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Cell communication across gap junctions: a historical perspective and current developments W. Howard Evans*1 *Wales Heart Research Institute and Institute of Infection and Immunity, Cardiff University School of Medicine, Cardiff, CF11 4XN Wales, U.K.

Biochemical Society Transactions

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Abstract Collaborative communication lies at the centre of multicellular life. Gap junctions (GJs) are surface membrane structures that allow direct communication between cells. They were discovered in the 1960s following the convergence of the detection of low-resistance electrical interactions between cells and anatomical studies of intercellular contact points. GJs purified from liver plasma membranes contained a 27 kDa protein constituent; it was later named Cx32 (connexin 32) after its full sequence was determined by recombinant technology. Identification of Cx43 in heart and later by a further GJ protein, Cx26 followed. Cxs have a tetraspan organization in the membrane and oligomerize during intracellular transit to the plasma membrane; these were shown to be hexameric hemichannels (connexons) that could interact end-to-end to generate GJs at areas of cell-to-cell contact. The structure of the GJ was confirmed and refined by a combination of biochemical and structural approaches. Progress continues towards obtaining higher atomic 3D resolution of the GJ channel. Today, there are 20 and 21 highly conserved members of the Cx family in the human and mouse genomes respectively. Model organisms such as Xenopus oocytes and zebra fish are increasingly used to relate structure to function. Proteins that form similar large pore membrane channels in cells called pannexins have also been identified in chordates. Innexins form GJs in prechordates; these two other proteins, although functionally similar, are very different in amino acid sequence to the Cxs. A time line tracing the historical progression of wide ranging research in GJ biology over 60 years is mapped out. The molecular basis of channel dysfunctions in disease is becoming evident and progress towards addressing Cx channel-dependent pathologies, especially in ischaemia and tissue repair, continues.

Introduction The progression from unicellular to multicellular life was a major step in evolution. It required the interdependent metabolic activities of cell assemblies to be co-ordinated and controlled. To achieve this goal, intercellular junctions weave together the activities of trillions of cells. Anatomists have described various categories of cell junctions in vertebrates such as adhesion junctions, desmosomes and tight junctions; septate junctions are present in prechordates. However, the discovery of a cell-to-cell junction that could provide a passageway enabling direct communication between attached cells, later to be named the gap junction (GJ), was a relatively late discovery in the 1960s and 1970s of the twentieth century. Contributing to the delay were three major concepts in biology. First, the cell theory promulgated in the nineteenth century by Schleiden and Schwann emphasized the dominating doctrine that cells in animals and plants behaved as independent units. Second, the growth of endocrinology at the turn of the last century provided mechanisms underpinning mainly long-distance communication between cells using chemical

Key words: biogenesis and turnover, channel structure and isolation, connexin (Cx) diseases, control by Ca2 + , channel manipulation by connexin (Cx) mimetic peptides. Abbreviations: Cx, connexin; CxHc, connexin hemichannel; GJ, gap junction. email [email protected].

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messengers (hormones) that interact specifically with cell surface ‘receptors’. Thirdly, acceptance of contact-dependent direct cell coupling across GJs was retarded by a dispute about how this communication occurred, especially in the nervous system, viz, chemical compared with electrical mechanisms. This controversy was won at the time by proponents that claimed that communication was effected by chemical messengers released across synaptic clefts; Henry Dale and Otto Loewi were awarded the Nobel Prize in 1936 for their work that gave birth to neuropharmacology. Today it is generally accepted that these two mechanisms of cell communication are complimentary, especially in the brain, and operates widely, especially between glial cells [1,2]. Evidence pointing to direct electrical intercellular communication began to appear in the 1950s (Table 1) notably from the laboratory of the Swiss physiologist Silvio Weidmann [3]. Subsequently, a more sympathetic acceptance of the idea that cells were directly coupled electrically by ‘low-resistance junctions’ emerged; for example, the studies of Loewenstein and Kanno [4] of insect salivary glands were convincing. In reality, there was a supporting cast whose papers provided compelling evidence supporting this novel concept [5–7], with much of the early electrophysiological work carried out using invertebrate cells. The geographical location and morphological features of a junctional site at the cell surface enabling direct intercellular communication is often credited Biochem. Soc. Trans. (2015) 43, 450–459; doi:10.1042/BST20150056

