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Gap junctions confer interconnectivity of the cytoplasm in neighboring cells via docking of two connexons expressed in each of the adjacent membranes. Undocked connexons, referred to as hemichannels, may open and connect the cytoplasm with the extracellular fluid. The hemichannel configuration of connexins (Cxs) displays isoform-specific permeability profiles that are not directly determined by the size and charge of the permeant. To further explore Ca2+-mediated gating and permeability features of connexin hemichannels, we heterologously expressed Cx30 hemichannels in Xenopus laevis oocytes. The sensitivity toward divalent cation-mediated gating differed between small atomic ions (current) and fluorescent dye permeants, indicating that these permeants are distinctly gated. Three aspartate residues in Cx30 (Asp-50, Asp-172, and Asp-179) have been implicated previously in the Ca2+ sensitivity of other hemichannel isoforms. Although the aspartate at position Asp-50 was indispensable for divalent cation-dependent gating of Cx30 hemichannels, substitutions of the two other residues had no significant effect on gating, illustrating differences in the gating mechanisms between connexin isoforms. Using the substituted cysteine accessibility method (SCAM), we evaluated the role of possible pore-lining residues in the permeation of ions and ethidium through Cx30 hemichannels. Of the cysteine-substituted residues, interaction of a proposed pore-lining cysteine at position 37 with the positively charged compound [2-(trimethylammonium)ethyl] methane thiosulfonate bromide (MTS-ET) increased Cx30-mediated currents with unperturbed ethidium permeability. In summary, our results demonstrate that the permeability of hemichannels is regulated in a permeant-specific manner and underscores that hemichannels are selective rather than non-discriminating and freely diffusable pores.

We previously demonstrated that altering extracellular sodium (Nao) and calcium (Cao) can modulate a form of electrical communication between cardiomyocytes termed “ephaptic coupling” (EpC), especially during loss of gap junction coupling. We hypothesized that altering Nao and Cao modulates conduction velocity (CV) and arrhythmic burden during ischemia. Electrophysiology was quantified by optically mapping Langendorff-perfused guinea pig ventricles with modified Nao (147 or 155 mM) and Cao (1.25 or 2.0 mM) during 30 min of simulated metabolic ischemia (pH 6.5, anoxia, aglycemia). Gap junction-adjacent perinexal width ( WP), a candidate cardiac ephapse, and connexin (Cx)43 protein expression and Cx43 phosphorylation at S368 were quantified by transmission electron microscopy and Western immunoblot analysis, respectively. Metabolic ischemia slowed CV in hearts perfused with 147 mM Nao and 2.0 mM Cao; however, theoretically increasing EpC with 155 mM Nao was arrhythmogenic, and CV could not be measured. Reducing Cao to 1.25 mM expanded WP, as expected during ischemia, consistent with reduced EpC, but attenuated CV slowing while delaying arrhythmia onset. These results were further supported by osmotically reducing WP with albumin, which exacerbated CV slowing and increased early arrhythmias during ischemia, whereas mannitol expanded WP, permitted conduction, and delayed the onset of arrhythmias. Cx43 expression patterns during the various interventions insufficiently correlated with observed CV changes and arrhythmic burden. In conclusion, decreasing perfusate calcium during metabolic ischemia enhances perinexal expansion, attenuates conduction slowing, and delays arrhythmias. Thus, perinexal expansion may be cardioprotective during metabolic ischemia. NEW & NOTEWORTHY This study demonstrates, for the first time, that modulating perfusate ion composition can alter cardiac electrophysiology during simulated metabolic ischemia.

Connexins are integral membrane building blocks that form gap junctions, enabling direct cytoplasmic exchange of ions and low-molecular-mass metabolites between adjacent cells. In the heart, gap junctions mediate the propagation of cardiac action potentials and the maintenance of a regular beating rhythm. A number of connexin interacting proteins have been described and are known gap junction regulators either through direct effects (e.g., kinases) or the formation of larger multifunctional complexes (e.g., cytoskeleton scaffold proteins). Most connexin partners can be categorized as either proteins promoting coupling by stimulating forward trafficking and channel opening or inhibiting coupling by inducing channel closure, internalization, and degradation. While some interactions have only been implied through co-localization using immunohistochemistry, others have been confirmed by biophysical methods that allow detection of a direct interaction. Our understanding of these interactions is, by far, most well developed for connexin 43 (Cx43) and the scope of this review is to summarize our current knowledge of their functional and regulatory roles. The significance of these interactions is further exemplified by demonstrating their importance at the intercalated disc, a major hub for Cx43 regulation and Cx43 mediated effects.

