Differential expression of connexins during histogenesis of the chick retina

Gap junction (GJ) channels couple adjacent cells, allowing transfer of second messengers, ions, and molecules up to 1 kDa. These channels are composed by a multigene family of integral membrane proteins called connexins (Cx). In the retina, besides being essential circuit element in the visual processing, GJ channels also play important roles during its development. Herein, we analyzed Cx43, Cx45, Cx50, and Cx56 expression during chick retinal histogenesis. Cx exhibited distinct expression profiles during retinal development, except for Cx56, whose expression was not detected. Cx43 immunolabeling was observed at early development, in the transition of ventricular zone and pigmented epithelium. Later, Cx43 was seen in the outer plexiform and ganglion cell layers, and afterwards also in the inner plexiform layer. We observed remarkable changes in the phosphorylation status of this protein, which indicated modifications in functional properties of this Cx during retinal histogenesis. By contrast, Cx45 showed stable gene expression levels throughout development and ubiquitous immunoreactivity in progenitor cells. From later embryonic development, Cx45 was mainly observed in the inner retina, and it was expressed by glial cells and neurons. In turn, Cx50 was virtually absent in the chick retina at initial embryonic phases. Combination of PCR, immunohistochemistry and Western blot indicated that this Cx was present in differentiated cells, arising in parallel with the formation of the visual circuitry. Characterization of Cx expression in the developing chick retina indicated particular roles for these proteins and revealed similarities and differences when compared to other species.     

Protein kinase A-mediated phosphorylation of connexin36 in mouse retina results in decreased gap junctional communication between AII amacrine cells

Gap junctions in AII amacrine cells of mammalian retina participate in the coordination of the rod and cone signaling pathway involved in visual adaptation. Upon stimulation by light, released dopamine binds to D(1) receptors on AII amacrine cells leading to increased intracellular cAMP (cyclic adenosine monophosphate) levels. AII amacrine cells express the gap junctional protein connexin36 (Cx36). Phosphorylation of Cx36 has been hypothesized to regulate gap junctional activity of AII amacrine cells. However, until now in vivo phosphorylation of Cx36 has not been reported. Indeed, it had been concluded that Cx36 in bovine retina is not phosphorylated, but in vitro phosphorylation for Cx35, the bass ortholog of Cx36, had been shown. To clarify this experimental discrepancy, we examined protein kinase A (PKA)-induced phosphorylation of Cx36 in mouse retina as a possible mechanism to modulate the extent of gap junctional coupling. The cytoplasmic domains of Cx36 and the total Cx36 protein were phosphorylated in vitro by PKA. Mass spectroscopy revealed that all four possible PKA consensus motifs were phosphorylated; however, domains point mutated at the sites in question showed a prevalent usage of Ser-110 and Ser-293. Additionally, we demonstrated that Cx36 was phosphorylated in cultured mouse retina. Furthermore, activation of PKA increased the level of phosphorylation of Cx36. cAMP-stimulated, PKA-mediated phosphorylation of Cx36 protein was accompanied by a decrease of tracer coupling between AII amacrine cells. Our results link increased phosphorylation of Cx36 to down-regulation of permeability through gap junction channels mediating light adaptation in the retina.     

Reciprocal Regulation of Epileptiform Neuronal Oscillations and Electrical Synapses in the Rat Hippocampus

Gap junction (GJ) channels have been recognized as an important mechanism for synchronizing neuronal networks. Herein, we investigated the participation of GJ channels in the pilocarpine-induced status epilepticus (SE) by analyzing electrophysiological activity following the blockade of connexins (Cx)-mediated communication. In addition, we examined the regulation of gene expression, protein levels, phosphorylation profile and distribution of neuronal Cx36, Cx45 and glial Cx43 in the rat hippocampus during the acute and latent periods. Electrophysiological recordings revealed that the GJ blockade anticipates the occurrence of low voltage oscillations and promotes a marked reduction of power in all analyzed frequencies.Cx36 gene expression and protein levels remained stable in acute and latent periods, whereas upregulation of Cx45 gene expression and protein redistribution were detected in the latent period. We also observed upregulation of Cx43 mRNA levels followed by changes in the phosphorylation profile and protein accumulation. Taken together, our results indisputably revealed that GJ communication participates in the epileptiform activity induced by pilocarpine. Moreover, considering that specific Cxs undergo alterations through acute and latent periods, this study indicates that the control of GJ communication may represent a focus in reliable anti-epileptogenic strategies.     

