Multiple connexin proteins in single intercellular channels: Connexin compatibility and functional consequences

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 consequencesin 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 observedin vivo.     

Molecular organization of gap junction membrane channels

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 formingin 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.     

The cellular internet: On-line with connexins

Most cells communicate with their immediate neighbors through the exchange of cytosolic molecules such as ions, second messengers and small metabolites. This activity is made possible by clusters of intercellular channels called gap junctions, which connect adjacent cells. In terms of molecular architecture, intercellular channels consist of two channels, called connexons, which interact to span the plasma membranes of two adjacent cells and directly join the cytoplasm of one cell to another. Connexons are made of structural proteins named connexins, which compose a multigene family. Connexin channels participate in the regulation of signaling between developing and differentiated cell types, and recently there have been some unexpected findings. First, unique ionic- and size-selectivities are determined by each connexin; second, the establishment of intercellular communication is defined by the expression of compatible connexins; third, the discovery of connexin mutations associated with human diseases and the study of knockout mice have illustrated the vital role of cell-cell communication in a diverse array of tissue functions.     

Gap-junction-mediated cell-to-cell communication

Cells of multicellular organisms need to communicate with each other and have evolved various mechanisms for this purpose, the most direct and quickest of which is through channels that directly connect the cytoplasms of adjacent cells. Such intercellular channels span the two plasma membranes and the intercellular space and result from the docking of two hemichannels. These channels are densely packed into plasma-membrane spatial microdomains termed “gap junctions” and allow cells to exchange ions and small molecules directly. A hemichannel is a hexameric torus of junctional proteins around an aqueous pore. Vertebrates express two families of gap-junction proteins: the well-characterized connexins and the more recently discovered pannexins, the latter being related to invertebrate innexins (“invertebrate connexins”). Some gap-junctional hemichannels also appear to mediate cell-extracellular communication. Communicating junctions play crucial roles in the maintenance of homeostasis, morphogenesis, cell differentiation and growth control in metazoans. Gap-junctional channels are not passive conduits, as previously long regarded, but use “gating” mechanisms to open and close the central pore in response to biological stimuli (e.g. a change in the transjunctional voltage). Their permeability is finely tuned by complex mechanisms that have just begun to be identified. Given their ubiquity and diversity, gap junctions play crucial roles in a plethora of functions and their dysfunctions are involved in a wide range of diseases. However, the exact mechanisms involved remain poorly understood.     

Endocytosis and post-endocytic sorting of connexins

The connexins constitute a family of integral membrane proteins that form intercellular channels, enabling adjacent cells in solid tissues to directly exchange ions and small molecules. These channels assemble into distinct plasma membrane domains known as gap junctions. Gap junction intercellular communication plays critical roles in numerous cellular processes, including control of cell growth and differentiation, maintenance of tissue homeostasis and embryonic development. Gap junctions are dynamic plasma membrane domains, and there is increasing evidence that modulation of endocytosis and post-endocytic trafficking of connexins are important mechanisms for regulating the level of functional gap junctions at the plasma membrane. The emerging picture is that multiple pathways exist for endocytosis and sorting of connexins to lysosomes, and that these pathways are differentially regulated in response to physiological and pathophysiological stimuli. Recent studies suggest that endocytosis and lysosomal degradation of connexins is controlled by a complex interplay between phosphorylation and ubiquitination. This review summarizes recent progress in understanding the molecular mechanisms involved in endocytosis and post-endocytic sorting of connexins, and the relevance of these processes to the regulation of gap junction intercellular communication under normal and pathophysiological conditions. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Gap junctions: structure and function (Review)

