40. Similarly, antibody against Cx40 coimmunoprecipitated Cx43 from the same connexon fraction but only Cx40 from Cx (monomer) fractions. These results indicate that heteromeric connexons are formed by these Cxs in the A7r5 cells. The gap junction channels formed in the A7r5 cells display many unitary conductances distinct from homomeric/homotypic Cx43 or Cx40 channels. Voltage-dependent gating parameters in the A7r5 cells are also quite variable compared with cells that express only Cx40 or Cx43. These data indicate that Cxs 43 and 40 form functional heteromeric channels with unique gating and conductance properties.

• Yamasaki H, Omori Y, Zaidan-Dagli ML, Mironov N, Mesnil M, Krutovskikh V
• Genetic and epigenetic changes of intercellular communication genes during multistage carcinogenesis.
• Cancer Detect Prev. 1999; 23: 273-9
• Display abstract

• During multistage carcinogenesis, the functions of several key genes involved in cell growth control must be damaged. Such genes include not only those involved in cell cycle control of individual cells, but also those involved in the coordination of cell growth throughout a given tissue through cell-cell communication. The most intimate form of intercellular communication is mediated by gap junctions. Gap junctional intercellular communication (GJIC) is known to transfer small water soluble molecules, including cAMP and IP3, from the cytoplasm of one cell to that of its neighbors; the growth of a given GJIC-associated cell is thus kept in check by other GJIC-connected cells. Most tumor cells have a reduced ability to communicate among themselves and/or with surrounding normal cells, confirming the importance of intact GJIC in growth control. When connexin (gap junction protein) genes are transfected into such cells, normal cell growth control is often recovered. Certain dominant-negative mutant connexin genes can reverse such tumor suppression. While these results suggest that connexin genes form a family of tumor suppressor genes, so far we have found no connexin gene mutations in human tumors; only two connexin gene mutations were found in chemically induced rat tumors. On the other hand, our recent studies suggest that connexin genes may be inactivated by hypermethylation of their promoter regions, suggesting that epigenetic inactivation of connexin genes may be a mechanism of GJIC disturbance in certain tumors. However, in many tumor cells connexins are normally expressed but aberrantly localized. The mechanisms of aberrant localization of connexins include lack of an appropriate cell-cell recognition apparatus and aberrant phosphorylation of connexins. These results suggest that GJIC disorders may occur not only because of aberrant expression of connexin genes themselves, but also as a result of disruption of various control mechanisms of the protein functions.

• Yeager M, Unger VM, Falk MM
• Synthesis, assembly and structure of gap junction intercellular channels.
• Curr Opin Struct Biol. 1998; 8: 517-24
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• Gap junction membrane channels assemble as dodecameric complexes, in which a hexameric hemichannel (connexon) in one plasma membrane docks end to end with a connexon in the membrane of a closely apposed cell. Steps in the synthesis, assembly and turnover of gap junction channels appear to follow the general secretory pathway for membrane proteins. In addition to homo-oligomeric connexons, different connexin polypeptide subunits can also assemble as hetero-oligomers. The ability to form homotypic and heterotypic channels that consist of two identical or two different connexons, respectively, adds even greater versatility to the functional modulation of gap junction channels. Electron cryocrystallography of recombinant gap junction channels has recently provided direct evidence for alpha-helical folding of at least two of the transmembrane domains within each connexin subunit. The potential to correlate the structure and biochemistry of gap junction channels with recently identified human diseases involving connexin mutations makes this a particularly exciting area of research.

• Perkins GA, Goodenough DA, Sosinsky GE
• Formation of the gap junction intercellular channel requires a 30 degree rotation for interdigitating two apposing connexons.
• J Mol Biol. 1998; 277: 171-7
• Display abstract

• 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.

• Phelan P, Stebbings LA, Baines RA, Bacon JP, Davies JA, Ford C
• Drosophila Shaking-B protein forms gap junctions in paired Xenopus oocytes.
• Nature. 1998; 391: 181-4
• Display abstract

• In most multicellular organisms direct cell-cell communication is mediated by the intercellular channels of gap junctions. These channels allow the exchange of ions and molecules that are believed to be essential for cell signalling during development and in some differentiated tissues. Proteins called connexins, which are products of a multigene family, are the structural components of vertebrate gap junctions. Surprisingly, molecular homologues of the connexins have not been described in any invertebrate. A separate gene family, which includes the Drosophila genes shaking-B and l(1)ogre, and the Caenorhabditis elegans genes unc-7 and eat-5, encodes transmembrane proteins with a predicted structure similar to that of the connexins. shaking-B and eat-5 are required for the formation of functional gap junctions. To test directly whether Shaking-B is a channel protein, we expressed it in paired Xenopus oocytes. Here we show that Shaking-B localizes to the membrane, and that its presence induces the formation of functional intercellular channels. To our knowledge, this is the first structural component of an invertebrate gap junction to be characterized.

• Steinberg TH
• Gap junction function: the messenger and the message.
• Am J Pathol. 1998; 152: 851-4

• Yeager M
• Structure of cardiac gap junction intercellular channels.
• J Struct Biol. 1998; 121: 231-45
• Display abstract

• Gap junction proteins, termed connexins, constitute a multigene family of polytopic membrane channel proteins that have four hydrophobic transmembrane domains with the N- and C-termini located on the cytoplasmic membrane face. The principal gap junction protein in the heart, alpha 1 connexin (also designated Cx43), mediates action potential propagation between cells in order to synchronize cardiac contraction. alpha 1 connexin channels are concentrated in gap junction plaques located in the intercalated disks. The intercellular channel is formed by the docking of two hemi-channels, termed connexons, formed by a ring of six 43-kDa alpha 1 connexin subunits. Each subunit is asymmetric with an axial ratio of 4-5:1 with approximately 20 A extending into the extracellular gap approximately 50 A spanning the lipid bilayer and approximately 50 A extending into the cytoplasmic space. We have recently grown two-dimensional crystals of a recombinant C-terminal truncation mutant of alpha 1 connexin (designated alpha 1Cx263T) that are ordered to better than 7 A resolution. Projection density maps derived by electron cryocrystallography revealed that the intercellular channel is lined by six alpha-helices, and there is a second ring of six alpha-helices at the interface with the membrane lipids. These rings of alpha-helices are staggered by 30 degrees, which predicts that the two connexons in the channel are staggered by 30 degrees such that each connexin subunit in one connexon interacts with two subunits in the apposed connexon. Such a quaternary arrangement may confer stability in the docking of the connexons to form a tight electrical seal for intercellular current flow during cardiac conduction.

• Dermietzel R, Spray DC
• From neuro-glue ('Nervenkitt') to glia: a prologue.
• Glia. 1998; 24: 1-7

• Bevans CG, Kordel M, Rhee SK, Harris AL
• Isoform composition of connexin channels determines selectivity among second messengers and uncharged molecules.
• J Biol Chem. 1998; 273: 2808-16
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• Intercellular connexin channels (gap junction channels) have long been thought to mediate molecular signaling between cells, but the nature of the signaling has been unclear. This study shows that connexin channels from native tissue have selective permeabilities, partially based on pore diameter, that discriminate among cytoplasmic second messenger molecules. Permeability was assessed by measurement of selective loss/retention of tracers from liposomes containing reconstituted connexin channels. The tracers employed were tritiated cyclic nucleotides and a series of oligomaltosaccharides derivatized with a small uncharged fluorescent moiety. The data define different size cut-off limits for permeability through homomeric connexin-32 channels and through heteromeric connexin-32/connexin-26 channels. Connexin-26 contributes to a narrowed pore. Both cAMP and cGMP were permeable through the homomeric connexin-32 channels. cAMP was permeable through only a fraction of the heteromeric channels. Surprisingly, cGMP was permeable through a substantially greater fraction of the heteromeric channels than was cAMP. The data suggest that isoform stoichiometry and/or arrangement within a connexin channel determines whether cyclic nucleotides can permeate, and which ones. This is the first evidence for connexin-specific selectivity among biological signaling molecules.