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to a key paper appearing in 1967 by Revel and Karnovsky [8]. The term GJ soon came into common parlance to denote a discrete intercellular contact point characterized by hexagonal arrays tightly and regularly packed and catalysing a growing view that this communication junction or nexus was made up of conglomerates of hexameric structures. This junction was morphologically and functionally different from other junctions (such as epithelial tight junctions). Hence the conception of a new field in cell biology and it grew rapidly. The first international conference devoted mainly to ‘gap or communicating junctions’ was held at a Cambridge College in 1976 and the proceedings published in 1978 [9]. This developed into biennial international conferences at which devotees describe the latest advances in GJs and cell communication.

Structural analysis of gap junctions; identification and purification of the major channel forming protein Cx Biochemists soon joined the fray as they sought to define the molecular makeup of a junction that allowed cells to ‘talk’ directly to one another without any infringements from the extracellular milieu. Subcellular fractionation techniques were used to prepare fractions enriched in GJs. However, many problems had to be confronted and overcome. The GJ accounts for a minute area of the plasma membrane and ‘bucket chemistry’ methods were used to purify sufficient material for analysis. For example, tissue homogenates derived from up to 200 mouse livers were fractionated by centrifugation using large zonal rotors (developed by Norman G. Anderson at Oak Ridge National laboratories in Tennessee in a megaproject to separate and analyse subcellular components). Large amounts of liver plasma membranes could be prepared by using these zonal rotors [10]. Extraction with mild detergents, especially n-lauryl sarcosinate, solubilized non-junctional plasma membranes producing minute pellets enriched GJs. However, samples were contaminated by extracellular matrix debris and greater morphological purity was achieved by addition of collagenases and hyaluronidases. However, these products contained proteases whose actions complicated identification of the major protein(s) of the junction. The tendency of the GJ protein to dimerize when separated during PAGE was a further difficulty [11–13]. The development of an alkaline extraction method sidelined many of these earlier problems [14]. The absence of enzymic biochemical markers meant that enrichment had to be monitored by EM, especially of negatively stained samples. Today, GJ proteins of high purity that are used for structural analysis are produced by recombinant methods [15]. There were other rival proteins proposed as GJ constituents at this time but they soon fell by the way. A major 26 kDa protein in eye lens fibres was considered as a likely ‘communication’ protein but its structure was subsequently shown to be unrelated to the liver 27 kDa protein [16];

however, other connexins (Cxs) have since been identified in the lens [17,73]. Ductins comprised another group of proteins proposed as GJ constituents and present in a wide range of cells but these were later shown to be subcomponents of a proton ATPase [18].

Antibodies to gap junctions as experimental tools GJs have proven to be poorly immunogenic structures. However, the affinity-purified, 27 kDa protein present in liver GJs allowed the generation of powerful antibodies. A dramatic result was obtained in 1984 that highlighted GJs as key determinants in embryonic development. The data showed that injection of this antibody, raised to a 27 kDa liver protein into eight-cell Xenopus embryos, led to deranged oneeyed tadpoles [19]. This antibody also disrupted embryonic development in eight-cell mouse embryos thus pointing strongly to universal roles for GJs in developmental processes across species [20]. Surprisingly, the antibody also disrupted patterning development in the cnidarian hydra, an invertebrate later shown to utilize unrelated proteins called innexins to construct GJs [21]. Nevertheless, a key role for GJs and direct cell communication in early stages of animal development was strongly established. It soon became apparent that gap junctional communication at post implantation stages of embryogenesis is a far more complex issue than earlier thought and featured several GJ proteins [22,23] One explanation for the broad disruptive actions of this antibody is that it blocked a large pore channel structure present in both connexin (Cx) and innexin GJs. Thus, two unrelated ‘GJ’ proteins, with differing primary sequences shared a common ‘shape’ epitope to which the antibodies bound. This can be considered an example of convergent evolution that has occurred in chordates and prechordates to fulfil a broad and fundamental cell communication function. These results also, in retrospect, indicate that caution should always be exercized when using antibodies to study complex cellular systems. The advent of a range of well-defined sitespecific antibodies raised to short, highly purified synthetic peptides corresponding to amino acid sequences in a wide range of Cxs were used to raise antibodies emerged as a highly successful approach to analyse GJ distribution and functions [56].