Gap junctions are plasma membrane spatial microdomains constructed of assemblies of channel proteins called connexins in vertebrates and innexins in invertebrates. The channels provide direct intercellular communication pathways allowing rapid exchange of ions and metabolites up to ~1 kD in size. Approximately 20 connexins are identified in the human or mouse genome, and orthologues are increasingly characterized in other vertebrates. Most cell types express multiple connexin isoforms, making likely the construction of a spectrum of heteromeric hemichannels and heterotypic gap junctions that could provide a structural basis for the charge and size selectivity of these intercellular channels. The precise nature of the potential signalling information traversing junctions in physiologically defined situations remains elusive, but extensive progress has been made in elucidating how connexins are assembled into gap junctions. Also, participation of gap junction hemichannels in the propagation of calcium waves via an extracellular purinergic pathway is emerging. Connexin mutations have been identified in a number of genetically inherited channel communication-opathies. These are detected in connexin 32 in Charcot Marie Tooth-X linked disease, in connexins 26 and 30 in deafness and skin diseases, and in connexins 46 and 50 in hereditary cataracts. Biochemical approaches indicate that many of the mutated connexins are mistargeted to gap junctions and/or fail to oligomerize correctly into hemichannels. Genetic ablation approaches are helping to map out a connexin code and point to specific connexins being required for cell growth and differentiation as well as underwriting basic intercellular communication.     

Gap junctions: structure and function (Review)

Gap junctions are plasma membrane spatial microdomains constructed of assemblies of channel proteins called connexins in vertebrates and innexins in invertebrates. The channels provide direct intercellular communication pathways allowing rapid exchange of ions and metabolites up to approximately 1 kD in size. Approximately 20 connexins are identified in the human or mouse genome, and orthologues are increasingly characterized in other vertebrates. Most cell types express multiple connexin isoforms, making likely the construction of a spectrum of heteromeric hemichannels and heterotypic gap junctions that could provide a structural basis for the charge and size selectivity of these intercellular channels. The precise nature of the potential signalling information traversing junctions in physiologically defined situations remains elusive, but extensive progress has been made in elucidating how connexins are assembled into gap junctions. Also, participation of gap junction hemichannels in the propagation of calcium waves via an extracellular purinergic pathway is emerging. Connexin mutations have been identified in a number of genetically inherited channel communication-opathies. These are detected in connexin 32 in Charcot Marie Tooth-X linked disease, in connexins 26 and 30 in deafness and skin diseases, and in connexins 46 and 50 in hereditary cataracts. Biochemical approaches indicate that many of the mutated connexins are mistargeted to gap junctions and/or fail to oligomerize correctly into hemichannels. Genetic ablation approaches are helping to map out a connexin code and point to specific connexins being required for cell growth and differentiation as well as underwriting basic intercellular communication.     

Reciprocal influence of connexins and apical junction proteins on their expressions and functions

Membranes of adjacent cells form intercellular junctional complexes to mechanically anchor neighbour cells (anchoring junctions), to seal the paracellular space and to prevent diffusion of integral proteins within the plasma membrane (tight junctions) and to allow cell-to-cell diffusion of small ions and molecules (gap junctions). These different types of specialised plasma membrane microdomains, sharing common adaptor molecules, particularly zonula occludens proteins, frequently present intermingled relationships where the different proteins co-assemble into macromolecular complexes and their expressions are co-ordinately regulated. Proteins forming gap junction channels (connexins, particularly) and proteins fulfilling cell attachment or forming tight junction strands mutually influence expression and functions of one another.     

The gap junction cellular internet: connexin hemichannels enter the signalling limelight

Cxs (connexins), the protein subunits forming gap junction intercellular communication channels, are transported to the plasma membrane after oligomerizing into hexameric assemblies called connexin hemichannels (CxHcs) or connexons, which dock head-to-head with partner hexameric channels positioned on neighbouring cells. The double membrane channel or gap junction generated directly couples the cytoplasms of interacting cells and underpins the integration and co-ordination of cellular metabolism, signalling and functions, such as secretion or contraction in cell assemblies. In contrast, CxHcs prior to forming gap junctions provide a pathway for the release from cells of ATP, glutamate, NAD+ and prostaglandin E2, which act as paracrine messengers. ATP activates purinergic receptors on neighbouring cells and forms the basis of intercellular Ca2+ signal propagation, complementing that occuring more directly via gap junctions. CxHcs open in response to various types of external changes, including mechanical, shear, ionic and ischaemic stress. In addition, CxHcs are influenced by intracellular signals, such as membrane potential, phosphorylation and redox status, which translate external stresses to CxHc responses. Also, recent studies demonstrate that cytoplasmic Ca2+ changes in the physiological range act to trigger CxHc opening, indicating their involvement under normal non-pathological conditions. CxHcs not only respond to cytoplasmic Ca2+, but also determine cytoplasmic Ca2+, as they are large conductance channels, suggesting a prominent role in cellular Ca2+ homoeostasis and signalling. The functions of gap-junction channels and CxHcs have been difficult to separate, but synthetic peptides that mimic short sequences in the Cx subunit are emerging as promising tools to determine the role of CxHcs in physiology and pathology.     