• Martin PE et al.
• Assembly of chimeric connexin-aequorin proteins into functional gap junction channels. Reporting intracellular and plasma membrane calcium environments.
• J Biol Chem. 1998; 273: 1719-26
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• Chimeric proteins comprising connexins 26, 32, and 43 and aequorin, a chemiluminescent calcium indicator, were made by fusing the amino terminus of aequorin to the carboxyl terminus of connexins. The retention of function by the chimeric partners was investigated. Connexin 32-aequorin and connexin 43-aequorin retained chemiluminescent activity whereas that of connexin 26-aequorin was negligible. Immunofluorescent staining of COS-7 cells expressing the chimerae showed they were targeted to the plasma membrane. Gap junction intercellular channel formation by the chimerae alone and in combination with wild-type connexins was investigated. Stable HeLa cells expressing connexin 43-aequorin were functional, as demonstrated by Lucifer yellow transfer. Paris of Xenopus oocytes expressing connexin 43-aequorin were electrophysiologically coupled, but those expressing chimeric connexin 26 or 32 showed no detectable levels of coupling. The formation of heteromeric channels constructed of chimeric connexin 32 or connexin 43 and the respective wild-type connexins was inferred from the novel voltage gating properties of the junctional conductance. The results show that the preservation of function by each partner of the chimeric protein is dictated mainly by the nature of the connexin, especially the length of the cytoplasmic carboxyl-terminal domain. The aequorin partner of the connexin 43 chimera reported calcium levels in COS-7 cells in at least two different calcium environments.

• Saez JC, Martinez AD, Branes MC, Gonzalez HE
• Regulation of gap junctions by protein phosphorylation.
• Braz J Med Biol Res. 1998; 31: 593-600
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• Gap junctions are constituted by intercellular channels and provide a pathway for transfer of ions and small molecules between adjacent cells of most tissues. The degree of intercellular coupling mediated by gap junctions depends on the number of gap junction channels and their activity may be a function of the state of phosphorylation of connexins, the structural subunit of gap junction channels. Protein phosphorylation has been proposed to control intercellular gap junctional communication at several steps from gene expression to protein degradation, including translational and post-translational modification of connexins (i.e., phosphorylation of the assembled channel acting as a gating mechanism) and assembly into and removal from the plasma membrane. Several connexins contain sites for phosphorylation for more than one protein kinase. These consensus sites vary between connexins and have been preferentially identified in the C-terminus. Changes in intercellular communication mediated by protein phosphorylation are believed to control various physiological tissue and cell functions as well as to be altered under pathological conditions.

• Dhein S
• Gap junction channels in the cardiovascular system: pharmacological and physiological modulation.
• Trends Pharmacol Sci. 1998; 19: 229-41
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• Intercellular communication provides the basis for the intact functioning of tissue and for various organs and tissue types in an organism to work together. It is the crucial difference between isolated cells and intact tissue. Cells communicate in various ways with each other; these include the release of chemical transmitters, hormones and mediators as well as direct electrical and chemical intercellular communication via gap junction channels. The gap junction coupling is important for the organization of the tissue as an electrical syncytium and for accurate development. Pharmacological modulation of these channels could be important in the fields of arrhythmogenesis, vasomotion and cell differentiation. In this review, Stefan Dhein outlines the structure, synthesis and function of gap junction channels. Since their physiology and pharmacology are best investigated in the cardiovascular system, the second part of the article focuses on the role of gap junctions in the heart and vasculature, with special emphasis on the regulation of the channels by physiological stimuli such as ions, pH mediators and transjunctional voltage as well as their pharmacological modulation.

• Bozhkova VP, Rozanova NV
• [Current status of the gap junction problems and and views on their role in development]
• Ontogenez. 1998; 29: 5-20
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• This review presents published data on various aspects of gap junction functioning during development. The process of formation of these junctions and their structure, along with relationships between molecular characteristics of connexins and properties of gap junctions channels are considered in detail. Some newly discovered functions of gap junctions in early embryonic development, their participation in the formation of various cellular compartments and in coordinated behavior are discussed; their relationship with other types of cell interactions is also reviewed. Promising problems for further research are outlined.

• Foote CI, Zhou L, Zhu X, Nicholson BJ
• The pattern of disulfide linkages in the extracellular loop regions of connexin 32 suggests a model for the docking interface of gap junctions.
• J Cell Biol. 1998; 140: 1187-97
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• Connexins, like true cell adhesion molecules, have extracellular domains that provide strong and specific homophilic, and in some cases, heterophilic interactions between cells. Though the structure of the binding domains of adhesion proteins have been determined, the extracellular domains of connexins, consisting of two loops of approximately 34-37 amino acids each, are not easily studied in isolation from the rest of the molecule. As an alternative, we used a novel application of site-directed mutagenesis in which four of the six conserved cysteines in the extracellular loops of connexin 32 were moved individually and in all possible pairwise and some quadruple combinations. This mapping allowed us to deduce that all disulfides form between the two loops of a single connexin, with the first cysteine in one loop connected to the third of the other. Furthermore, the periodicity of movements that produced functional channels indicated that these loops are likely to form antiparallel beta sheets. A possible model that could explain how these domains from apposed connexins interact to form a complete channel is discussed.

• Boitano S, Dirksen ER, Evans WH
• Sequence-specific antibodies to connexins block intercellular calcium signaling through gap junctions.
• Cell Calcium. 1998; 23: 1-9
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• Mechanical stimulation of a single cell in primary airway epithelial cell cultures induces an intercellular Ca2+ wave that has been proposed to be mediated via gap junctions. To investigate directly the role of gap junctions in this multicellular response, the effects of intracellularly-loaded sequence-specific connexin (gap junction) antibodies on the propagation of intercellular Ca2+ waves were evaluated. Electroporation of antibodies to the cytosolic loop (Des 1, generated to amino acids 102-112 + 116-124; and Des 5, amino acids 108-119), or to the carboxyl tail (Gap 9, amino acids 264-283) of connexin 32 inhibited the propagation of intercellular Ca2+ waves. The inhibitory effect of Des 1 antibody was competitively reversed by the co-loading of a peptide derived from a similar cytosolic loop sequence (Des 5 peptide). Conversely, the inhibitory effects on intercellular Ca2+ wave propagation of Gap 9 antibody was not altered by co-loading with the Des 5 peptide. Antibodies raised to peptide sequences within the extracellular loop (Gap 11, amino acids 151-187), or the cytoplasmically located amino terminus (Gap 10, amino acids 1-21) of connexin 32 did not inhibit mechanically-induced intercellular communication. Also ineffective in perturbing intercellular communication were antibodies raised to peptide sequences of the cytosolic loops of connexin 43 (Gap 15, amino acids 131-142) or connexin 26 (Des 3, amino acids 106-119). These data suggest that mechanically-induced Ca2+ waves in airway cell cultures are propagated through gap junctions made up of connexin 32 proteins.