Connexins, the building blocks of vertebrate gap junctions are born The identification of a 27 kDa liver GJ protein was soon followed by a 47 kDa homologous protein in heart membranes enriched in intercalated discs. The deduction by recombinant methods of the full sequences of these two GJ proteins [24,25] constituted one of the most important advances in the GJ field. Beyer et al. [24] proposed that the term Cx be applied generally to GJ proteins. Subsequently, the number of Cxs increased quickly and a highly conserved  C The

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Cx family now exists with 20 in humans and 21 in mouse. Cloning of a 21 kDa protein, first thought to be a degradation product of the 28 kDa liver protein, rapidly followed [26]. The distribution of Cxs in various tissues and organs grew with most expressing two or more Cxs. It appeared that the initial nomenclature that is based on the molecular size of the cloned Cxs product would soon become problematic as the number of Cxs increased but it has been remained unchanged. Where necessary, reference can also be made to a genetically based nomenclature system in which for example Cx32 is GJβ1, Cx26 is GJβ2 and Cx43 is GJα1. Hydropathy plots of the protein sequences indicated that Cxs were most likely to be tetraspan membrane with four hydrophobic transmembrane sequences, two of which were domains possibly lining the channel wall. These are linked by two highly conserved extracellular loops each linked by three intramolecular disulfide bonds. At the cytoplasmic face of the protein, there is a short N-terminus stub, a highly variable intracellular loop connecting transmembrane segments 2 and 3 and finally a cytoplasmic tail that varies between Cxs in sequence and in length and that accounts for the variability in molecular size of Cx family members. The cytoplasmic tail has emerged without question as a major regulatory domain of the channel in Cx43 and is subject to extensive phosphorylation (occurring at two tyrosines and 21 serines) and modulated by range of protein kinases and phosphatases. Other post-translational modifications of Cxs described include ubiquitination, glutamate hydroxylation, symoylation, palmitoylation, methylation and S-nitrosylation [27]. Details of functions incurred by these Cx modifications are the subject of intensive research. Notably, Cxs are one of the very few proteins not subject to glycosylation, a further difference to pannexin 1. The predicted topography according to the hydropathy data of Cxs in the membrane has proved to be correct and extends to all members of the Cx family. However at the time, the overall details of Cx topography in the membrane needed to be confirmed. Conventional approaches were applied, especially to Cx32, Cx26 and Cx43, involving the use of antibodies raised to specific sequences on the outer and inner membrane regions of Cxs, proteolytic dissection of intact GJs and junctions split into component halves with urea and analysis by immunogold EM. [28,29]. Hexameric arrays denoting channel-like structures in isolated GJs stained with lanthanum indicated that progress towards obtaining high resolution 3D structures of GJ channels would be rapid and follow in the trail of the success achieved at the time with bacteriorhodopsin membranes. Early work proved promising with atomic resolution of the channel slowly increasing [30–33]. GJs constructed of Cx43 in which the carboxy tail had been enzymically removed were used in many investigations and these provided important 3D structural information of the dodecameric GJ double ˚ channel. An analysis of Cx26 has been produced at 3.5 A ˚ = 0.1 nm) resolution [34,35] and has further confirmed (1 A the intricate details obtained in earlier work. The Cx channel is formed by end-to-end docking of two hexameric connexon  C The

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or hemichannel components and consists of 24 rods of density inserted in the membrane and arranged around the central pore with a tight seal separating the channel from the extracellular milieu. This structure is similar overall to that deduced with Cx43 but, as might be expected, the increased resolution identifies some fine differences; for example, channel diameter that may account for selectivity differences. Other complementary approaches using, for example AFM, have added to the total picture of the structure of the GJ and its two component CxHcs (Cx hemichannels) [36].