Regulation of gap junction intercellular communication by the ubiquitin system

Intercellular communication via gap junctions plays a critical role in numerous cellular processes, including the control of cell growth and differentiation, maintenance of tissue homeostasis and embryonic development. Gap junctions are aggregates of intercellular channels that enable adjacent cells in solid tissues to directly exchange ions and small molecules. These channels are formed by a family of integral membrane proteins called connexins, of which the best studied is connexin43. Connexins have a high turnover rate in most tissue types, and degradation of connexins is considered to be a tightly regulated process. Post-translational modification of connexins by ubiquitin is emerging as an important event in the regulation of connexin degradation. Ubiquitination is involved in endoplasmic reticulum-associated degradation of connexins as well as in trafficking of connexins to lysosomes. At both the endoplasmic reticulum and the plasma membrane, ubiquitination of connexins is strongly affected by changes in the extracellular environment. There is increasing evidence that the regulation of connexin ubiquitination might be an important mechanism for rapidly modifying the level of functional gap junctions at the plasma membrane, under both normal and pathological conditions. This review discusses the current knowledge about the regulation of intercellular communication via gap junctions by ubiquitination of connexins.     

Cadherins mediate intercellular mechanical signaling in fibroblasts by activation of stretch-sensitive calcium-permeable channels

Cells in mechanically active environments form extensive, cadherin-mediated intercellular junctions that are important in tissue remodeling and differentiation. Currently, it is unknown whether adherens junctions in connective tissue fibroblasts transmit mechanical signals and coordinate multicellular adaptations to physical forces. We hypothesized that cadherins mediate intercellular mechanotransduction by activating calcium-permeable, stretch-sensitive channels. Human gingival fibroblasts in suspension were plated on established homotypic monolayer cultures. The cells formed intercellular adherens junctions. Controlled mechanical forces were applied to intercellular junctions by electromagnets acting on cells containing internalized magnetite beads. At early but not later stages of intercellular attachment, force application visibly displaced magnetite bead-loaded cells and induced robust Ca(2+) transients (65 +/- 9.4 nm above base line). Similar Ca(2+) transients were induced by force application to anti-N-cadherin antibody-coated magnetite beads. Ca(2+) responses depended on influx of extracellular Ca(2+) through mechanosensitive channels because both Ca(2+) chelation and gadolinium chloride abolished the response and MnCl(2) quenched fura-2 fluorescence after force application. Force application induced accumulation of microinjected rhodamine-actin at intercellular contacts; actin assembly was inhibited by buffering intracellular calcium fluxes. Our results indicate that mechanical forces applied to adherens junctions activate stretch-sensitive calcium-permeable channels and increase actin polymerization. We suggest that N-cadherins in fibroblasts are intercellular mechanotransducers.     