• Donaldson P, Eckert R, Green C, Kistler J
• Gap junction channels: new roles in disease.
• Histol Histopathol. 1997; 12: 219-31
• Display abstract

• The importance of intercellular communication to complex cellular processes such as development, differentiation, growth, propagation of electrical impulses and diffusional feeding has long been appreciated. The realization that intercellular communication is mediated by gap junction channels, which are in turn comprised of a diverse family of proteins called the connexins, has provided new tools and avenues for studying the role of intercellular communication in these important cellular processes. The identification of different connexin isoforms has not only enabled the development of specific reagents to study connexin expression patterns, but has also allowed the functional properties of the different connexin isoforms and how they interact with each other, to be explored. Increasingly, the knowledge gained from studying connexin diversity is being used to investigate the role played by gap junction channels in a number of diseases. In this article we highlight selected cases where gap junction channels have been shown or are believed to be directly involved in the disease process.

• Falk MM, Buehler LK, Kumar NM, Gilula NB
• Cell-free synthesis and assembly of connexins into functional gap junction membrane channels.
• EMBO J. 1997; 16: 2703-16
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• Several different gap junction channel subunit isotypes, known as connexins, were synthesized in a cell-free translation system supplemented with microsomal membranes to study the mechanisms involved in gap junction channel assembly. Previous results indicated that the connexins were synthesized as membrane proteins with their relevant transmembrane topology. An integrated biochemical and biophysical analysis indicated that the connexins assembled specifically with other connexin subunits. No interactions were detected between connexin subunits and other co-translated transmembrane proteins. The connexins that were integrated into microsomal vesicles assembled into homo- and hetero-oligomeric structures with hydrodynamic properties of a 9S particle, consistent with the properties reported for hexameric gap junction connexons derived from gap junctions in vivo. Further, cell-free assembled homo-oligomeric connexons composed of beta1 or beta2 connexin were reconstituted into synthetic lipid bilayers. Single channel conductances were recorded from these bilayers that were similar to those measured for these connexons produced in vivo. Thus, this is the first direct evidence that the synthesis and assembly of a gap junction connexon can take place in microsomal membranes. Finally, the cell-free system has been used to investigate the properties of alpha1, beta1 and beta2 connexin to assemble into hetero-oligomers. Evidence has been obtained for a selective interaction between individual connexin isotypes and that a signal determining the potential hetero-oligomeric combinations of connexin isotypes may be located in the N-terminal sequence of the connexins.

• Nicholson SM, Bruzzone R
• Gap junctions: getting the message through.
• Curr Biol. 1997; 7: 3404-3404
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• Connexin proteins make intercellular channels - gap junctions - which provide a direct pathway for cell-cell signaling in vertebrates. Studies of mice lacking connexin genes have demonstrated the need for intercellular transfer of messenger molecules and are uncovering the specific functions of each connexin.

• Krutovskikh V, Yamasaki H
• The role of gap junctional intercellular communication (GJIC) disorders in experimental and human carcinogenesis.
• Histol Histopathol. 1997; 12: 761-8
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• There is a growing body of evidence supporting the etiologic implication of gap junctional intercellular communication disorders in carcinogenesis. Substantial progress has recently been made both in molecular biology of gap junction and in the field of cancer research. They provide new insights and conceptions of gap junctional disorders in tumor pathology. Modern understanding of the structure, function and regulation of gap junctions, as well as putative mechanisms of its disorders in human and experimental carcinogenesis are discussed in this review with particular emphasis on fast-moving aspects of this problem.

• Perkins G, Goodenough D, Sosinsky G
• Three-dimensional structure of the gap junction connexon.
• Biophys J. 1997; 72: 533-44
• Display abstract

• The gap junction membrane channel is composed of macular aggregations of intercellular channels permitting the direct intercellular transfer of ions and small molecules. Each intercellular channel is formed by the apposition of two hexameric transmembrane channels (connexons), one from each cell. The interlocking of the two channels occurs extracellularly in a narrow 2.5-nm "gap" separating the junctional membranes. The channel-channel interaction is known to be selective between members of the family of proteins, called connexins, which oligomerize into the connexons. In addition to selectivity, the molecular interfaces involved in the extracellular interactions between connexons must be very congruent, since the intercellular channel must provide high resistances to the leakage of small ions between the channel lumen and the extracellular space. By using a recently developed biochemical procedure for obtaining ordered arrays of connexons from gap junctions split in the extracellular gap, (Ghoshroy, S., D. A. Goodenough, and G. E. Sosinsky. 1994. Preparation, characterization, and structure of half gap junctional layers spit with urea and EGTA. J. Membr. Biol. 146:15-28) a three-dimensional reconstruction of a connexon has been obtained by electron crystallographic methods. This reconstruction emphasizes the structural asymmetry between the extracellular and cytoplasmic domains and assigns lobed structural features to the extracellular domains of the connexon. The implication of our hemichannel structure is discussed in relation to the in vivo state of unpaired connexons, which have been shown to exist in the plasma membrane.

• Willecke K, Haubrich S
• Connexin expression systems: to what extent do they reflect the situation in the animal?
• J Bioenerg Biomembr. 1996; 28: 319-26
• Display abstract

• Intercellular communication is mediated by specialized cell-cell contact areas known as gap junctions. Connexins are the constitutive proteins of gap junction intercellular channels. Various cell expression systems are used to express connexins and, in turn, these expression systems can then be tested for their ability to form functional cell-cell channels. In this review, expression of murine endogenous connexins in primary cells and established cell lines is compared with results obtained by expression of exogenous connexins in Xenopus oocytes and cultured mammalian cells. In addition, first reports on characterization of connexin-deficient mice are discussed.

• Yeager M, Nicholson BJ
• Structure of gap junction intercellular channels.
• Curr Opin Struct Biol. 1996; 6: 183-92
• Display abstract

• Gap junctions are formed by a multigene family of polytopic membrane channel proteins, connexins, that have four hydrophobic transmembrane domains and their N and C termini located on the cytoplasmic membrane face. The C-terminal tail plays important roles in channel regulation by pH and phosphorylation. Conserved cysteine residues stabilize the conformation of the extracellular loops that mediate the 'docking' between connexons in the intercellular channel. Over the past year, electron cryocrystallography of two-dimensional crystals of a truncated recombinant alpha 1 (Cx43) has revealed that the transmembrane boundary of the intercellular channel is lined with alpha helices. Furthermore, a ring of alpha helices resides at the interface with the membrane lipids. A three-dimensional analysis based on images recorded from tilted crystals should reveal the location and secondary structure of additional transmembrane domains, as well as provide important structural details about the interactions between connexins within a hemi-channel and connexon-connexon interactions in the extracellular gap.