Gap junctions are essential to the function of multicellular animals, which require a high degree of coordination between cells. In vertebrates, gap junctions comprise connexins and currently 21 connexins are known in humans. The functions of gap junctions are highly diverse and include exchange of metabolites and electrical signals between cells, as well as functions, which are apparently unrelated to intercellular communication. Given the diversity of gap junction physiology, regulation of gap junction activity is complex. The structure of the various connexins is known to some extent; and structural rearrangements and intramolecular interactions are important for regulation of channel function. Intercellular coupling is further regulated by the number and activity of channels present in gap junctional plaques. The number of connexins in cell-cell channels is regulated by controlling transcription, translation, trafficking, and degradation; and all of these processes are under strict control. Once in the membrane, channel activity is determined by the conductive properties of the connexin involved, which can be regulated by voltage and chemical gating, as well as a large number of posttranslational modifications. The aim of the present article is to review our current knowledge on the structure, regulation, function, and pharmacology of gap junctions. This will be supported by examples of how different connexins and their regulation act in concert to achieve appropriate physiological control, and how disturbances of connexin function can lead to disease. © 2012 American Physiological Society. Compr Physiol 2:1981-2035, 2012.

Gαq-stimulation reduces intercellular coupling within 10 min via a decrease in the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2), but the mechanism is unknown. Here we show that uncoupling in rat cardiomyocytes after stimulation of α-adrenergic Gαq-coupled receptors with norepinephrine is prevented by proteasomal and lysosomal inhibitors, suggesting that internalization and possibly degradation of connexin43 (Cx43) is involved. Uncoupling was accompanied by increased Triton X-100 solubility of Cx43, which is considered a measure of the non-junctional pool of Cx43. However, inhibition of the proteasome and lysosome further increased solubility while preserving coupling, suggesting that communicating gap junctions can be part of the soluble fraction. Ubiquitination of Cx43 was also increased, and Cx43 co-immunoprecipitated with the ubiquitin ligase Nedd4.Norepinephrine increases ubiquitination of Cx43 in cardiomyocytes, possibly via Nedd4. We suggest that Cx43 is subsequently internalized, which is preceded by acquired solubility in Triton X-100, which does not lead to uncoupling per se.

Much of our current knowledge about the physiological and pathophysiological role of gap junctions is based on experiments where coupling has been reduced by either chemical agents or genetic modification. This has brought evidence that gap junctions are important in many physiological processes. In a number of cases, gap junctions have been implicated in the initiation and progress of disease, and experimental uncoupling has been used to investigate the exact role of coupling. The inverse approach, i.e., to increase coupling, has become possible in recent years and represents a new way of testing the role of gap junctions. The aim of this review is to summarize the current knowledge obtained with agents that selectively increase gap junctional intercellular coupling. Two approaches will be reviewed: increasing coupling by the use of antiarrhythmic peptide and its synthetic analogs and by interfering with the gating of gap junctional channels.

This synopsis covers the main results and conclusions from the platform presentations during the International Gap Junction Conference. More detailed information is provided in the mini reviews on controversial scientific issues, short reports of research results and conference abstracts published in this issue of Cell Communication and Adhesion.

Connexin43 (Cx43) generates intercellular gap junction channels involved in, among others, cardiac and brain function. Gap junctions are formed by the docking of two hemichannels from neighbouring cells. Undocked Cx43 hemichannels can upon different stimuli open towards the extracellular matrix and allow transport of molecules such as fluorescent dyes and ATP. A range of phosphorylated amino acids have been detected in the C-terminus of Cx43 and their physiological role has been intensively studied both in the gap junctional form of Cx43 and in its hemichannel configuration. We present the current knowledge of protein kinase C (PKC)-dependent regulation of Cx43 and discuss the divergent results.

