Evolutionary selection pressure and family relationships among connexin genes

We suggest an extension of connexin orthology relationships across the major vertebrate lineages. We first show that the conserved domains of mammalian connexins (encoding the N-terminus, four transmembrane domains and two extracellular loops) are subjected to a considerably more strict selection pressure than the full-length sequences or the variable domains (the intracellular loop and C-terminal tail). Therefore, the conserved domains are more useful for the study of family relationships over larger evolutionary distances. The conserved domains of connexins were collected from chicken, Xenopus tropicalis, zebrafish, pufferfish, green spotted pufferfish, Ciona intestinalis and Halocynthia pyriformis (two tunicates). A total of 305 connexin sequences were included in this analysis. Phylogenetic trees were constructed, from which the orthologies and the presumed evolutionary relationships between the sequences were deduced. The tunicate connexins studied had the closest, but still distant, relationships to vertebrate connexin 36, 39.2, 43.4, 45 and 47. The main structure in the connexin family known from mammals pre-dates the divergence of bony fishes, but some additional losses and gains of connexin sequences have occurred in the evolutionary lineages of subsequent vertebrates. Thus, the connexin gene family probably originated in the early evolution of chordates, and underwent major restructuring with regard to gene and subfamily structures (including the number of genes in each subfamily) during early vertebrate evolution.     