The intercellular synchronization of ca(2+) oscillations evaluates cx36-dependent coupling

Connexin36 (Cx36) plays an important role in insulin secretion by controlling the intercellular synchronization of Ca(2+) transients induced during stimulation. The lack of drugs acting on Cx36 channels is a major limitation in further unraveling the molecular mechanism underlying this effect. To screen for such drugs, we have developed an assay allowing for a semi-automatic, fluorimetric quantification of Ca(2+) transients in large populations of MIN6 cells. Here, we show that (1) compared to control cells, MIN6 cells with reduced Cx36 expression or function showed decreased synchrony of glucose-induced Ca(2+) oscillations; (2) glibenclamide, a sulphonylurea which promotes Cx36 junctions and coupling, increased the number of synchronous MIN6 cells, whereas quinine, an antimalarial drug which inhibits Cx36-dependent coupling, decreased this proportion; (3) several drugs were identified that altered the intercellular Ca(2+) synchronization, cell coupling and distribution of Cx36; (4) some of them also affected insulin content. The data indicate that the intercellular synchronization of Ca(2+) oscillations provides a reliable and non-invasive measurement of Cx36-dependent coupling, which is useful to identify novel drugs affecting the function of β-cells, neurons, and neuron-related cells that express Cx36.     

Impaired β-cell-β-cell coupling mediated by Cx36 gap junctions in prediabetic mice

Gap junctional intercellular communication between β-cells is crucial for proper insulin biosynthesis and secretion. The aim of this work was to investigate the expression of connexin (Cx)36 at the protein level as well as the function and structure of gap junctions (GJ) made by this protein in the endocrine pancreas of prediabetic mice. C57BL/6 mice were fed a high-fat (HF) or regular chow diet for 60 days. HF-fed mice became obese and prediabetic, as shown by peripheral insulin resistance, moderate hyperglycemia, hyperinsulinemia, and compensatory increase in endocrine pancreas mass. Compared with control mice, prediabetic animals showed a significant decrease in insulin-secretory response to glucose and displayed a significant reduction in islet Cx36 protein. Ultrastructural analysis further showed that prediabetic mice had GJ plaques about one-half the size of those of the control group. Microinjection of isolated pancreatic islets with ethidium bromide revealed that prediabetic mice featured a β-cell-β-cell coupling 30% lower than that of control animals. We conclude that β-cell-β-cell coupling mediated by Cx36 made-channels is impaired in prediabetic mice, suggesting a role of Cx36-dependent cell-to-cell communication in the pathogenesis of the early β-cell dysfunctions that lead to type 2-diabetes.     

Cx36 expression in the AII‐mediated rod pathway is activity dependent in the developing rabbit retina

Gap junctions are composed of connexin 36 (Cx36) and play a critical role in the rod photoreceptor signaling pathways of the vertebrate retina. Despite the fact that their connection and modulation in various rod pathways have been extensively studied in adult animals, little is known about the contribution and regulation of gap junctions to the development of the AII amacrine cell (AC)‐mediated rod pathway. Using immunohistochemistry and microinjection, this study demonstrates a steady increase in relative Cx36 protein expression in both plexiform layers of the rabbit retina at around the time of eye opening. However, immediately after eye opening, most Cx36 immunoreactive AII ACs show no gap junction coupling pattern to neighboring cells and it is not until the third postnatal week that AII cells begin to exhibit an adult‐like tracer‐coupling pattern. Moreover, studies using dark‐rearing and AMPA receptor blockade during postnatal development both revealed that relative levels of Cx36 immunoreactivity in AII ACs were increased when neural activity was inhibited . Our findings suggest that Cx36 expression in the AII‐mediated rod pathway is activity dependent in the developing rabbit retina . © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 473–486, 2016     

Properties of Connexin26 Hemichannels Expressed in Xenopus Oocytes

1. Hemichannels formed by connexin26 (Cx26) on the horizontal cell dendrites that invaginate cone terminals in the vertebrate retina have been implicated in the feedback mechanism by which horizontal cells regulate transmitter release from cone photoreceptors. However, their membrane properties had not been studied previously, and it was unclear whether they could subserve their purported function at the membrane potentials over which horizontal cells operate. 2. We used the two-electrode voltage clamp technique to record the membrane currents and pharmacological properties of Cx26 hemichannels formed in the Xenopus oocyte expression system. 3. Oocytes expressing Cx26 exhibited large membrane conductances over a broad range of hyperpolarizing and depolarizing membrane potentials, and displayed little evidence of voltage-dependent gating, indicating that the hemichannels are constitutively open. The Cx26-mediated nonjunctional currents were relatively insensitive to quinine, a cinchona alkaloid that opens hemichannels formed by several other connexins. However, the hemichannel currents were blocked by carbenoxolone, a rise in extracellular calcium, or lowering intracellular pH. The currents could also be suppressed by reducing extracellular pH, and by the chloride channel blocker NPPB through its direct interaction with Cx26 hemichannels. 4. These findings provide a basis with which to evaluate the in situ pharmacological studies that attempt to assess the putative role of Cx26 hemichannels in the feedback pathway in the distal retina.     