Astrocytes and neurons express several large pore (hemi)channels that may open in response to various stimuli, allowing fluorescent dyes, ions, and cytoplasmic molecules such as ATP and glutamate to permeate. Several of these large pore (hemi)channels have similar characteristics with regard to activation, permeability, and inhibitor sensitivity. Consequently, their behaviors and roles in astrocytic and neuronal (patho)physiology remain undefined. We took advantage of the Xenopus laevis expression system to determine the individual characteristics of several large pore channels in isolation. Expression of connexins Cx26, Cx30, Cx36, or Cx43, the pannexins Px1 or Px2, or the purinergic receptor P2X7 yielded functional (hemi)channels with isoform-specific characteristics. Connexin hemichannels had distinct sensitivity to alterations of extracellular Ca(2+) and their permeability to dyes and small atomic ions (conductance) were not proportional. Px1 and Px2 exhibited conductance at positive membrane potentials, but only Px1 displayed detectable fluorescent dye uptake. P2X7, in the absence of Px1, was permeable to fluorescent dyes in an agonist-dependent manner. The large pore channels displayed overlapping sensitivity to the inhibitors Brilliant Blue, gadolinium, and carbenoxolone. These results demonstrated isoform-specific characteristics among the large pore membrane channels; an open (hemi)channel is not a nonselective channel. With these isoform-specific properties in mind, we characterized the divalent cation-sensitive permeation pathway in primary cultured astrocytes. We observed no activation of membrane conductance or Cx43-mediated dye uptake in astrocytes nor in Cx43-expressing C6 cells. Our data underscore that although Cx43-mediated transport is observed in overexpressing cell systems, such transport may not be detectable in native cells under comparable experimental conditions.

Background Diabetes increases the risk of cardiovascular complications including arrhythmias, but the underlying mechanisms remain to be established. Decreased conduction velocity (CV), which is an independent risk factor for re-entry arrhythmias, is present in models with streptozotocin (STZ) induced type 1 diabetes. Whether CV is also disturbed in models of type 2 diabetes is currently unknown. Methods We used Zucker Diabetic Fatty (ZDF) rats, as a model of type 2 diabetes, and their lean controls Zucker Diabetic Lean (ZDL) rats to investigate CV and its response to the anti-arrhythmic peptide analogue AAP10. Gap junction remodeling was examined by immunofluorescence and western blotting. Cardiac histomorphometry was examined by Masson`s Trichrome staining and intracellular lipid accumulation was analyzed by Bodipy staining. Results CV was significantly slower in ZDF rats (56±1.9 cm/s) compared to non-diabetic controls (ZDL, 66±1.6 cm/s), but AAP10 did not affect CV in either group. The total amount of Connexin43 (C×43) was identical between ZDF and ZDL rats, but the amount of lateralized C×43 was significantly increased in ZDF rats (42±12 %) compared to ZDL rats (30±8%), p Conclusion CV is reduced in type 2 diabetic ZDF rats. The CV disturbance may be partly explained by increased lateralization of C×43, but other factors are likely also involved. Our data indicates that lipotoxicity potentially may play a role in development of conduction disturbances and arrhythmias in type 2 diabetes.

t f f t c c i o s g i p c w t l a It has long been recognized that some states of cardiac disease are associated with a dislocation of gap junctions from the intercalated disc (ID) to the lateral sides, known as gap junction lateralization or disarray (for recent review, see Fontes et al). Lateralization was first directly linked to arrhythmia in the border zone of healing infarcts, where Peters et al showed that areas prone to inducible arrhythmia also exhibited gap junction lateralization. It is, however, still uncertain what causes the dislocation of gap junctions, how they get there, and whether lateralization is arrhythmogenic per se. In this issue of HeartRhythm, Chkourko et al show that pulmonary hypertension is associated with the lateralization of not only the connexins making up the gap junction channels but also a number of structural proteins that are normally located at the ID and proteins involved in the transport of connexin 43 (Cx43, the most abundant cardiac gap junctional protein) to the plasma membrane. Cx43 can be transported along microtubules, and recently it was shown that the microtubule-associated protein EB1 is essential for correct trafficking of Cx43 to the ID. The study also showed that EB1 disappears from the ID during ischemia, abrogating the trafficking of Cx43 to the disc. The study by Chkourko et al shows that EB1 is most ikely responsible for redirecting Cx43 to the lateral memranes during remodeling in pulmonary hypertension. The inking of EB1 to the membrane is reported to involve everal components ending with N-cadherin as the memrane anchor. Does this imply that N-cadherin is the first omponent to arrive at the lateral membrane? Maybe in the ase of pulmonary hypertension, but in some models of gap unction lateralization, N-cadherin stays at the ID (eg, see olussi et al). Still, N-cadherin remains an interesting andidate, raising the question why it translocates. Recent vidence implies that cadherins are involved in mechanoensing at cell-to-cell interfaces and maybe cadherin-de