Emerging issues of connexin channels: biophysics fills the gap

This summary is a proposed synthesis of available information for the non-specialist. It does not incorporate all the published data, is inconsistent with some, and reflects the biases of the author. Connexin proteins have a common transmembrane topology, with four alpha-helical transmembrane domains, two extracellular loops, a cytoplasmic loop, and cytoplasmic N- and C-terminal domains. The sequences are most conserved in the transmembrane and extracellular domains, yet many of the key functional differences between connexins are determined by amino-acid differences in these largely conserved domains. Each extracellular loop contains three cysteines with invariant spacing (save one isoform) that are required for channel function. The junctional channel is composed of two end-to-end hemichannels, each of which is a hexamer of connexin subunits. Hemichannels formed by some connexin isoforms can function as well-behaved, single-membrane-spanning channels in plasma membrane. In junctional channels, the cysteines in the extracellular loops form intra-monomer disulfide bonds between the two loops, not intermonomer or inter-hemichannel bonds. The end-to-end homophilic binding between hemichannels is via non-covalent interactions. Mutagenesis studies suggest that the docking region contains beta structures, and may resemble to some degree the beta-barrel structure of porin channels. The two hemichannels that compose a junctional channel are rotationally staggered by approximately 30 degrees relative to each other so that the alpha-helices of each connexin monomer are axially aligned with the alpha-helices of two adjacent monomers in the apposed hemichannel. At present there is a published 3D map with 7.5 A resolution in the plane of the membrane, based on electron cryomicroscopy of 2D crystals of junctional channels formed by C-terminal truncated Cx43. The correspondence between the imaged transmembrane alpha-helices and the known transmembrane amino-acid sequences is a matter of debate. Each of the approximately 20 connexin isoforms produces channels with distinct unitary conductances, molecular permeabilities, and electrical and chemical gating sensitivities. The channels can be heteromeric, and subfamilies among connexins largely determine heteromeric specificity, similar to the specificities within the voltage-dependent potassium channel superfamily. The second extracellular loop contains the primary determinants of the specificity of hemichannel-hemichannel docking (analogous to the tetramerization domain of potassium channels). The 7.5 A map shows that each monomer exposes only two transmembrane alpha-helices to the pore lumen. However the conductance state of the imaged structure and the effects of the C-terminal truncation are unknown, so it is possible that other transmembrane domains contribute to the lumen in other functional states of the channel. In the transmembrane region, SCAM and mutagenesis data suggest that parts of the first three transmembrane alpha-helices are exposed to the lumen. Some of these data are contradictory, but may reflect conformational or isoform differences. There is reason to think that the first part of the N-terminal cytoplasmic domain can line the pore in some conformations. In the extracellular part of junctional channels, the N-terminal portion of the first extracellular loop is exposed to the lumen. The unitary conductances through connexin channels vary over an order of magnitude, from 15 pS to over 300 pS. There is a range of charge selectivities among atomic ions, from slightly anion selective to highly cation selective, which does not correlate with unitary conductance. There appear to be substantial ion-ion interactions within the pore, making the GHK model of assessing selectivities of limited value. Pores formed by different connexins have a range of limiting diameters as assessed by uncharged and charged probes, which also does not correlate with unitary conductance (i.e. some have high conductance but have a narrow limiting diameter, and vice versa). Channels formed by different connexins have different permeabilities to various cytoplasmic molecules. Where it has been assessed, the selectivity among cytoplasmic molecules is substantial and does not correlate in an obvious manner with the size selectivity data derived from fluorescent tracer studies, suggesting there are chemical specificities within the pore that enhance or reduce permeability to specific cytoplasmic molecules, functionally analogous to the ability of some porins to facilitate transport of specific substrates. For example, heteromeric channels with different stoichiometries or arrangements of isoforms can distinguish among second messengers. The differences in permeability to cytoplasmic molecules have biological consequences; in most cases one connexin cannot fully substitute for another. Voltage and chemical gating mechanisms largely operate within each hemichannel, though there is evidence for inter-hemichannel allosteric effects as well. There are at least two distinct gating mechanisms. One (Vj-gating) is a voltage-driven mechanism that governs rapid transitions between conducting states. Its voltage sensor involves charges in the first several positions of the cytoplasmic N-terminal domain and possibly in the N-terminal part of the first extracellular loop, which may both be exposed to the lumen of the pore in some states. The polarity of Vj-gating sensitivity is connexin-specific, closing with depolarization for some connexins and with hyperpolarization for others. The polarity can be reversed by point mutations at the second position. The lower conductance states induced by Vj-gating correspond to physical restrictions of the pore, and thus restricted or eliminated molecular permeation. Since the channels are not fully closed by Vj-gating, it can be seen as a way to eliminate molecular signaling while leaving electrical signaling operational. A second, independent gating mechanism mediates slow transitions (approximately 10-30 ms) into and out of non-conducting state(s). These transitions can occur in response to voltage ('loop gating'), chemical factors such as pH and lipophiles ('chemical gating'), and the docking of two hemichannels (sometimes called the 'docking gate'). These slow transitions may reflect a common structural change induced by these several effectors (electrical, chemical and homodimerization). Alternatively, they could reflect distinct gating processes responding to one or more of these effectors, that are indistinguishable at the single-channel level and have yet to be resolved mechanistically. The slow or loop gate closes with hyperpolarization. As a result, where Vj-gating closes with depolarization, individual hemichannels can close in response to both polarities of voltage (but only to a subconductance state for the Vj-gating polarity). Because of this, it is difficult to assign a macroscopic voltage sensitivity, or its modification due to mutagenesis, chemical modification or heteromeric interactions, to one or the other of these very distinct voltage-sensitive processes. This distinction can be made reliably only at the single-channel level. The Vj-gating voltage sensor and the loop-gating voltage sensor appear to be independent structures, since the Vj-gating voltage sensitivity can modified without effect on loop gating. For some connexins, certain modifications of the C-terminal domain seem to interfere with the operation of the Vj-gate while leaving loop gating unaffected. In some connexins, but not all, the chemical sensitivity to pH can involve interactions between regions of the C-terminal domain and cytoplasmic loop. Whether these regions exert their effects directly by physically blocking the pore, or by allosteric mechanisms (which may be more consistent with the relatively long time-course of closure) is not clear. For several connexins, truncation of the C-terminal domain eliminates the pH sensitivity, and co-expressing the domain with the truncated connexin restores the pH sensitivity. This has a functional resemblance to the particle-receptor mechanism for N-type inactivation of Shaker channels. What is being protonated is not clear, and may involve cytoplasmic factors, such as endogenous aminosulfonates. For other connexins, the action of pH does not involve the C-terminal domain and seems due to direct protonation of connexin. PKC phosphorylation of serine(s) in the C-terminal domain can affect the substate occupancy of at least one connexin. Phosphorylation of series in the C-terminal domain by MAP kinase appears to facilitate an interaction between it and an unknown receptor domain to eliminate coupling. This process has yet to be studied at the single-channel level. It also has a functional analogy to the particle-receptor model of channel inactivation. Both MAP kinase phosphorylation-induced and pH-induced inhibition can be mediated in truncated connexins by the corresponding free peptide. However, the relation between these two mechanisms are unexplored, as are specific mechanisms of direct endogenous regulation of connexin channel activity. (ABSTRACT TRUNCATED)     