Protective role of gap junctions in preconditioning against myocardial infarction

The aim of the present study was to examine the hypothesis that acceleration of gap junction (GJ) closure during ischemia contributes to anti-infarct tolerance afforded by preconditioning (PC). First, the effects of PC on GJ communication during ischemia were assessed. Isolated buffer-perfused rabbit hearts were subjected to 5-min global ischemia with or without PC with two cycles of 5-min ischemia/5-min reperfusion or a GJ blocker (2 mM heptanol), and then the tissue excised from the ischemic region was incubated in anoxic buffer containing lucifer yellow (LY; 2.5 mg/ml), a tracer of GJ permeability, for 20 min at 37 degrees C. PC and heptanol significantly reduced the area to which LY was transported in the ischemic myocardium by 39% and by 54%, respectively. In the second series of experiments, three GJ blockers (heptanol, 18beta-glycyrrhetinic acid, and 2,3-butanedione monoxime) infused after the onset of ischemia reduced infarct size after 30-min ischemia/2-h reperfusion to an extent equivalent to that in the case of PC. In the third series of experiments, Western blotting for connexin43 (Cx43) showed that PC shortened the time to the onset of ischemia-induced Cx43 dephosphorylation but reduced the extent of Cx43 dephosphorylation during a 30-min period of ischemia. Calphostin C, a protein kinase C (PKC) inhibitor, abolished preservation of phosphorylated Cx43 but not the early onset of Cx43 dephosphorylation after ischemia in the preconditioned myocardium. These results suggest that PC-induced reduction of GJ permeability during ischemia, presumably by PKC-mediated Cx43 phosphorylation, contributes to infarct size limitation.     

Gap Junctions Contribute to the Regulation of Walking-Like Activity in the Adult Mudpuppy (Necturus Maculatus)

Although gap junctions are widely expressed in the developing central nervous system, the role of electrical coupling of neurons and glial cells via gap junctions in the spinal cord in adults is largely unknown. We investigated whether gap junctions are expressed in the mature spinal cord of the mudpuppy and tested the effects of applying gap junction blocker on the walking-like activity induced by NMDA or glutamate in an in vitro mudpuppy preparation. We found that glial and neural cells in the mudpuppy spinal cord expressed different types of connexins that include connexin 32 (Cx32), connexin 36 (Cx36), connexin 37 (Cx37), and connexin 43 (Cx43). Application of a battery of gap junction blockers from three different structural classes (carbenexolone, flufenamic acid, and long chain alcohols) substantially and consistently altered the locomotor-like activity in a dose-dependent manner. In contrast, these blockers did not significantly change the amplitude of the dorsal root reflex, indicating that gap junction blockers did not inhibit neuronal excitability nonselectively in the spinal cord. Taken together, these results suggest that gap junctions play a significant modulatory role in the spinal neural networks responsible for the generation of walking-like activity in the adult mudpuppy.     

Gating properties of heterotypic gap junction channels formed of connexins 40, 43, and 45

Connexins (Cxs) 40, 43, and 45 are expressed in many different tissues, but most abundantly in the heart, blood vessels, and the nervous system. We examined formation and gating properties of heterotypic gap junction (GJ) channels assembled between cells expressing wild-type Cx40, Cx43, or Cx45 and their fusion forms tagged with color variants of green fluorescent protein. We show that these Cxs, with exception of Cxs 40 and 43, are compatible to form functional heterotypic GJ channels. Cx40 and Cx43 hemichannels are unable or effectively impaired in their ability to dock and/or assemble into junctional plaques. When cells expressing Cx45 contacted those expressing Cx40 or Cx43 they readily formed junctional plaques with cell-cell coupling characterized by asymmetric junctional conductance dependence on transjunctional voltage, V(j). Cx40/Cx45 heterotypic GJ channels preferentially exhibit V(j)-dependent gating transitions between open and residual states with a conductance of approximately 42 pS; transitions between fully open and closed states with conductance of approximately 52 pS in magnitude occur at substantially lower ( approximately 10-fold) frequency. Cx40/Cx45 junctions demonstrate electrical signal transfer asymmetry that can be modulated between unidirectional and bidirectional by small changes in the difference between holding potentials of the coupled cells. Furthermore, both fast and slow gating mechanisms of Cx40 exhibit a negative gating polarity.     