Gap junction synthesis, turnover and degradation GJ proteins turn over rapidly within a few hours and occurs about 10 times faster than membrane proteins in general. After transcription, Cxs are co-translationally inserted into the endoplasmic reticulum although Cx26 can be posttranslationally inserted directly into plasma membranes [37]. The intracellular trafficking routes of various Cxs to and from the plasma membrane also appear to differ. Cx32 and Cx43 progressing along the conventional secretory pathway whereas Cx26 behaves differently [38–40]. Cxs need to oligomerize to generate a hexameric channel and an unexpected finding was that this process occurs with Cx43 in the Golgi apparatus in contrast with most membrane proteins moving along the secretory pathway to the plasma membrane [41,42]. During transit, extensive phosphorylation occurs at the cytoplasmic tail. This process has been extensively studied with Cx43. The Cx “half” channels (also referred to as connexons) then adhere to partners on adjacent cells and these coalesce laterally to form GJ plaques that vary in size between tissues. GJs are removed as units from the centre of the plaques and are degraded mainly in the endolysosomal networks [43,44]. Clearly, assembly and the fast turnover of Cxs need tight regulation (Figure 1). Cxs are also broken down in proteosomes, organelles that normally destroy abnormal proteins.

Calcium and cell communication via Cx channels Ever since the prescient work of Loewenstein [45], it was likely that Ca2 + , a key player in intracellular signalling, would also feature centrally in regulating intercellular coupling across GJs. But how? The propagation of increases in Ca2 + across groups of cells visualized using Ca2 + sensitive reporter dyes provided a convincing demonstration that these changes that could be induced by gentle mechanical stimulation at the cell surface were capable of being propagated to neighbouring cells via GJs [46–50]. Some earlier examples of intercellular Ca2 + wave propagation were observed in airway epithelial cells [47] and Ca2 + waves have since been studied extensively in a wide range of cells such as glia, retina etc. The Ca2 + waves approach cell surface contact regions at GJs and after a momentary delay, the Ca2 + wave spreads

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Figure 1 Assembly and breakdown of GJs emphasizing the genesis and trafficking of CxHcs Formation of CxHcs occurs early in the secretory pathway (stage 1) and they are transported along the secretory pathway (stages 2 and 3), implicating regions of the endoplasmic reticulum and the Golgi apparatus. A second assembly mechanism that is poorly characterized also operates with Cx26 and possibly other Cxs has been reported which does not directly feature the Golgi apparatus. CxHcs, after insertion into the plasma membrane (stage 4), dock with partners (stage 5) located on an attached cell and gate to an open configuration (stage 6), a process that may occur concurrently with the aggregation of the GJ channels into large adhesive plaques (stage 7). Stages 7 and 8 involve internalization of GJs into one of the attached cells and break down by hydrolysis in lysosomes (stage 9). Reproduced from [60] with permission from the authors.

and continues to generate Ca2 + changes in neighbouring cells. Ca2 + is regarded mainly as a local rather than a global signalling messenger and it was unlikely to be a signal moving between cells across GJs due, not least, to its high avidity for calmodulin. Cxs possess three calmodulin-binding sites located on cytoplasmic regions [51–53]. IP3 and possibly ATP emerged as the likely signalling vehicles traversing the GJ and enabling the propagation across cell conglomorates of the Ca2 + wave [49,54]. Roles for GJs in the intercellular spread of Ca2 + waves was re-enforced by induction of wave blockage by the newly introduced Cx mimetic peptides [55– 57] (see below). Explanations that proposed an exclusive role for GJs in Ca2 + wave propagation however proved to be an over-simplification for purinergic signalling mechanisms were also involved [60]. A dual mechanism for communication involving GJs and/or CxHc emerged (Figure 2). HeLa cells transfected with recombinant Cx43, Cx32 or Cx26 labelled with

GFP allowed the precise position of GJs to be seen by confocal microscopy. Ca2 + oscillations, visualized using a fluorophore, were shown to spread from cell-to-cell through membrane apposition areas containing the GFP-labelled GJs. However, when apyrase (an enzyme that hydrolyses ATP) was omitted, Ca2 + waves also spread across cell layers via non junctional surface membranes. Thus, Ca2 + wave spread between cell populations occurred by more than one route with a non-junctional route facilitating release from the cell of ATP into the cell exterior probably across open CxHc [58]. As shown in Figure 2, purinergic receptors on neighbouring cells underpin the spread of Ca2 + across groups of cells that are also linked by GJs. As well as ATP, other tri-nucleotides, glutamate, glutathione and prostaglandins are released by cells across hemichannels. The involvement of CxHcs in cell communication utilizing purinergic paracellular mechanisms gained acceptance and highlighted the implication of CxHcs. At first, it had  C The

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Table 1 Timeline: some major milestones in research on GJs and intercellular communication

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Event

1952

Cells are shown to communicate across ‘low-resistance’ electrical junctions in heart, salivary glands and brains. Intercellular contact (junctional) candidates explored by morphological approaches.