Crucial motifs and residues in the extracellular loops influence the formation and specificity of connexin docking

Most of the early studies on gap junction (GJ) channel function and docking compatibility were on rodent connexins, while recent research on GJ channels gradually shifted from rodent to human connexins largely due to the fact that mutations in many human connexin genes are found to associate with inherited human diseases. The studies on human connexins have revealed some key differences from those found in rodents, calling for a comprehensive characterization of human GJ channels. Functional studies revealed that docking and formation of functional GJ channels between two hemichannels are possible only between docking-compatible connexins. Two groups of docking-compatible rodent connexins have been identified. Compatibility is believed to be due to their amino acid residue differences at the extracellular loop domains (E1 and E2). Sequence alignment of the E1 and E2 domains of all connexins known to make GJs revealed that they are highly conserved and show high sequence identity with human Cx26, which is the only connexin with near atomic resolution GJ structure. We hypothesize that different connexins have a similar structure as that of Cx26 at the E1 and E2 domains and use the corresponding residues in their E1 and E2 domains for docking. Based on the Cx26 GJ structure and sequence analysis of well-studied connexins, we propose that the E1-E1 docking interactions are staggered with each E1 interacting with two E1s on the docked connexon. The putative E1 docking residues are conserved in both docking-compatible and -incompatible connexins, indicating that E1 does not likely serve a role in docking compatibility. However, in the case of E2-E2 docking interactions, the putative docking residues are only conserved within the docking-compatible connexins, suggesting the E2 is likely to serve the function of docking compatibility. Docking compatibility studies on human connexins have attracted a lot of attention due to the fact that putative docking residues are mutational hotspots for several connexin-linked human diseases. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

SCAM analysis of Panx1 suggests a peculiar pore structure

Vertebrates express two families of gap junction proteins: the well-characterized connexins and the pannexins. In contrast to connexins, pannexins do not appear to form gap junction channels but instead function as unpaired membrane channels. Pannexins have no sequence homology to connexins but are distantly related to the invertebrate gap junction proteins, innexins. Despite the sequence diversity, pannexins and connexins form channels with similar permeability properties and exhibit similar membrane topology, with two extracellular loops, four transmembrane (TM) segments, and cytoplasmic localization of amino and carboxy termini. To test whether the similarities extend to the pore structure of the channels, pannexin 1 (Panx1) was subjected to analysis with the substituted cysteine accessibility method (SCAM). The thiol reagents maleimidobutyryl-biocytin and 2-trimethylammonioethyl-methanethiosulfonate reacted with several cysteines positioned in the external portion of the first TM segment (TM1) and the first extracellular loop. These data suggest that portions of TM1 and the first extracellular loop line the outer part of the pore of Panx1 channels. In this aspect, the pore structures of Panx1 and connexin channels are similar. However, although the inner part of the pore is lined by amino-terminal amino acids in connexin channels, thiol modification was detected in carboxyterminal amino acids in Panx1 channels by SCAM analysis. Thus, it appears that the inner portion of the pores of Panx1 and connexin channels may be distinct.     