Functional role of gap junctions in cytokine-induced leukocyte adhesion to endothelium in vivo

To assess the hypothesis that gap junctions (GJs) participate on leukocyte-endothelium interactions in the inflammatory response, we compared leukocyte adhesion and transmigration elicited by cytokine stimulation in the presence or absence of GJ blockers in the hamster cheek pouch and also in the cremaster muscle of wild-type (WT) and endothelium-specific connexin 43 (Cx43) null mice (Cx43e(-/-)). In the cheek pouch, topical tumor necrosis factor-alpha (TNF-alpha; 150 ng/ml, 15 min) caused a sustained increment in the number of leukocytes adhered to venular endothelium (LAV) and located at perivenular regions (LPV). Superfusion with the GJ blockers 18-alpha-glycyrrhetinic acid (AGA; 75 microM) or 18-beta-glycyrrhetinic acid (50 microM) abolished the TNF-alpha-induced increase in LAV and LPV; carbenoxolone (75 microM) or oleamide (100 microM) reduced LAV by 50 and 75%, respectively, and LPV to a lesser extent. None of these GJ blockers modified venular diameter, blood flow, or leukocyte rolling. In contrast, glycyrrhizin (75 microM), a non-GJ blocker analog of AGA, was devoid of effect. Interestingly, when AGA was removed 90 min after TNF-alpha stimulation, LAV started to rise at a similar rate as in control. Conversely, application of AGA 90 min after TNF-alpha reduced the number of previously adhered cells. In WT mice, intrascrotal injection of TNF-alpha (0.5 microg/0.3 ml) increased LAV (fourfold) and LPV (threefold) compared with saline-injected controls. In contrast to the observations in WT animals, TNF-alpha stimulation did not increase LAV or LPV in Cx43e(-/-) mice. These results demonstrate an important role for GJ communication in leukocyte adhesion and transmigration during acute inflammation in vivo and further suggest that endothelial Cx43 is key in these processes.     

Multiple Neuronal Connexins in the Mammalian Retina

Gap junctions are abundant in the mammalian retina and many neuronal types form neural networks. Several different neuronal connexins have now been identified in the mammalian retina. Cx36 supports coupling in the AII amacrine cell network and is essential for processing rod signals. Cx36 is probably also responsible for photoreceptor coupling. Horizontal cells appear to be extensively coupled by either Cx50 or Cx57. These results indicate that multiple neuronal connexins are expressed in the mammalian retina and that different cell types express different connexins.     

Multiple neuronal connexins in the mammalian retina

Gap junctions are abundant in the mammalian retina and many neuronal types form neural networks. Several different neuronal connexins have now been identified in the mammalian retina. Cx36 supports coupling in the AII amacrine cell network and is essential for processing rod signals. Cx36 is probably also responsible for photoreceptor coupling. Horizontal cells appear to be extensively coupled by either Cx50 or Cx57. These results indicate that multiple neuronal connexins are expressed in the mammalian retina and that different cell types express different connexins.     

Inhibition of Cx43 mediates protective effects on hypoxic/reoxygenated human neuroblastoma cells

Olfactory ensheathing cells (OECs), a special population of glial cells, are able to synthesise several trophic factors exerting a neuroprotective action and promoting growth and functional recovery in both in vitro and in vivo models. In the present work, we investigated the neuroprotective effects of OEC‐conditioned medium (OEC‐CM) on two different human neuron‐like cell lines, SH‐SY5Y and SK‐N‐SH (neuroblastoma cell lines), under normoxic and hypoxic conditions. In addition, we also focused our attention on the role of connexins (Cxs) in the neuroprotective processes. Our results confirmed OEC‐CM mediated neuroprotection as shown by cell adherence, proliferation and cellular viability analyses. Reduced connexin 43 (Cx43) levels in OEC‐CM compared to unconditioned cells in hypoxic conditions prompted us to investigate the role of Cx43‐Gap junctions (GJs) and Cx43‐hemichannels (HCs) in hypoxic/reoxygenation injury using carbenoxolone (non‐selective GJ inhibitor), ioxynil octanoato (selective Cx43‐GJ inhibitor) and Gap19 (selective Cx43‐HC inhibitor). We found that Cx43‐GJ and Cx43‐HC inhibitors are able to protect SH‐SY5Y and allow to these cultures to overcome the injury. Our findings support the hypothesis that both OEC‐CM and the inhibition of Cx43‐GJs and Cx43‐HCs offer a neuroprotective effect by reducing Cx43‐mediated cell‐to‐cell and cell‐to‐extracellular environment communications.     