Objective A recently developed dye-based assay to study gap junction permeability is analysed. The assay is based on electroporation of dye into a large number of connexin 43 expressing cells, grown to confluency on electrically conductive slides. The subsequent intercellular spread of dye to non-electroporated parts of the monolayer enables estimation of the intercellular coupling. So far, the extent of dye spread has been analyzed in qualitative terms only and not in a manner based directly on the physics of the underlying diffusion process. Methods We apply a continuum approximation assuming that the observed dye spread can be described by Fick’s law of diffusion. Deduced from Fick’s law, new measures are presented which directly relate the observed spread of dye to the diffusion coefficient. Results The theoretical framework enables the estimation of an effective diffusion coefficient from Fick’s law independently of the specific indicator substance used in the assay. For Lucifer Yellow, diffusion stops within few minutes after the electroporation. Therefore only an order-of-magnitude estimate of the diffusion coefficient can be given for this dye. Conclusion In terms of the underlying diffusion coefficient, the hitherto used measures give a relatively poor degree of quantification. In contrast, the present methods may yield direct information on the effective intercellular diffusion coefficient and hence provide additional and more precise information as to the permeability modulating effect of various substances.

Intercellular communication via gap junction channels can be quantified by several methods based on diffusion of fluorescent dyes or metabolites. Given the variation in intercellular coupling of cells, even under untreated control conditions, it is of essence to quantify the coupling between numerous cells to obtain reliable estimates of metabolic coupling. Quantification is often based on manual counting of fluorescent cells, which is time consuming and may include some degree of subjectivity. In this report, we introduce a technique based on digital image analysis, and the software for the analysis is presented together with a detailed protocol in the online supplemental material ( http://bmi.ku.dk/matlab_program/ ). Fluorescent dye was introduced in connexin 43-expressing C6 glioma cells by in situ electroporation, and fluorescence intensity was measured in the electroporated cells and in cells receiving dye by intercellular diffusion. The analysis performed is semiautomatic, and comparison with traditional cell counting shows that this method reliably determines the effect of uncoupling by several interventions. This new method of analysis yields a rapid and objective quantification process with a high degree of reproducibility.

Gap junctions provide a low-resistance pathway for cardiac electric propagation. The role of GJ regulation in arrhythmia is unclear, partly because of limited availability of pharmacological tools. Recently, we showed that a peptide called “RXP-E” binds to the carboxyl terminal of connexin43 and prevents chemically induced uncoupling in connexin43-expressing N2a cells. Here, pull-down experiments show RXP-E binding to adult cardiac connexin43. Patch-clamp studies revealed that RXP-E prevented heptanol-induced and acidification-induced uncoupling in pairs of neonatal rat ventricular myocytes. Separately, RXP-E was concatenated to a cytoplasmic transduction peptide (CTP) for cytoplasmic translocation (CTP–RXP-E). The effect of RXP-E on action potential propagation was assessed by high-resolution optical mapping in monolayers of neonatal rat ventricular myocytes, containing ≈20% of randomly distributed myofibroblasts. In contrast to control experiments, when heptanol (2 mmol/L) was added to the superfusate of monolayers loaded with CTP–RXP-E, action potential propagation was maintained, albeit at a slower velocity. Similarly, intracellular acidification (pH i 6.2) caused a loss of action potential propagation in control monolayers; however, propagation was maintained in CTP–RXP-E–treated cells, although at a slower rate. Patch-clamp experiments revealed that RXP-E did not prevent heptanol-induced block of sodium currents, nor did it alter voltage dependence or amplitude of Kir2.1/Kir2.3 currents. RXP-E is the first synthetic molecule known to: (1) bind cardiac connexin43; (2) prevent heptanol and acidification-induced uncoupling of cardiac gap junctions; and (3) preserve action potential propagation among cardiac myocytes. RXP-E can be used to characterize the role of gap junctions in the function of multicellular systems, including the heart.