• Trexler EB, Bennett MV, Bargiello TA, Verselis VK
• Voltage gating and permeation in a gap junction hemichannel.
• Proc Natl Acad Sci U S A. 1996; 93: 5836-41
• Display abstract

• Gap junction channels are formed by members of the connexin gene family and mediate direct intercellular communication through linked hemichannels (connexons) from each of two adjacent cells. While for most connexins, the hemichannels appear to require an apposing hemichannel to open, macroscopic currents obtained from Xenopus oocytes expressing rat Cx46 suggested that some hemichannels can be readily opened by membrane depolarization [Paul, D. L., Ebihara, L., Takemoto, L. J., Swenson, K. I. & Goodenough, D. A. (1991), J. Cell Biol. 115, 1077-1089]. Here we demonstrate by single channel recording that hemichannels comprised of rat Cx46 exhibit complex voltage gating consistent with there being two distinct gating mechanisms. One mechanism partially closes Cx46 hemichannels from a fully open state, gammaopen, to a substate, gammasub, about one-third of the conductance of gammaopen; these transitions occur when the cell is depolarized to inside positive voltages, consistent with gating by transjunctional voltage in Cx46 gap junctions. The other gating mechanism closes Cx46 hemichannels to a fully closed state, gammaclosed, on hyperpolarization to inside negative voltages and has unusual characteristics; transitions between gammaclosed and gammaopen appear slow (10-20 ms), often involving several transient substates distinct from gammasub. The polarity of activation and kinetics of this latter form of gating indicate that it is the mechanism by which these hemichannels open in the cell surface membrane when unapposed by another hemichannel. Cx46 hemichannels display a substantial preference for cations over anions, yet have a large unitary conductance (approximately 300 pS) and a relatively large pore as inferred from permeability to tetraethylammonium (approximately 8.5 angstroms diameter). These hemichannels open at physiological voltages and could induce substantial cation fluxes in cells expressing Cx46.

• Yamasaki H, Naus CC
• Role of connexin genes in growth control.
• Carcinogenesis. 1996; 17: 1199-213

• Mazzoleni G et al.
• Effect of tumor-promoting and anti-promoting chemicals on the viability and junctional coupling of human HeLa cells transfected with DNAs coding for various murine connexin proteins.
• Comp Biochem Physiol C Pharmacol Toxicol Endocrinol. 1996; 113: 247-56
• Display abstract

• Gap-junctional intercellular communication is thought to be essential for maintaining cellular homeostasis and growth control. Its perturbation entails toxicological implications and it has been correlated with the in vivo tumor-promoting potential of chemicals. Little is known about the mechanism(s) responsible for the tumor promoters interference with the cellular coupling. Moreover, nongenotoxic carcinogens, as well as connexins (gap-junctional protein subunits), are known to be organ-/tissue-specific; this implies that the effect of different agents should be evaluated on their specific target, that is, connexin. To investigate the role of different connexins in regulating gap-junctional gating and to compare the properties of homotypic junctional channels, we evaluated the effects of tissue-specific tumor promoters and anti-promoters on the viability and intercellular coupling (dye-transfer) of HeLa cells stably transfected with cDNAs coding for connexin(cx)43, cx40, cx26 and cx32. The results demonstrate that the transfectants possess individual junctional permeabilities, differentially affected by the chemicals, they also show different sensitivities to the cytotoxic effect of the compounds. These findings confirm that connexin diversity may be responsible for the different gating properties of gap-junctional channels, being also suggestive for their separate functions and independent regulatory mechanisms.

• Rousset B
• [Introduction to the structure and functions of junction communications or gap junctions]
• Ann Endocrinol (Paris). 1996; 57: 476-80
• Display abstract

• Cell-to-cell communication through gap junctions (GJ) represents a direct route of exchange of informations between neighboring cells within tissues and organs. GJ are formed from the assembly of a large number of channels that differ from the other known channels because they connect the cytoplasm of adjacent cells. The GJ channel is built from two parts: the connexons. A connexon inserted into the plasma membrane of a cell interacts with another connexon belonging to an adjacent cell. Connexons are composed of proteins with four transmembrane domains that are named connexins (Cx). Six Cx form a connexon. Cx belong to a protein family with 13 known members at present. Each Cx is defined by its molecular mass in kDa (ex: Cx32, Cx43...). A given cell type expresses one or several Cx. The cell to cell transfer of molecules through GJ channels exhibit a size selectivity; only molecules with a molecular mass lower than 1000 Da such as ions and second messengers freely pass through GJ. Depending on the Cx they are made of, GJ seem to differ somewhat in their permeability properties. Cell-to-cell communication via GJ is a regulated process. GJ channels can be either open or closed. GJ mediated cell-to-cell communication or junctional coupling can be detected and quantified by visualization of the cell to cell transfer of a fluorescent probe (such as Lucifer Yellow...) previously introduced in a single cell by microinjection. The presence of GJ channels can also be identified by recording the passage of an electric current between contiguous cells. GJ are involved in numerous fundamental biological processes from the embryonic development to the homeostasis in adult tissues and organs. GJ coordinate cell activities and sometimes synchronize cell behaviour. This is the case for the propagation of the excitation wave in the cardiac muscle and smooth muscle. GJ mediate metabolic cooperation between cells; they represent a way of supply of nutrients for tissues that are weakly or not vascularized. GJ take part in the control of cell proliferation. The loss of GJ-mediated cell-to-cell communication is a common feature of transformed cells and the re-establishment of junctional coupling is associated with a decrease of tumoregenicity. Allowing the cell-to-cell transfer of second messengers, GJ participate (and sometimes control) the response of a cell population to signalling molecules. It is known for example that hormones influence the expression of Cx and thus the level of the junctional coupling and that communication via GJ has an effect on the type and extent of action of hormones.

• Meda P
• [Role of intercellular communication via gap junctions in insulin secretion]
• Ann Endocrinol (Paris). 1996; 57: 481-3
• Display abstract

• Alike most other cell types of eukaryotic organisms, the insulin-producing beta-cells of pancreatic islets are functionally interconnected by specialized regions of their cell membrane, referred to as gap junctions. These structures represent concentrations of channels which are formed by the proteins of the connexin family, and that are specialized for the exchange between neighboring cells of cytoplasmic ions and small molecules. This exchange is referred to as junctional coupling. Increasing evidence indicates that a normal expression of connexins and junctional coupling is essential to ensure proper insulin biosynthesis and secretion.

• Zhang JT, Chen M, Foote CI, Nicholson BJ
• Membrane integration of in vitro-translated gap junctional proteins: co- and post-translational mechanisms.
• Mol Biol Cell. 1996; 7: 471-82
• Display abstract

• Connexins (Cx) are protein components of gap junction channels that permit the passage of small molecules between neighboring cells. cDNAs of a large family of connexins have been isolated and sequenced. A gap junction channel consists of two connexons, one from each cell in contact, composed of six connexin subunits. It has been suggested by Musil and coworkers that the oligomerization of formation of a connexon occurs at the level of the trans-Golgi network. In the present study, we initiated an analysis of the early stages of protein synthesis and membrane insertion of Cx32 and Cx26, two connexins that we have demonstrated are co-expressed in the same junctions in hepatocytes. Using an in vitro transcription and a coupled cell-free translation and translocation system, we observed that both Cx32 and Cx26 could insert into microsome membranes co-translationally, producing a topological structure indistinguishable from that in isolated gap junctions. To our surprise, Cx26 could also insert into membranes post-translationally with a native orientation. This post-translational membrane insertion process is dependent on nucleotides but not their hydrolysis. Cx32, on the other hand, could not insert into membranes post-translationally. These disparate properties of Cx32 and Cx26 are not due to the significant difference in the lengths of their C-terminal domains, but rather to their internal amino acid sequences. These observations raise the possibility that there may be another pathway for Cx26 to insert into membranes in cells and this feature may be important for the regulation of its functions. These findings may also lead us to a new approach to reconstitution without detergent extraction.