Cx23, a connexin with only four extracellular-loop cysteines, forms functional gap junction channels and hemichannels

Gap junction channels may be comprised of either connexin or pannexin proteins (innexins and pannexins). Membrane topologies of both families are similar, but sequence similarity is lacking. Recently, connexin-like sequences have been identified in mammalian and zebrafish genomes that have only four conserved cysteines in the extracellular domains (Cx23), a feature of the pannexins. Phylogenetic analyses of the non-canonical "C4" connexins reveal that these sequences are indeed connexins. Functional assays reveal that the Cx23 gap junctions are capable of sharing neurobiotin, and further, that Cx23 connexins form hemichannels in vitro.     

Role of connexin (gap junction) genes in cell growth control and carcinogenesis

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.     

Doubly mutant mice, deficient in connexin32 and -43, show normal prenatal development of organs where the two gap junction proteins are expressed in the same cells

The connexins are a family of proteins that form the intercellular membrane channels of gap junctions. Genes encoding 13 different rodent connexins have been cloned and characterized to date. Connexins vary both in their distribution among adult cell types and in the properties of the channels that they form. In order to explore the functional significance of connexin diversity, several mouse connexin-encoding genes have been disrupted by homologous recombination in embryonic stem cells. Although those experiments have illuminated specific physiological roles for individual connexins, the results have also raised the possibility that connexins may functionally compensate for one another in cells where they are coexpressed. In the present study, we have tested this hypothesis by interbreeding mice carrying null mutations in the genes (Gjb1 and Gja1) encoding connexin32 (beta 1 connexin) and connexin43 (alpha 1 connexin), respectively. We found that fetuses lacking both connexins survive to term but, as expected, the pups die soon thereafter from the cardiac abnormality caused by the absence of connexin43. A survey of the major organ systems of the doubly mutant fetuses, including the thyroid gland, developing teeth, and limbs where these two connexins are coexpressed, failed to reveal any morphological abnormalities not already seen in connexin43 deficient fetuses. Furthermore, the production of thyroxine by doubly mutant thyroids was confirmed by immunocytochemistry. We conclude that, at least as far as the prenatal period is concerned, the normal development of those three organs in fetuses lacking connexin43 cannot simply be explained by the additional presence of connexin32 and vice-versa. Either gap junctional coupling is dispensable in embryonic and fetal cells in which these two connexins are coexpressed, or coupling is provided by yet another connexin when both are absent.     

Formation of the gap junction nexus: binding partners for connexins.

Gap junctions are the morphological correlates of direct cell-cell communication and are formed of hexameric assemblies of gap junction proteins (connexins) into hemichannels (or connexons) provided by each coupled cell. Gap junction channels formed by each of the connexin subtypes (of which there are as many as 20) display different properties, which have been attributed to differences in amino acid sequences of gating domains of the connexins. Recent studies additionally indicate that connexin proteins interact with other cellular components to form a protein complex termed the Nexus. This review summarizes current knowledge regarding the protein-protein interactions involving of connexin proteins and proposes hypothesized functions for these interactions.     

Functional analysis of selective interactions among rodent connexins.

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.     

Connexin and pannexin as modulators of myocardial injury

Multicellular organisms have developed a variety of mechanisms that allow communication between their cells. Whereas some of these systems, as neurotransmission or hormones, make possible communication between remote areas, direct cell-to-cell communication through specific membrane channels keep in contact neighboring cells. Direct communication between the cytoplasm of adjacent cells is achieved in vertebrates by membrane channels formed by connexins. However, in addition to allowing exchange of ions and small metabolites between the cytoplasms of adjacent cells, connexin channels also communicate the cytosol with the extracellular space, thus enabling a completely different communication system, involving activation of extracellular receptors. Recently, the demonstration of connexin at the inner mitochondrial membrane of cardiomyocytes, probably forming hemichannels, has enlarged the list of actions of connexins. Some of these mechanisms are also shared by a different family of proteins, termed pannexins. Importantly, these systems allow not only communication between healthy cells, but also play an important role during different types of injury. The aim of this review is to discuss the role played by both connexin hemichannels and pannexin channels in cell communication and injury. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Connexin- and Pannexin-Based Channels in Normal Skeletal Muscles and Their Possible Role in Muscle Atrophy