Changes in the lipid composition of cardiac myocytes have been reported during cardiac hypertrophy, cardiomyopathy, and infarction. Because a recent study indicates a relation between low phosphatidylinositol-bisphosphate (PIP(2)) levels and reduced intercellular coupling, we tested the hypothesis that agonist-induced changes in PIP(2) can result in a reduction of the functional coupling of cardiomyocytes and, consequently, in changes in conduction velocity. Intercellular coupling was measured by Lucifer Yellow dye transfer in cultured neonatal rat cardiomyocytes. Conduction velocity was measured in cardiomyocytes grown on microelectrode arrays. Intercellular coupling was reduced by angiotensin II (43.7 +/- 9.3%, N = 11) and noradrenaline (58.0 +/- 10.7%, N = 11). To test if reduced intercellular coupling after agonist stimulation was caused by PIP(2)-depletion, myocytes were stimulated by angiotensin II (57.3 +/- 5.7%, N = 14) and then allowed to recover in medium with or without wortmannin (an inhibitor of PIP(2) synthesis). Intercellular coupling fully recovered in control medium (102.1 +/- 8.9%, N = 10), whereas no recovery occurred in the presence of wortmannin (69.3 +/- 7.8%, N = 12). Inhibition of PKC, calmodulin, or arachidonic acid production did not affect the response to either angiotensin II or noradrenaline. Furthermore, decreasing or increasing PIP(2) also decreased and increased intercellular coupling, respectively. This supports the role of PIP(2) in the regulation of intercellular coupling. In beating myocytes, conduction velocity was reduced by angiotensin II stimulation, and recovery after wash out was prevented by inhibition of PIP(2) production. Reductions in PIP(2) inhibit intercellular coupling in cardiomyocytes, and stimulation by physiologically relevant agonists reduces intercellular coupling by this mechanism. The reduction in intercellular coupling lowered conduction velocity.

Treatment with non-selective drugs (eg, long-chain alcohols, halothane) that reduce gap junction intercellular communication (GJIC) is associated with reduced infarct size after myocardial infarction (MI). Therefore, it has been suggested that gap junction intercellular communication stimulating compounds may increase infarct size. The antiarrhythmic peptide analogue rotigaptide (ZP123) increases cardiac gap junction intercellular communication and the purpose of the present study was to examine the effects of rotigaptide treatment on infarct size. Myocardial infarction was induced in male rats by ligation of the left anterior descending artery (LAD). Rats (n = 156) were treated with rotigaptide at three dose levels or vehicle from the onset of ischemia and for 3 weeks following LAD occlusion. Infarct size was determined using histomorphometry after 3 weeks treatment. Rotigaptide treatment producing steady state plasma levels of 0.8 +/- 0.1, 5.5 +/- 0.5, and 86 +/- 8 nmol/L had no effect on mortality, but reduced infarct size to 90 +/- 10% (P = 0.41), 67 +/- 7% (P = 0.005), and 82 +/- 7% (P = 0.13), respectively relative to vehicle-treated myocardial infarction rats (100 +/- 12%). In contrast to what was predicted, our data demonstrates that rotigaptide treatment was associated with a significant infarct size reduction. We conclude that whereas treatment with non-selective inhibitors of gap junction intercellular communication cause a reduction in infarct size, this information cannot be extrapolated to the effects of compounds that selectively increase gap junction intercellular communication.