• Gros DB, Jongsma HJ
• Connexins in mammalian heart function.
• Bioessays. 1996; 18: 719-30
• Display abstract

• In heart, the propagation of electrical activity is mediated by intercellular channels, referred to as junctional channels, aggregated into gap junctions and localised between myocytes. These channels consist of structurally related transmembrane proteins, the connexins, three of which (CX43, CX40 and CX45) have been shown to be associated with the myocytes of mammalian heart; a fourth, CX37, was detected exclusively in endothelial cells. In this paper, we review the recent data dealing with the topographical heterogeneity of expression of these connexins in the different cardiac tissues and the unique conductance properties of the channels they form, and attempt to assess the role played by each connexin and the consequences of their multiplicity in the propagation of action potentials.

• Sosinsky GE
• Molecular organization of gap junction membrane channels.
• J Bioenerg Biomembr. 1996; 28: 297-309
• Display abstract

• Gap junctions regulate a variety of cell functions by creating a conduit between two apposing tissue cells. Gap junctions are unique among membrane channels. Not only do the constituent membrane channels span two cell membranes, but the intercellular channels pack into discrete cell-cell contact areas forming in vivo closely packed arrays. Gap junction membrane channels can be isolated either as two-dimensional crystals, individual intercellular channels, or individual hemichannels. The family of gap junction proteins, the connexins, create a family of gap junctions channels and structures. Each channel has distinct physiological properties but a similar overall structure. This review focuses on three aspects of gap junction structure: (1) the molecular structure of the gap junction membrane channel and hemichannel, (2) the packing of the intercellular channels into arrays, and (3) the ways that different connexins can combine into gap junction channel structures with distinct physiological properties. The physiological implications of the different structural forms are discussed.

• Laird DW
• The life cycle of a connexin: gap junction formation, removal, and degradation.
• J Bioenerg Biomembr. 1996; 28: 311-8
• Display abstract

• Gap junction proteins, connexins, possess many properties that are atypical of other well-characterized integral membrane proteins. Oligomerization of connexins into hemichannels (connexons) has been shown to occur after the protein exits the endoplasmic reticulum. Once delivered to the cell surface, connexons from one cell pair with connexons from a neighboring cell, a process that is facilitated by calcium-dependent cell adhesion molecules. Channels cluster into defined plasma membrane domains to form plaques. Unexpectedly, gap junctions are not stable (half-life < 5 h) and are thought to be retrieved back into the cell in the form of double membrane structures when one cell internalizes the entire gap junction through endocytosis. Evidence exists for both proteasomal and lysosomal degradation of gap junctions, and it remains possible that both mechanisms are involved in connexin degradation. In addition to opening and closing of gap junction channels (gating), the formation and removal of gap junctions play an essential role in regulating the level of intercellular communication.

• Veenstra RD
• Size and selectivity of gap junction channels formed from different connexins.
• J Bioenerg Biomembr. 1996; 28: 327-37
• Display abstract

• Gap junction channels have long been viewed as static structures containing a large-diameter, aqueous pore. This pore has a high permeability to hydrophilic molecules of approximately 900 daltons in molecular weight and a weak ionic selectivity. The evidence leading to these conclusions is reviewed in the context of more recent observations primarily coming from unitary channel recordings from transfected connexin channels expressed in communication-deficient cell lines. What is emerging is a more diverse view of connexin-specific gap junction channel structure and function where electrical conductance, ionic selectivity, and dye permeability vary by one full order of magnitude or more. furthermore, the often held contention that channel conductance and ionic or molecular selectivity are inversely proportional is refuted by recent evidence from five distinct connexin channels. The molecular basis for this diversity of channel function remains to be identified for the connexin family of gap junction proteins.

• White TW, Bruzzone R
• Multiple connexin proteins in single intercellular channels: connexin compatibility and functional consequences.
• J Bioenerg Biomembr. 1996; 28: 339-50
• Display abstract

• In vertebrates, the protein subunits of intercellular channels found in gap junctions are encoded by a family of genes called connexins. These channels span two plasma membranes and result from the association of two half channels, or connexons, which are hexameric assemblies of connexins. Physiological analysis of channel formation and gating has revealed unique patterns of connexin-connexin interaction, and uncovered novel functional characteristics of channels containing more than one type of connexin protein. Structure-function studies have further demonstrated that unique domains within connexins participate in the regulation of different functional properties of intercellular channels. Thus, gap junctional channels can contain more than one connexin, and this structural heterogeneity has functional consequences in vitro. Moreover, emerging evidence for the existence of intercellular channels containing multiple connexins in native tissues suggests that the functional diversity generated by connexin-connexin interaction could contribute to complex communication patterns that have been observed in vivo.

• Meda P
• The role of gap junction membrane channels in secretion and hormonal action.
• J Bioenerg Biomembr. 1996; 28: 369-77
• Display abstract

• Connexins, gap junctions, and coupling are obligatory features of both endocrine and exocrine glandular epithelia. Evidence from these two types of tissues, and particularly from pancreatic islets and acini, indicates that cell-to-cell communication via gap junction channels is required for proper biosynthesis, storage, and release of specific secretory products. However, endocrine and exocrine glands express a different set of connexins and show opposite connexin and coupling changes in relation with the activation and inhibition of their secretory function. Also, several hormones modulate connexin and coupling expression, and junctional coupling affects hormonal stimulation. These observations indicate that gap junction channels play an important role in the control of secretion and hormonal action.

• Goliger JA, Bruzzone R, White TW, Paul DL
• Dominant inhibition of intercellular communication by two chimeric connexins.
• Clin Exp Pharmacol Physiol. 1996; 23: 1062-7
• Display abstract

• 1. The physiological significance of communication through gap junction channels has been difficult to assess because channel activity cannot be experimentally modulated in a specific manner. To address this problem we have constructed chimeric connexins that function as dominant-negative inhibitors of intercellular channel activity.

• Davies TC, Barr KJ, Jones DH, Zhu D, Kidder GM
• Multiple members of the connexin gene family participate in preimplantation development of the mouse.
• Dev Genet. 1996; 18: 234-43
• Display abstract

• The connexin gene family, of which there are at least 12 members in rodents, encodes the protein subunits intercellular membrane channels (gap junction channels). Because of the diverse structural and biophysical properties exhibited by the different connexins, it has been proposed that each may play a unique role in development or homeostasis. We have begun to test this hypothesis in the preimplantation mouse embryo in which de novo gap junction assembly is a developmentally regulated event. As a first step, we have used reverse transcription-polymerase chain reaction (RT-PCR) to determine the connexin mRNA phenotype of mouse blastocysts, and have identified transcripts of connexins 30.3, 31, 31.1, 40, 43, and 45. Quantitative measurements indicated that all six of these connexin genes are transcribed after fertilization. They can be divided into two groups with respect to the timing of mRNA accumulation: Cx31, Cx43, and Cx45 mRNAs accumulate continuously from the two- or four-cell stage, whereas Cx30.3, Cx31.1, and Cx40 mRNAs accumulate beginning in the eight-cell stage. All six mRNAs were found to co-sediment with polyribosomes from their time of first appearance, indicating that all six are translated. The expression of Cx31.1 and Cx40 was examined by confocal immunofluorescence microscopy; whereas both could be detected in compacting embryos, only Cx31.1 could be seen in punctate membrane foci indicative of gap junctions. Taken together with other results (published or submitted), our findings indicate that at least four connexins (Cx31, 31.1, 43 and 45) contribute to gap junctions in preimplantation development. The expression of multiple connexin genes during this early period of embryogenesis (when there are only two distinct cell types) raises questions about the functional significance of connexin diversity in this context.