1967

Tentative identification of ‘communication’ junctions in liver plasma membranes; negative staining shows hexagonal lattices suggestive of membrane pores. GJs named (or misnamed) as the likely site of electrical continuity between cells in tissues and organs.

1969 1972 1974

GJ fractions isolated from liver and heart tissue and composition examined major 25000–30000 protein identified as a possible channel component in liver, a 54000 protein in heart (later named Cx 32 and Cx43). Roles of GJs in embryonic development studied; pitfalls.

1979 1980 1986–87

Structure of GJs obtained at 18 Å resolution. Crucial role of calcium in channel operation and later in cellular synchronization by GJs delineated. Cx32 (liver), Cx26 (heart) and Cx26 sequences obtained from cDNA, a crucial advance. Cx coined as the principal GJ protein, one of a family that expanded to 21/22. Topography in membrane established. Cx knockout genetic approaches applied extensively and outlines importance of Cxs in tissues and organs. Confocal immunofluorescence and immunogold microscopy using anti-peptide antibodies map out Cx distribution in, e.g., heart.

1990 1993

Importance of Cx43/40 in the immune system shown. Mutations in Cx32 in CMT X-linked disease Charcot Marie Tooth X-determined; mutations in non-syndromic hearing described later.

1995 1997

Amino acid motifs required for GJ assembly and channel function identified. Development of Cx-mimetic peptides, Gap 26, 27 and others used to dissect role of GJ in initially vascular endothelium.

2000 2004 2007

Pannexin channel proteins ‘cousins’ of Cxs discovered. Explosion in pannexin research. Comparisons with innexins and Cxs investigated. Cx43 detected in mitochondrial inner membrane; nitrosylation of Cx43 described

2009

Roles from connexins in DNA change by nanoparticles confined to cellular bilayers discovered. High resolution structure of Cx26 gap junctions at 3.5 Å resolution reported. Roles of Cxs in non-channel communication functions emerge.

2010 2012

Identification of Cxs in platelets and eosinophils and roles in thrombosis reported. Exploitation of Gap 19, a Cx mimetics specific for Cx43 hemichannels. Use of Cx mimetic peptides in translational research expands. Non-canonical roles for Cxs in migration, adhesion begin to build up.

Figure 2 CxHcs and Ca2 + signalling (A) CxHcs open in response to cytoplasmic Ca2 + changes and thereby form a conduit for the release of messengers such as ATP and others; CxHcs only open in so-called ’trigger cells’. (B) ATP diffuses into the extracellular space and activates G-protein-coupled serpentine receptors on neighbouring cells. This results in the activation of phospholipase C, the formation of InsP3 and the release of Ca2 + from the endoplamic reticulum. This pathway underlies paracrine cell–cell communication of Ca2 + signals. (C) Ca2 + signals can also be communicated by the diffusion of InsP3 or Ca2 + via GJs connecting cells. (D) The extracellular ATP concentration gradually decreases and the communication of Ca2 + signals stops unless another trigger cell is encountered that regenerates the ATP signal. (E) Ca2 + -triggered ATP release via CxHcs may also be involved in Ca2 + oscillations in the cell via an autocrine signalling path (see the text). (F) Cytoplasmic Ca2 + changes can trigger CxHc opening and conversely open CxHcs may magnify Ca2 + changes by Ca2 + entry from the extracellular space. InsP3 moves directly across GJs. Reproduced from [60] with permission from the authors.