Fine structure of the rectal pads in the desert locust Schistocerca gregaria with reference to the mechanism of water uptake

The rectal pads of Schistocerca gregaria are composed of three different cell types: epithelial, secondary and junctional cells. The rectal pads are interconnected by simple rectal cells and both are lined internally by a articular intima. The epithelial cells exhibit extensive infoldings of the apical plasma membranes that are closely associated with mitochondria. Their lateral plasma membranes are highly folded around large mitochondria and enclose intercellular channels and spaces. They are united by belt and spot desmosomes, septate junctions, gap junctions and scalariform junctions, but terminate in a basal syncytium without contacting the basal plasma membranes. The apical and basal cytoplasm contain coated vesicles, dense tubular elements, multivesicular bodies and lysosomes, suggesting receptor-mediated endocytosis of small peptide molecules into the epithelial cells. The apical membrane infoldings of the secondary cells are also associated with large mitochondria. Their basal plasma membranes are covered by connective cell processes and connected with them by spot desmosomes which may be involved in solute recycling. The presence of neurosecretory-like axons near the secondary cells suggests that they exert local control on the function of these cells. The ultrastructural details are examined in relation to their role in solute and water transport.     

Desmosomal cadherins utilize distinct kinesins for assembly into desmosomes

The desmosomal cadherins, desmogleins (Dsgs) and desmocollins (Dscs), comprise the adhesive core of intercellular junctions known as desmosomes. Although these adhesion molecules are known to be critical for tissue integrity, mechanisms that coordinate their trafficking into intercellular junctions to regulate their proper ratio and distribution are unknown. We demonstrate that Dsg2 and Dsc2 both exhibit microtubule-dependent transport in epithelial cells but use distinct motors to traffic to the plasma membrane. Functional interference with kinesin-1 blocked Dsg2 transport, resulting in the assembly of Dsg2-deficient junctions with minimal impact on distribution of Dsc2 or desmosomal plaque components. In contrast, inhibiting kinesin-2 prevented Dsc2 movement and decreased its plasma membrane accumulation without affecting Dsg2 trafficking. Either kinesin-1 or -2 deficiency weakened intercellular adhesion, despite the maintenance of adherens junctions and other desmosome components at the plasma membrane. Differential regulation of desmosomal cadherin transport could provide a mechanism to tailor adhesion strength during tissue morphogenesis and remodeling.     

Opening of single gap junction channels during formation of electrical coupling between embryonic muscle cells

Gap junctions, which are low-resistance intercellular pathways, may contribute to normal embryogenesis by allowing cell-to-cell passage of as yet unidentified regulatory or inductive signals. But little is known about the properties of newly formed single junctional channels which are the basis of the communicating junctions. Reported here are the first direct measurements of current passing through single junctional channels as they form. Individual pairs of embryonic Xenopus muscle cells in culture were manipulated into contact, allowing control of the onset time and area of cellular contact, and current was recorded with the patch clamp technique. The opening of single channels which pass current between the two cells at a conductance of about 100 pS was observed within minutes of cell-cell contact. The channels opened one-at-a-time, and once formed, remained open for long periods of time, with infrequent brief closures. This suggests that formation of electrical coupling between two cells proceeds by addition of single conducting junctional channels one channel-at-a-time.     

The carboxy-tail of connexin-43 localizes to the nucleus and inhibits cell growth

Gap junctions are plasma membrane intercellular communication channels that in addition to ensuring electrical coupling and coordinated mechanical activity, can act as growth suppressors. To define the role of a non-channel forming domain of connexin-43 (Cx43), the main constituent of cardiomyocyte gap junctions, on growth regulation, we expressed its C-terminal portion (CT-Cx43) in cardiomyocytes and HeLa cells. In addition to broad cytoplasmic localization, CT-Cx43 was also localized to the nucleus of both cell types, detected by immunofluorescence as well as immunoblotting of subcellular fractions. Furthermore, stable expression of CT-Cx43 in HeLa cells induced a significant decrease in proliferation. It is therefore suggested that plasma membrane localization and formation of channels are not required for growth inhibition by Cx43, and that nuclear localization of CT-Cx43 may exert effects on gene expression and growth.     