• Paul DL, Yu K, Bruzzone R, Gimlich RL, Goodenough DA
• Expression of a dominant negative inhibitor of intercellular communication in the early Xenopus embryo causes delamination and extrusion of cells.
• Development. 1995; 121: 371-81
• Display abstract

• A chimeric construct, termed 3243H7, composed of fused portions of the rat gap junction proteins connexin32 (Cx32) and connexin43 (Cx43) has been shown to have selective dominant inhibitory activity when tested in the Xenopus oocyte pair system. Co-injection of mRNA coding for 3243H7 together with mRNAs coding for Cx32 or Cx43 completely blocked the development of channel conductances, while the construct was ineffective at blocking intercellular channel assembly when coinjected with rat connexin37 (Cx37). Injection of 3243H7 into the right anterodorsal blastomere of 8-cell-stage Xenopus embryos resulted in disadhesion and delamination of the resultant clone of cells evident by embryonic stage 8; a substantial number, although not all, of the progeny of the injected cell were eliminated from the embryo by stage 12. A second construct, 3243H8, differing from 3243H7 in the relative position of the middle splice, had no dominant negative activity in the oocyte pair assay, nor any detectable effects on Xenopus development, even when injected at four-fold higher concentrations. The 3243H7-induced embryonic defects could be rescued by coinjection of Cx37 with 3243H7. A blastomere reaggregation assay was used to demonstrate that a depression of dye-transfer could be detected in 3243H7-injected cells as early as stage 7; Lucifer yellow injections into single cells also demonstrated that injection of 3243H7 resulted in a block of intercellular communication. These experiments indicate that maintenance of embryonic cell adhesion with concomitant positional information requires gap junction-mediated intercellular communication.

• White TW, Bruzzone R, Paul DL
• The connexin family of intercellular channel forming proteins.
• Kidney Int. 1995; 48: 1148-57

• Paul DL
• New functions for gap junctions.
• Curr Opin Cell Biol. 1995; 7: 665-72
• Display abstract

• The most significant finding of the past year in gap junction research has been the association of connexin defects with human diseases. Connexin32 mutations cause X-linked Charcot-Marie-Tooth disease, a demyelinating peripheral neuropathy. Mutations in connexin43 may underlie cardiac malformations in visceroatrial heterotaxia syndromes. Genetic approaches and gene targeting have provided new insights, but also raise new questions concerning connexin function, the significance of connexin diversity and the regulation of intercellular communication.

• Wolburg H, Rohlmann A
• Structure--function relationships in gap junctions.
• Int Rev Cytol. 1995; 157: 315-73
• Display abstract

• Gap junctions are metabolic and electrotonic pathways between cells and provide direct cooperation within and between cellular nets. They are among the cellular structures most frequently investigated. This chapter primarily addresses aspects of the assembly of the gap junction channel, considering the insertion of the protein into the membrane, the importance of phosphorylation of the gap junction proteins for coupling modulation, and the formation of whole channels from two hemichannels. Interactions of gap junctions with the subplasmalemmal cytoplasm on the one side and with tight junctions on the other side are closely considered. Furthermore, reviewing the significance and alterations of gap junctions during development and oncogenesis, respectively, including the role of adhesion molecules, takes up a major part of the chapter. Finally, the literature on gap junctions in the central nervous system, especially between astrocytes in the brain cortex and horizontal cells in the retina, is summarized and new aspects on their structure-function relationship included.

• Sosinsky G
• Mixing of connexins in gap junction membrane channels.
• Proc Natl Acad Sci U S A. 1995; 92: 9210-4
• Display abstract

• Gap junctions are plaque-like clusters of intercellular channels that mediate intercellular communication. Each of two adjoining cells contains a connexon unit which makes up half of the whole channel. Gap junction channels are formed from a multigene family of proteins called connexins, and different connexins may be coexpressed by a single cell type and found within the same plaque. Rodent gap junctions contain two proteins, connexins 32 and 26. Use of a scanning transmission electron microscope for mass analysis of rodent gap junction plaques and split gap junctions prvided evidence consistent with a model in which the channels may be made from (i) solely connexin 26, (ii) solely connexin 32, or (iii) mixtures of connexin 26 and connexin 32 in which the two connexons are made entirely of connexin 26 and connexin 32. The different types of channels segregate into distinct domains, implying tha connexon channels self-associate to give a non-random distribution within tissues. Since each connexin confers distinct physiological properties on its membrane channels, these results imply that the physiological properties of channels can be tailored by mixing the constituent proteins within these macromolecular structures.

• White TW, Paul DL, Goodenough DA, Bruzzone R
• Functional analysis of selective interactions among rodent connexins.
• Mol Biol Cell. 1995; 6: 459-70
• Display abstract

• One consequence of the diversity in gap junction structural proteins is that cells expressing different connexins may come into contact and form intercellular channels that are mixed in connexin content. We have systematically examined the ability of adjacent cells expressing different connexins to communicate, and found that all connexins exhibit specificity in their interactions. Two extreme examples of selectivity were observed. Connexin40 (Cx40) was highly restricted in its ability to make heterotypic channels, functionally interacting with Cx37, but failing to do so when paired with Cx26, Cx32, Cx43, Cx46, and Cx50. In contrast, Cx46 interacted well with all connexins tested except Cx40. To explore the molecular basis of connexin compatibility and voltage gating, we utilized a chimera consisting of Cx32 from the N-terminus to the second transmembrane domain, fused to Cx43 from the middle cytoplasmic loop to the C-terminus. The chimeric connexin behaved like Cx43 with regard to selectivity and like Cx32 with regard to voltage dependence. Taken together, these results demonstrate that the second but not the first extracellular domain affects compatibility, whereas voltage gating is strongly influenced by sequences between the N-terminus and the second transmembrane domain.

• Bruzzone R, White TW, Yoshizaki G, Patino R, Paul DL
• Intercellular channels in teleosts: functional characterization of two connexins from Atlantic croaker.
• FEBS Lett. 1995; 358: 301-4
• Display abstract

• Gap junction channels, composed of protein subunits termed connexins, are believed to play a critical role in the process of oocyte differentiation and maturation. We have used the paired Xenopus oocyte assay to characterize functionally two connexin genes, connexin-32.2 and connexin-32.7, recently cloned from the ovary of the Atlantic croaker (Micropogonia undulatus), a species that has emerged as a useful model to study the process of maturation of the ovarian follicle. We have found that, while both connexin proteins were expressed at comparable levels in Xenopus oocytes, only one, connexin-32.2, was functionally competent to induce the formation of intercellular channels. Connexin-32.2 channels exhibited voltage-dependent closure that was similar to, but distinct from that of previously characterized mammalian connexins. In addition, the silent connexin-32.7 was unable to functionally interact with connexin-32.2, either in heterotypic channels or as dominant negative inhibitor. Because connexin-32.2 expression is strikingly regulated during oocyte maturation, these data provide further evidence for a role of intercellular channels in the control of oocyte-follicular cell interactions.