Precursor cells of skeletal muscles express connexins 39, 43 and 45 and pannexin1. In these cells, most connexins form two types of membrane channels, gap junction channels and hemichannels, whereas pannexin1 forms only hemichannels. All these channels are low-resistance pathways permeable to ions and small molecules that coordinate developmental events. During late stages of skeletal muscle differentiation, myofibers become innervated and stop expressing connexins but still express pannexin1 hemichannels that are potential pathways for the ATP release required for potentiation of the contraction response. Adult injured muscles undergo regeneration, and connexins are reexpressed and form membrane channels. In vivo, connexin reexpression occurs in undifferentiated cells that form new myofibers, favoring the healing process of injured muscle. However, differentiated myofibers maintained in culture for 48 h or treated with proinflammatory cytokines for less than 3 h also reexpress connexins and only form functional hemichannels at the cell surface. We propose that opening of these hemichannels contributes to drastic changes in electrochemical gradients, including reduction of membrane potential, increases in intracellular free Ca²⁺ concentration and release of diverse metabolites (e.g., NAD⁺ and ATP) to the extracellular milieu, contributing to multiple metabolic and physiologic alterations that characterize muscles undergoing atrophy in several acquired and genetic human diseases. Consequently, inhibition of connexin hemichannels expressed by injured or denervated skeletal muscles might reduce or prevent deleterious changes triggered by conditions that promote muscle atrophy.     

Inhibition of gap junction and adherens junction assembly by connexin and A-CAM antibodies

We examined the roles of the extracellular domains of a gap junction protein and a cell adhesion molecule in gap junction and adherens junction formation by altering cell interactions with antibody Fab fragments. Using immunoblotting and immunocytochemistry we demonstrated that Novikoff cells contained the gap junction protein, connexin43 (Cx43), and the cell adhesion molecule, A-CAM (N-cadherin). Cells were dissociated in EDTA, allowed to recover, and reaggregated for 60 min in media containing Fab fragments prepared from a number of antibodies. We observed no cell-cell dye transfer 4 min after microinjection in 90% of the cell pairs treated with Fab fragments of antibodies for the first or second extracellular domain of Cx43, the second extracellular domain of connexin32 (Cx32) or A-CAM. Cell-cell dye transfer was detected within 30 s in cell pairs treated with control Fab fragments (pre-immune serum, antibodies to the rat major histocompatibility complex or the amino or carboxyl termii of Cx43). We observed no gap junctions by freeze-fracture EM and no adherens junctions by thin section EM between cells treated with the Fab fragments that blocked cell-cell dye transfer. Gap junctions were found on approximately 50% of the cells in control samples using freeze-fracture EM. We demonstrated with reaggregated Novikoff cells that: (a) functional interactions of the extracellular domains of the connexins were necessary for the formation of gap junction channels; (b) cell interactions mediated by A-CAM were required for gap junction assembly; and (c) Fab fragments of antibodies for A-CAM or connexin extracellular domains blocked adherens junction formation.     

Molecular cloning and developmental expression of two chick embryo gap junction proteins

The connexins are a family of related gap junction proteins which contain conserved transmembrane and extracellular domains but unique cytoplasmic regions. To identify connexins with potential roles in development, a chick embryo cDNA library was screened by hybridization at low stringency with a cDNA for rat connexin-43. cDNA clones for two previously undescribed connexins were isolated. Chick connexin-45 has a predicted molecular mass of 45,376 daltons; connexin-42 has a predicted molecular mass of 41,748 daltons. Both of these predicted connexin proteins share the homologous regions noted in other members of this family, and each has its own unique regions. Southern blots of chicken genomic DNA suggest that each connexin is encoded by a distinct single copy gene. RNA blots demonstrate that while chick connexin-43, -42, and -45 are each expressed in a number of chick organs, they each have a unique tissue distribution. Each connexin mRNA is present in heart. Blots of total RNA isolated from hearts of chick embryos of different ages demonstrate that the abundance of connexin-42 and -43 mRNAs varies no more than 2-fold between the embryo and the adult. However, connexin-45 mRNA shows a dramatic change, falling 10-fold from the 6-day embryonic heart to the adult. These multiple connexins are likely to have different physiological properties and may account for the multiple physiologically distinct gap junction channels which have been observed in cardiac myocytes. They may provide a mechanism for the formation of communication compartments in the developing myocardium.     