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been thought that CxHcs resident in non-junctional plasma membrane areas were precursors en-route for assembly into GJs. CxHc, in an open configuration were regarded as laboratory induced artefacts, although they had been reported to be present and functional in horizontal cells of the retina [59]. CxHc could be induced experimentally to open in cell cultures by low Ca2 + media, by osmotic differences or by hypoxic conditions [60–63]. CxHc have now emerged as one of the ever widening properties of Cxs. Detailed work to analyse the operation of CxHc, in comparison with GJs, was aided by the realization that Cx mimetic peptides (see below) were rapid and efficient inhibitors of CxHcs. Elevation of intracellular Ca2 + induces closure of GJs and made it unlikely that Ca2 + could act as an intercellular signal. However, it was shown subsequently that intracellular Ca2 + levels that closed GJ had a different effect on CxHc causing them to open [64,65]. These observations underlined the fact that the two types of channels, both Cx channel constructs, have different Ca2 + -dependent conductance properties probably reflecting different functions in the cell.

Connexins and disease GJs have always been considered as likely candidates in instigating disease processes from cancer [66] to Huntington’s [67]. Subak-Sharpe and Pits [68], in the 1980s, had predicted the potential roles of cell junctions in intercellular metabolic co-operation and dis-regulation in metastasis leading to the use of terms such as the kiss of life or death that depended on exchange of metabolites via cell communication junctions. Extensive studies followed over decades that considered the possible implication of GJ malfunction as a critical factor in metastatic behaviour and although progress continues incisive advances involving precise association of Cx functions and cancer, progress has been thin on the ground [69,70]. A red letter day in 1993 (Table 1) saw the first report of mutations in Cx32 in peripheral nerves of patients with X-linked Charcot Marie Tooth syndrome, a rare peripheral demyelinating neuropathy. [71]. Today well over 100 Cx mutations have been associated with this syndrome although many are polymorphisms. Of more practical medical application was the detection of numerous point mutations in Cx26 associated with deafness and skin problems [72]. Routine sequencing of the Cx26 gene in new-born children can now alert parents of potential hearing difficulties likely to become prominent later in life. Mutations in Cx30 and Cx31 are also associated with skin disorders. Mutations in Cx26 are also found in Vohwinkel syndrome that affects epidermal integrity [73]. Mutations in Cx47 in the central nervous system are found in patients with Merzbacher-like disease [74]. Cataracts are commonly associated with mutations in Cx46 and Cx47 in the eye lens [73]. Relatively few mutations in Cx43, the most-widely expressed Cx, have been found. They are however detected in occulodentodigital dysplasia [75]. However, the widespread distribution of Cx43 and the comparative rarity of mutated

forms emphasize the central importance of Cx43 and was exemplified many years ago by the fact that Cx43 genetic deletion experiments in mice lead to death in utero due to the onset of cardio-pulmonary problems.

Blocking Cx channels: mimetic peptides Gap 19, 24, 26 and 27 provide new experimental tools and opportunities using translational approaches Pharmacologists gave research on GJs a wide berth because junctional areas were fairly inaccessible to inhibitory reagents hopelessly lacking in specificity [76]. This has now changed with the introduction of the Cx mimetic peptides that correspond to specific short sequences copied from the outer and inner sides of the Cx channel proteins. Their introduction as research tools was prompted by the shortcomings of antibodies as agents to block GJs, linked to their often unreliable specificity and limitations imposed by their size. By testing the efficacy of a range of short Cx peptides in retarding the co-ordinated beating of chick cells or in mouse embryos, the best regions to make synthetic peptide candidates that block a GJ-dependent processes were selected [77,78]. By the early 1990s, the widely expressed Cx43 appeared as the best candidate to use as a template and two peptides were designed that have now featured widely in GJ research (Table 1). They were designated Gap 26 (extracellular loop 1) and Gap 27 (extracellular loop 2; Figure 3). Earliest results showed that these mimetic peptides inhibited endothelial-dependent relaxations in blood vessels, a series of studies that pointed to a central role for GJs in this process [79]. Since ATP release was at the time beginning to be accepted as a characteristic feature of open CxHc [80,81], it was shown later that the peptides in addition to their action in blocking GJs, also inhibited CxHcs. Subsequently their predominant CxHc inhibitory action occurred within minutes. Indeed, approaching a 100 reports have now appeared and the blocking properties of Gap 26 and 27 on GJs and or CxHcs in a wide range of cells and tissues has been documented [82,83]. Importantly, their actions appear to be largely reversible when tested in experimental situations. They have proved to be highly selective in blocking movement through channels in both directions but the use of optimum peptide concentrations needs to be monitored. A drawback that can also prove an advantage is they appear to act on most Cxs owing to the high conservation of amino acid sequences in the two Cx extracellular loops. For targeting specific Cxs, the introduction of peptides selective towards Cx32 or Cx43 has been facilitated by using Gap 24 and 19 respectively; these correspond to short sequences on the cytoplasmic intracellular loops, an area where sequences between various Cxs differ widely (Figure 3). These new mimetics have been effective in blocking CxHc with minimal effects on GJ and, importantly, they do not inhibit pannexin channels. Gap 19 mimetic has been shown in HeLa cells and in pig ventricular cardiomyocytes to enter cells by an  C The