• Ghoshroy S, Goodenough DA, Sosinsky GE
• Preparation, characterization, and structure of half gap junctional layers split with urea and EGTA.
• J Membr Biol. 1995; 146: 15-28
• Display abstract

• Gap junctions, collections of membrane channels responsible for intercellular communication, contain two paired hemichannels (also called connexons). We have investigated conditions for splitting the membrane pair using urea. We have developed a protocol which consistently splits the gap junction samples with 60-90% efficiency. Our results indicate that hydrophobic forces are important in holding the two connexons together but that Ca2+ ions are also important in the assembly of the membrane pair. Greater yields and better structural integrity of split junctions were obtained with a starting preparation of gap junctions which had been detergent treated. Image analysis of edge views of single connexon layers reveal an asymmetry in the appearance of the cytoplasmic and extracellular surface. Cryo-electron microscopy and image analysis of split junctions show that the packing and structural detail of membranes containing arrays of single connexons are the same as for intact junctions, and that the urea treatment causes no gross structural changes in the connexon assembly.

• Beyer EC, Davis LM, Saffitz JE, Veenstra RD
• Cardiac intercellular communication: consequences of connexin distribution and diversity.
• Braz J Med Biol Res. 1995; 28: 415-25
• Display abstract

• Gap junctions contain channels which allow the exchange of ions and small molecules between adjacent cells. In the heart, these channels are crucial for normal intercellular current flow and the propagation of action potentials throughout the myocardium. Molecular cloning studies have demonstrated that these channels are formed by members of a family of related proteins called connexins each containing conserved and unique regions. There are several consequences of this multiplicity of connexins. Multiple connexins are expressed in differing, but sometimes overlapping, distributions within cardiovascular and other tissues. Connexin40, connexin43, and connexin45 are all found in cardiac myocytes, but their abundance differs in specialized cardiac regions with disparate conductive properties. Individual connexins form channels with differing voltage-dependence, conductance, and permeability properties, as demonstrated by functional expression of the cloned sequences. Connexins differ in their modification by phosphorylation, which may contribute to physiological regulation of intercellular communication. Expression of multiple connexins may lead to the formation of multiple channel types in a single tissue or cell and potentially allows mixing to form heterotypic and/or heteromeric channels. Thus, multiple connexins may contribute to the differences in intercellular resistance in cardiac regions with differing conductive properties and possibly may allow differences in the signalling molecules that pass between cells.

• Dunina-Barkovskaia AI, Sharovskaia II, Eckert R, Hulser DF
• [Regulation of intercellular contacts in cultured cells transfected with the connexin Cx32 gene]
• Mol Biol (Mosk). 1995; 29: 1349-58

• Yamasaki H, Mesnil M, Omori Y, Mironov N, Krutovskikh V
• Intercellular communication and carcinogenesis.
• Mutat Res. 1995; 333: 181-8
• Display abstract

• Two types of intercellular communication (humoral and cell contact-mediated) are involved in control of cellular function in multicellular organisms, both of them mediated by membrane-embedded proteins. Involvement of aberrant humoral communication in carcinogenesis has been well documented and genes coding for some growth factors and their receptors have been classified as oncogenes. More recently, cell contact-mediated communication has been found to have an important role in carcinogenesis, and some genes coding for proteins involved in this type of communication appear to form a family of tumor-suppressor genes. Both homologous (among normal or (pre-)cancerous cells) as well as heterologous (between normal and (pre)cancerous cells) communications appear to play important roles in cell growth control. Gap junctional intercellular communication (GJIC) is the only means by which multicellular organisms can exchange low molecular weight signals directly from within one cell to the interior of neighboring cells. GJIC is altered by many tumor-promoting agents and in many human and rodent tumors. We have recently shown that liver tumor-promoting agents inhibit GJIC in the rat liver in vivo. Molecular mechanisms which could lead to aberrant GJIC include: (1) mutation of connexin genes; (2) reduced and/or aberrant expression of connexin mRNA; (3) aberrant localization of connexin proteins, i.e., intracytoplasmic rather than in the cytoplasmic membrane; and (4) modulation of connexin functions by other proteins, such as those involved in extracellular matrix and cell adhesion. Whilst mutations of the cx 32 gene appear to be rare in tumors, cx 37 gene mutations have been reported in a mouse lung tumor cell line. Our results suggest that aberrant connexin localization is rather common in cancer cells and that possible molecular mechanisms include aberrant phosphorylation of connexin proteins and lack of cell adhesion molecules. Studies on transfection of connexin genes into tumor cells suggest that certain connexin genes (e.g., cx 26, cx 43 and cx 32) act as tumor-suppressor genes.

• Yeager M
• Electron microscopic image analysis of cardiac gap junction membrane crystals.
• Microsc Res Tech. 1995; 31: 452-66
• Display abstract

• Cardiac gap junctions play an important functional role in the myocardium by electrically coupling adjacent cells, thereby providing a low resistance pathway for cell-to-cell propagation of the action potential. Two-dimensional crystallization of biochemically isolated rat ventricular gap junctions has been accomplished by an in situ method in which membrane suspensions are sequentially dialyzed against low concentrations of deoxycholate and dodecyl-beta-D-maltoside. Lipids are partially extracted without solubilizing the protein, and the increased protein concentration facilitates two-dimensional crystallization in the native membrane environment. The two-dimensional crystals have a nominal resolution of 16 A and display plane group symmetry p6 with a = b = 85 A and gamma = 120 degrees. Projection density maps show that the connexons in cardiac gap junctions are formed by a hexameric cluster of alpha 1 connexin subunits. Protease cleavage of alpha 1 connexin from 43 to 30 kDa releases approximately 13kDa from the carboxy-tail, and the projection density maps are not significantly altered. Uranyl acetate stain penetrates the ion channel, whereas phosphotungstic acid is preferentially deposited over the lipid regions. This differential staining can be used to selectively probe the central channel of the connexon and the interface between the connexon and the lipid. The hexameric design of alpha 1 connexons appears to be a recurring quaternary motif for the multigene family of gap junction proteins.

• Stauffer KA
• The gap junction proteins beta 1-connexin (connexin-32) and beta 2-connexin (connexin-26) can form heteromeric hemichannels.
• J Biol Chem. 1995; 270: 6768-72
• Display abstract

• Two different types of gap junction proteins, beta 1- and beta 2-connexin, were expressed in insect cells, either singly or together, using infection with recombinant baculovirus. Membrane fractions enriched in gap junction proteins were isolated, and connexons (hemichannels) were solubilized with detergent. These solubilized connexons were then run out on a gel filtration column which was capable of partially separating the two homomeric connexons. It was found that connexons from cells co-infected with both types of baculovirus ran together on this column, whereas connexons from cells infected separately and mixed before solubilization did not, suggesting that in the co-infected cells the two types of connexin are assembled into heteromeric hemichannels.