Selective interactions among the multiple connexin proteins expressed in the vertebrate lens: the second extracellular domain is a determinant of compatibility between connexins

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.     

Nanomechanics of hemichannel conformations: connexin flexibility underlying channel opening and closing

Gap junctional hemichannels mediate cell-extracellular communication. A hemichannel is made of six connexin (Cx) subunits; each connexin has four transmembrane domains, two extracellular loops, and cytoplasmic amino- and carboxyl-terminals (CTs). The extracellular domains are arranged differently at non-junctional and junctional (gap junction) regions, although very little is known about their flexibility and conformational energetics. The cytoplasmic tail differs considerably in the size and amino acid sequence for different connexins and is predicted to be involved in the channel open and closed conformations. For large connexins, such as Cx43, the CT makes large cytoplasmic fuzz visible under electron microscopy. If this CT domain controls channel permeability by physical occlusion of the pore mouth, movement of this portion could open or close the channel. We used atomic force microscopy-based single molecule spectroscopy with antibody-modified atomic force microscopy tips and connexin mimetic peptide modified tips to examine the flexibility of extracellular loop and CT domains and to estimate the energetics of their movements. Antibody to the CT portion closer to the membrane stretches the tail to a shorter length, and the antibody to CT tail stretches the tail to a longer length. The stretch length and the energy required for stretching the various portions of the carboxyl tail support the ball and chain model for hemichannel conformational changes.     

Connexins in lymphatic vessel physiology and disease

Connexins are transmembrane proteins that form gap junction- and hemi-channels. Once inserted into the membrane, hemi-channels (connexons) allow for diffusion of ions and small molecules (<1 kDa) between the extracellular space and the cytosol. Gap junction channels allow diffusion of similar molecules between the cytoplasms of adjacent cells. The expression and function of connexins in blood vessels has been intensely studied in the last few decades. In contrast, only a few studies paid attention to lymphatic vessels; convincing in vivo data with respect to expression patterns of lymphatic connexins and their functional roles have only recently begun to emerge. Interestingly, mutations in connexin genes have been linked to diseases of lymphatic vasculature, most notably primary and secondary lymphedema. This review summarizes the available data regarding lymphatic connexins. More specifically it addresses (i) early studies aimed at presence of gap junction-like structures in lymphatic vessels, (ii) more recent studies focusing on lymphatic connexins using genetically engineered mice, and (iii) results of clinical studies that have reported lymphedema-linked mutations in connexin genes.     

Cloning and functional expression of a novel human connexin-25 gene

Gap junctions are intercellular, water-filled channels composed of transmembrane proteins called connexins, six of which are arranged radially and dock with six homologous proteins in an adjacent cell to form an approximate 16 A pore. Through this pore cell-to-cell transfer of small water-soluble molecules up to about 1000 daltons occurs along concentration gradients. Connexins comprise a multigene family that share consensus sequences in the trans-membrane domains and the first and second extracellular loops. Comparison of the protein sequences of known human connexins with the draft nucleotide sequence of the human genome revealed two clones from chromosome 6 which showed strong similarity to highly conserved connexin sequences. Detailed analysis revealed the presence of a 672 nt open reading frame in these clones, encoding a 223 amino acid polypeptide with a predicted molecular weight of about 25 kD. This is smaller than other known human connexins. The ORF of the potential connexin25 was amplified by semi-nested PCR using human genomic DNA as a template. To confirm that this new gene encodes a connexin, Cx25 was transfected into a gap junction deficient subclone of the human HeLa cell line. After selection of transformants, cells were microinjected with the fluorescent dye Lucifer yellow. Transfectants but not controls successfully transferred dye, demonstrating that this new gene encodes a functional connexin.     