Pannexin channels are not gap junction hemichannels

Pannexins, a class of membrane channels, bear significant sequence homology with the invertebrate gap junction proteins, innexins and more distant similarities in their membrane topologies and pharmacological sensitivities with the gap junction proteins, connexins. However, the functional role for the pannexin oligomers, or pannexons, is different from connexin oligomers, the connexons. Many pannexin publications have used the term "hemichannels" to describe pannexin oligomers while others use the term "channels" instead. This has led to confusion within the literature about the function of pannexins that promotes the idea that pannexons serve as gap junction hemichannels and thus have an assembly and functional state as gap junctional intercellular channels. Here we present the case that unlike the connexin gap junction intercellular channels, so far, pannexin oligomers have repeatedly been shown to be channels that are functional in single membranes, but not as intercellular channel in appositional membranes. Hence, they should be referred to as channels and not hemichannels. Thus, we advocate that in the absence of firm evidence that pannexins form gap junctions, the use of the term "hemichannel" be discontinued within the pannexin literature.     

Manipulating Connexin Communication Channels: Use of Peptidomimetics and the Translational Outputs

Gap junctions are key components underpinning multicellularity. They provide cell to cell channel pathways that enable direct intercellular communication and cellular coordination in tissues and organs. The channels are constructed of a family of connexin (Cx) membrane proteins. They oligomerize inside the cell, generating hemichannels (connexons) composed of six subunits arranged around a central channel. After transfer to the plasma membrane, arrays of Cx hemichannels (CxHcs) interact and couple with partners in neighboring attached cells to generate gap junctions. Cx channels have been studied using a range of technical approaches. Short peptides corresponding to sequences in the extra- and intracellular regions of Cxs were used first to generate epitope-specific antibodies that helped studies on the organization and functions of gap junctions. Subsequently, the peptides themselves, especially Gap26 and -27, mimetic peptides derived from each of the two extracellular loops of connexin43 (Cx43), a widely distributed Cx, have been extensively applied to block Cx channels and probe the biology of cell communication. The development of a further series of short peptides mimicking sequences in the intracellular loop, especially the extremity of the intracellular carboxyl tail of Cx43, followed. The primary inhibitory action of the peptidomimetics occurs at CxHcs located at unapposed regions of the cell’s plasma membrane, followed by inhibition of cell coupling occurring across gap junctions. CxHcs respond to a range of environmental conditions by increasing their open probability. Peptidomimetics provide a way to block the actions of CxHcs with some selectivity. Furthermore, they are increasingly applied to address the pathological consequences of a range of environmental stresses that are thought to influence Cx channel operation. Cx peptidomimetics show promise as candidates in developing new therapeutic approaches for containing and reversing damage inflicted on CxHcs, especially in hypoxia and ischemia in the heart and in brain functions.     

Gap junctions between sertoli and germ cells of rat seminiferous tubules

Ultrastructural observations of rat seminiferous tubules show clearly the presence of plasma membrane junctions between Sertoli and germ cells in the basal and adluminal compartments. Results obtained from the freeze fracture and thin section techniques were correlated in order to elucidate the nature of these intercellular junctions. We suggest that these intercellular membrane specializations are gap junctions which occur within regions of plasma membrane that also exhibit adherens-like modifications.     

Ultrastructure of the myocardial junctions of the chick embryo at an early stage in the organogenesis of the heart

The ultrastructural features of the myocardiocyte junctional systems have been studied in the heart right myocardium of 6 day chick embryos in order to analyze the relationships between the spatial arrangement of the myocardial fibres and the formation of the myocardiocyte junctions. The myocardiocytes are remarkably branched, joined in small fibres to form a loose network with large intercellular spaces and appear well differentiated, rich in organelles, myofibrils and glycogen. Adherent-type junctions and well differentiated desmosomes are detectable at the level of the termino-terminal contacts between myocardiocytes, where the plasma membranes appear more or less complicately interconnected; nexus-type junctions are seen in the latero-lateral contact regions. The results indicate that already at an early stage of the heart organogenesis the myocardium intercellular junctions are similar, on the whole, to those described in the adult, so that they would not seem to be transitory or modifiable structures. The changes in the spatial orientation of the myocardial fibres, which take place after the 6th incubation day, could be allowed by the network-like arrangement of the primitive myocardium and presumably conditioned by the blood flow in the developing heart.