• Ruch RJ
• The role of gap junctional intercellular communication in neoplasia.
• Ann Clin Lab Sci. 1994; 24: 216-31
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• Gap junctions are comprised of proteinaceous, plasma membrane channels that link the interiors of adjacent cells and permit cells to directly exchange small (< 1,000 Daltons) molecules and ions. This exchange, termed gap junctional intercellular communication (GJIC), appears to be involved in growth regulation. Growth controlling factors may pass between cells through the junctions. The loss of gap junctions or impairment of their permeability has been observed in many neoplastic cells and cells treated with growth promoting carcinogens and other agents. The loss of GJIC appears to be an important event in the conversion of a normal cell into a neoplastic one. On the other hand, the restoration of GJIC in neoplastic cells by transfection with gap junction protein (connexin) cyclic deoxyribonucleic acids (cDNAs) or by stimulating endogenous connexin gene expression has led to the reversal of the neoplastic phenotype. The biology of gap junctions and their role in growth regulation and neoplasia are reviewed.

• Bennett MV, Zheng X, Sogin ML
• The connexins and their family tree.
• Soc Gen Physiol Ser. 1994; 49: 223-33
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• The connexins, gap junction forming proteins, are encoded by a gene family. Sequence comparisons reveal regions of conservation with functional implications for voltage dependence of junctional conductance, junction formation and regulation by phosphorylation. The best connexin tree shows that most gene duplications giving rise to the family occurred early in or before vertebrate divergence. The topology of most deep branches of the tree is uncertain. Evolutionary rates vary for different paralogous connexin genes.

• White TW, Bruzzone R, Wolfram S, Paul DL, Goodenough DA
• Selective interactions among the multiple connexin proteins expressed in the vertebrate lens: the second extracellular domain is a determinant of compatibility between connexins.
• J Cell Biol. 1994; 125: 879-92
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• Gap junctions are collections of intercellular channels composed of structural proteins called connexins (Cx). We have examined the functional interactions of the three rodent connexins present in the lens, Cx43, Cx46, and Cx50, by expressing them in paired Xenopus oocytes. Homotypic channels containing Cx43, Cx46, or Cx50 all developed high conductance. heterotypic channels composed of Cx46 paired with either Cx43 or Cx50 were also well coupled, whereas Cx50 did not form functional channels with Cx43. We also examined the functional response of homotypic and heterotypic channels to transjunctional voltage and cytoplasmic acidification. We show that all lens connexins exhibited sensitivity to cytoplasmic acidification as well as to voltage, and that voltage-dependent closure of heterotypic channels for a given connexin was dramatically influenced by its partner connexins in the adjacent cell. Based on the observation that Cx43 can discriminate between Cx46 and Cx50, we investigated the molecular determinants that specify compatibility by constructing chimeric connexins from portions of Cx46 and Cx50 and testing them for their ability to form channels with Cx43. When the second extracellular (E2) domain in Cx46 was replaced with the E2 of Cx50, the resulting chimera could no longer form heterotypic channels with Cx43. A reciprocal chimera, where the E2 of Cx46 was inserted into Cx50, acquired the ability to functionally interact with Cx43. Together, these results demonstrate that formation of intercellular channels is a selective process dependent on the identity of the connexins expressed in adjacent cells, and that the second extracellular domain is a determinant of heterotypic compatibility between connexins.

• Zhang ZQ, Lin ZX
• [The connexin family, gap junctional intercellular communication and tumor suppression]
• Sheng Li Ke Xue Jin Zhan. 1994; 25: 121-5

• Dahl G, Nonner W, Werner R
• Attempts to define functional domains of gap junction proteins with synthetic peptides.
• Biophys J. 1994; 67: 1816-22
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• To map the binding sites involved in channel formation, synthetic peptides representing sequences of connexin 32 were tested for their ability to inhibit cell-cell channel formation. Both large peptides representing most of the two presumed extracellular loops of connexin32 and shorter peptides representing subsets of these larger peptides were found to inhibit cell-cell channel formation. The properties of the peptide inhibition suggested that the binding site is complex, involving several segments of both extracellular loops. One of the peptides (a 12-mer) did not inhibit but instead was found to form channels in membranes. Both in oocyte membranes and in bilayers, the channels formed by the peptide were asymmetrically voltage dependent. Their unit conductances ranged from 20 to 160 pS. These data are discussed in the form of a model in which the connexin sequence represented by the peptide is part of a beta structure providing the lining of the channel pore.

• Verselis VK, Ginter CS, Bargiello TA
• Opposite voltage gating polarities of two closely related connexins.
• Nature. 1994; 368: 348-51
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• The molecular mechanisms underlying the voltage dependence of intercellular channels formed by the family of vertebrate gap junction proteins (connexins) are unknown. All vertebrate gap junctions are sensitive to the voltage difference between the cells, defined as the transjunctional voltage, Vj (refs 1, 2), and most appear to gate by the separate actions of their component hemichannels. The heterotypic Cx32/Cx26 junction displays an unpredicted rectification that was reported to represent a novel Vj dependence created by hemichannel interactions, mediated in part by the first extracellular loop E1 (ref. 9). Here we show that aspects of the rectification of Cx32/Cx26 junctions are explained by opposite gating polarities of the component hemichannels, and that the opposite gating polarity of Cx32 and Cx26 results from a charge difference in a single amino-acid residue located at the second position in the N terminus. We also show that charge substitutions at the border of the first transmembrane (M1) and E1 domains can reverse gating polarity and suppress the effects of a charge substitution at the N terminus. We conclude that the combined actions of residues at the N terminus and M1/E1 border form a charge complex that is probably an integral part of the connexin voltage sensor. A consistent correlation between charge substitution and gating polarity indicates that Cx26 and Cx32 voltage sensors are oppositely charged and that both move towards the cytoplasm upon hemichannel closure.

• Goodenough DA, Musil LS
• Gap junctions and tissue business: problems and strategies for developing specific functional reagents.
• J Cell Sci Suppl. 1993; 17: 133-8
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• The complex and overlapping tissue distribution of different members of the gap junctional connexin protein family is reviewed. Intermixing of different connexins in the building of intercellular channels and translational and posttranslational regulation of gap junctional channels add additional challenges to the interpretation of the possible functions played by gap junction-mediated intercellular communication in tissue business.

• Bruzzone R, Haefliger JA, Gimlich RL, Paul DL
• Connexin40, a component of gap junctions in vascular endothelium, is restricted in its ability to interact with other connexins.
• Mol Biol Cell. 1993; 4: 7-20
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• The cellular distribution of connexin40 (Cx40), a newly cloned gap junction structural protein, was examined by immunofluorescence microscopy using two different specific anti-peptide antibodies. Cx40 was detected in the endothelium of muscular as well as elastic arteries in a punctate pattern consistent with the known distribution of gap junctions. However, it was not detected in other cells of the vascular wall. By contrast, Cx43, another connexin present in the cardiovascular system, was not detected in endothelial cells of muscular arteries but was abundant in the myocardium and aortic smooth muscle. We have tested the ability of these connexins to interact functionally. Cx40 was functionally expressed in pairs of Xenopus oocytes and induced the formation of intercellular channels with unique voltage dependence. Unexpectedly, communication did not occur when oocytes expressing Cx40 were paired with those expressing Cx43, although each could interact with a different connexin, Cx37, to form gap junction channels in paired oocytes. These findings indicate that establishment of intercellular communication can be spatially regulated by the selective expression of different connexins and suggest a mechanism that may operate to control the extent of communication between cells.

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