Heteromeric Mixing of Connexins: Compatibility of Partners and Functional Consequences

Cx43 is widely expressed in many different cell types, and many of these cells also express other connexins. If these connexins are capable of mixing, the functional properties of channels containing heteromeric connexons may substantially influence intercellular communication between such cells. We used biochemical strategies (sedimentation through sucrose gradients, co-immunoprecipitation, or co-purification by Ni-NTA chromatography) to examine heteromeric mixing of Cx43 with other connexins (including Cx26, Cx37, Cx40, Cx45, and Cx56) in transfected cells. These analyses showed that all of the tested connexins except Cx26 formed heteromeric connexons with Cx43. We used the double whole-cell patch-camp technique to analyze the electrophysiological properties of gap junction channels in pairs of co-expressing cells. Cx37 and Cx45 made a large variety of functional heteromeric combinations with Cx43 based on detection of many different single channel conductances. Most of the channel event sizes observed in cells co-expressing Cx40 and Cx43 were similar to those of homomeric Cx43 or Cx40 hemichannels in homo- or hetero-typic configurations. Our data suggest several different possible consequences of connexin co-expression: (1) some combinations of connexins may form heteromeric connexons with novel proeprties; (2) some connexins may form heteromeric channels that do not have unique properties, and (3) some connexins may be incompatible for heteromeric mixing.     

Heteromeric mixing of connexins: compatibility of partners and functional consequences

Cx43 is widely expressed in many different cell types, and many of these cells also express other connexins. If these connexins are capable of mixing, the functional properties of channels containing heteromeric connexons may substantially influence intercellular communication between such cells. We used biochemical strategies (sedimentation through sucrose gradients, co-immunoprecipitation, or co-purification by Ni-NTA chromatography) to examine heteromeric mixing of Cx43 with other connexins (including Cx26, Cx37, Cx40, Cx45, and Cx56) in transfected cells. These analyses showed that all of the tested connexins except Cx26 formed heteromeric connexons with Cx43. We used the double whole-cell patch-camp technique to analyze the electrophysiological properties of gap junction channels in pairs of co-expressing cells. Cx37 and Cx45 made a large variety of functional heteromeric combinations with Cx43 based on detection of many different single channel conductances. Most of the channel event sizes observed in cells co-expressing Cx40 and Cx43 were similar to those of homomeric Cx43 or Cx40 hemichannels in homo- or hetero-typic configurations. Our data suggest several different possible consequences of connexin co-expression: (1) some combinations of connexins may form heteromeric connexons with novel proeprties; (2) some connexins may form heteromeric channels that do not have unique properties, and (3) some connexins may be incompatible for heteromeric mixing.     

Cloning and Functional Expression of a Novel Human Connexin-25 Gene

Gap junctions arc intercellular, water-filled channels composed of transmembrane proteins called connexins, six of which are arranged radially and dock with six homologous proteins in an adjacent cell to form an approximate 16 A pore. Through this pore cell-to-cell transfer of small water-soluble molecules up to about 1000 daltons occurs along concentration gradients. Connexins comprise a multigene family that share consensus sequences in the trans-membrane domains and the first and second extracellular loops. Comparison of the protein sequences of known human connexins with the draft nucleotide sequence of the human genome revealed two clones from chromosome 6 which showed strong similarity to highly conserved connexin sequences. Detailed analysis revealed the presence of a 672 nt open reading frame in these clones, encoding a 223 amino acid polypeptide with a predicted molecular weight of about 25 kD. This is smaller than other known human connexins. The ORF of the potential connexin25 was amplified by semi-nested PCR using human genomic DNA as a template. To confirm that this new gene encodes a connexin, Cx25 was transfected into a gap junction deficient subclone of the human HeLa cell line. After selection of transformants, cells were microinjected with the fluorescent dye Lucifer yellow. Transfectants but not controls successfully transferred dye, demonstrating that this new gene encodes a functional connexin.