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Figure 3 Position of Cx mimetic peptides Gap 26 amino acids 63–75 (first extracellular loop) [82]. Gap 27 amino acids 204–214 (second exctracellular loop) [82]. Gap 19 amino acids 108–127 (intracellular loop of Cx43) [84]. Gap 24 amino acids 110–122 (intracellular loop of Cx32). Gap 26 and Gap 27 act on GJs and CxHC. Gap 19 and 24 are specific for CxHC.

unknown mechanism and bind to the C-terminus of Cx43 thus preventing intermolecular interactions that are central to channel opening as reflected by inhibition of channel unitary currents [84]. Gap 19 also inhibited Cx communication in astroglial cells [85]. These Cx mimetics as well as a range of other peptides acting at the intracellular loop and C-terminal region are being tested to repair ischaemic damage in the heart and in spinal cord injury [86–90].

Non-channel functions of connexins It has now become evident that Cxs fulfill roles in addition to their channel functions, often called non-canonical functions [91,92]. This applies especially to the involvement of Cxs in cell adhesion and migration. In glioma cells mutated extracellular Cx loops reduced adhesion and effects on migration were also affected by treatment with Cx mimetic. Gap 26 treatment increased the migration of epidermal cells [93]. Developmental processes as in the neural cortical and neural crest cells provide further examples where non-channel functions of Cxs are critical for migratory processes [94].

Charging ahead across the gaps Undoubtedly, a huge amount of information has been gleaned over the last 50 years on how cells communicate across GJs. The field exploded in the 1980s (Table 1) with the discovery and characterization of the large family of Cx proteins that form these channels and there have been many surprises along the way. Studies of various Cxs distributed across virtually all body tissues and organs have posed questions regarding the physiological significance and consequences of the differential channel porosities provided by Cxs and the  C The

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presence of homomeric and or heteromeric channels adds complexity while providing a potential for subtle channelcontrolled messaging between cells. There is paucity in knowledge of the biochemical messengers moving across Cx channels. For example, asymmetrical transfer of small nucleic acids across GJs so avoiding nuclease action and preserving sequence information is an avenue for future research. Progress has been hugely reliant on the exploitation of genetic engineering techniques [95] with wide ranging studies in mice containing a variety of Cx deletions, mutations and Cx switching in several biological settings, especially in heart and brain. The discovery of CxHc moved quickly from possible artefact to physiological and pathological reality and purinergic paracellular signalling via these channels has introduced new functional horizons; and there are other large pore channels. Pannexins are similar sized proteins to Cxs and are more related in sequence to innexins that form GJs in insects. They appear unable to couple up with partners to form a ‘GJ’ but pannexin channels also release ATP and participate in purinergic intercellular signalling [96]. Translational application of mimetic peptides specific to each channel type are being actively pursued with implications for treating cardiovascular disease, cerebral ischaemia and blood vessel damage induced by cryopreservation [97]. In the immune system, Cxs in a variety of settings play a role in inflammatory processes [98] exemplified by interactions of lymphocytes with each other and with endothelial cells lining blood vessel walls. The pathology of atheroma in this process has a Cx component [99,100]. Cxs have also been shown to be present in platelets posing roles in thrombolytic events [101]. Roles for Cxs in mediating the effects of nanoparticles that can lead to cellular DNA changes have been described and occur especially in cellular bilayer barriers in the body [102].

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Acknowledgements

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This is a personal account and by its nature is highly selective. I apologize to colleagues whose important contributions have been omitted. Support for several GJ research programmes and projects in my laboratories over a long period in London and Cardiff has been generously provided in the main by the MRC, Welcome Trust and BHF.

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