Introduction: The aim of this study was to determine if the stable antiarrhythmic peptide (AAP) analogue ZP123 increases gap junctional intercellular conductance and prevents reentrant ventricular tachycardia (VT) during coronary artery occlusion. Methods and Results: Voltage clamp experiments demonstrated that 10 nM ZP123 improved gap junctional intercellular conductance by69%± 20%in pairs of guinea pig ventricular myocytes. VT was induced by programmed stimulation in α-chloralose anaesthetized open chest dogs 1 to 4 hours after coronary artery occlusion. Three-dimensional activation mapping was done using six bipolar electrograms on each of 23 multipolar needles in the risk zone. When VT was reproducibly induced, dogs were randomly assigned to receive either saline or ZP123 cumulatively at three dose levels (intravenous bolus followed by 30-min infusion per dose). Attempts to induce VT were repeated in each infusion period. Mass spectrometry was used to measure ZP123 plasma concentrations. Twenty-six dogs with reentrant VT were included. ZP123 significantly prevented reentrant VT at all plasma concentrations vs saline:1.0 ± 0.2 nM: 6/12 vs 0/12; 7.7 ± 0.6 nM: 7/13 vs 1/12; and69.2 ± 5.4 nM: 9/13 vs 1/13. The preventive effect of ZP123 on reentrant VT was closely correlated to reversal of functional, unidirectional conduction block. ZP123 did not affect effective refractory period, surface ECG parameters, mean arterial pressure, or infarct size. Conclusion: The stable AAP analogue ZP123 increased gap junctional intercellular conductance and specifically prevented the induction of reentrant VT during ischemia in a broad dose range without proarrhythmic or hemodynamic side effects. ZP123 is a promising candidate for use in preventing ischemia-induced VT.(J Cardiovasc Electrophysiol, Vol. 14, pp. 510-520, May 2003)

BACKGROUND: Type 2 diabetes is associated with abnormal electrical conduction and sudden cardiac death, but the pathogenic mechanism remains unknown. This study describes electrophysiological alterations in a diet-induced pre-diabetic rat model and examines the underlying mechanism.METHODS: Sprague-Dawley rats were fed either high-fat diet and fructose water or normal chow and water for 6 weeks. The electrophysiological properties of the whole heart was analyzed by in vivo surface ECG recordings, as wells as ex vivo in Langendorff perfused hearts during baseline, ischemia and re-perfussion. Conduction velocity was examined in isolated tissue strips. Ion channel and gap junction conductances were analyzed by patch-clamp studies in isolated cardiomyocytes. Fibrosis was examined by Masson's Trichrome staining and thin-layer chromatography was used to analyze cardiac lipid content. Connexin43 (Cx43) expression and distribution was examined by western blotting and immunofluorescence respectively.RESULTS: Following 6 weeks of feeding, fructose-fat fed rats (FFFRs) showed QRS prolongation compared to controls (16.1 ± 0.51 (n = 6) vs. 14.7 ± 0.32 ms (n = 4), p CONCLUSION: Six weeks on a high fructose-fat diet cause electrophysiological changes, which leads to QRS prolongation, decreased conduction velocity and increased arrhythmogenesis during reperfusion. These alterations are not explained by altered gap junctional coupling, Na(+), or K(+) currents, differences in cell size or fibrosis.

Connexin 43 (Cx43) hemichannels may form open channels in the plasma membrane when exposed to specific stimuli, e.g. reduced extracellular concentration of divalent cations, and allow passage of fluorescent molecules and presumably a range of smaller physiologically relevant molecules. However, the permeability profile of Cx43 hemichannels remains unresolved. Exposure of Cx43-expressing Xenopus laevis oocytes to divalent cation free solution induced a gadolinium-sensitive uptake of the fluorescent dye ethidium. In spite thereof, a range of biological molecules smaller than ethidium, such as glutamate, lactate, and glucose, did not permeate the pore whereas ATP did. In contrast, permeability of glutamate, glucose and ATP was observed in oocytes expressing Cx30. Exposure to divalent cation free solutions induced a robust membrane conductance in Cx30-expressing oocytes but none in Cx43-expressing oocytes. C-terminally truncated Cx43 (M257) displayed increased dye uptake and, unlike wild type Cx43 channels, conducted current. Neither Cx30 nor Cx43 acted as water channels in their hemichannel configuration. Our results demonstrate that connexin hemichannels have isoform-specific permeability profiles and that dye uptake cannot be equaled to permeability of smaller physiologically relevant molecules in given settings.