Carbenoxolone inhibits volume-regulated anion conductance in cultured rat cortical astroglia

Accumulating evidence indicate that the gap-junction inhibitor carbenoxolone (CBX) regulates neuronal synchronization, depresses epileptiform activity and has a neuroprotective action. These CBX effects do not depend solely on its ability to inhibit gap junction channels formed by connexins (Cx), but the underlying mechanisms remain to be elucidated. Here we addressed the questions whether CBX modulates volume-regulated anion channels (VRAC) involved in the regulatory volume decrease and regulates the associated release of excitatory amino acids in cultured rat cortical astrocytes. We found that CBX inhibits VRAC conductance with potency comparable to that able to depress the activity of the most abundant astroglial gap junction protein connexin43 (Cx43). However, the knock down of Cx43 with small interfering RNA (siRNA) oligonucleotides and the use of various pharmacological tools revealed that VRAC inhibition was not mediated by interaction of CBX with astroglial Cx proteins. Comparative experiments in HEK293 cells stably expressing another putative target of CBX, the purinergic ionotropic receptor P2X7, indicate that the presence of this receptor was not necessary for CBX-mediated depression of VRAC. Finally, we show that in COS-7 cells, which are not endowed with pannexin-1 protein, another astroglial plasma membrane interactor of CBX, VRAC current retained its sensitivity to CBX. Complementary analyses indicate that the VRAC-mediated release of excitatory amino acid aspartate was decreased by CBX. Collectively, these findings support the notion that CBX could affect astroglial ability to modulate neuronal activity by suppressing excitatory amino acid release through VRAC. They also provide a possible mechanistic clue for the neuroprotective effect of CBX in vivo.     

Direct coupling through gap junctions is not involved in urethral neurotransmission

Interstitial cells of Cajal (ICC) are believed to participate in urethral neurotransmission and it was proposed that direct coupling of ICC and smooth muscle cells (SMC) through gap junctions (GJ) is involved, although this still remains unclear. Hence, we investigated the distribution of different connexins (Cx 43, Cx40, and Cx37) in the sheep and rat urethra, as well as their possible role in neurotransmission. Conventional PCR confirmed that three Cxs are expressed in the urethra. Moreover, both Cx43 and Cx37-immunoreactivity (-ir) were present in SMC, ICC, and the urothelium, although Cx37-ir was significantly weaker and Cx40-ir was limited to the endothelium. While these results indicate that GJ intercellular communication could occur between SMC and ICC, neither the contractile (noradrenergic) nor the relaxant (nitrergic) responses of the rat and sheep urethra to electrical field stimulation were significantly modified by two different GJ inhibitors: 18α-glycyrrhetinic acid and a cocktail of Cx mimetic peptides ((Cx43)Gap 26, (Cx37, Cx43)Gap 27, and (Cx40)Gap 27). By contrast, contractions induced by high K(+) were effectively reduced by both blockers, evidence that they effectively inhibit intercellular communication. These results indicate that GJ are not implicated in urethral neurotransmission, although the question of whether ICC modulate neurotransmission through some other mechanism remains to be determined.     

The First Extracellular Domain Plays an Important Role in Unitary Channel Conductance of Cx50 Gap Junction Channels

Gap junction (GJ) channels provide direct passage for ions and small molecules to be exchanged between neighbouring cells and are crucial for many physiological processes. GJ channels can be gated by transjunctional voltage (known as V_j-gating) and display a wide range of unitary channel conductance (γ_j), yet the domains responsible for V_j-gating and γ_j are not fully clear. The first extracellular domain (E1) of several connexins has been shown to line part of their GJ channel pore and play important roles in V_j-gating properties and/or ion permeation selectivity. To test roles of the E1 of Cx50 GJ channels, we generated a chimera, Cx50Cx36E1, where the E1 domain of Cx50 was replaced with that of Cx36, a connexin showing quite distinct V_j-gating and γ_j from those of Cx50. Detailed characterizations of the chimera and three point mutants in E1 revealed that, although the E1 domain is important in determining γ_j, the E1 domain of Cx36 is able to effectively function within the context of the Cx50 channel with minor changes in V_j-gating properties, indicating that sequence differences between the E1 domains in Cx36 and Cx50 cannot account for their drastic differences in V_j-gating and γ_j. Our homology models of the chimera and the E1 mutants revealed that electrostatic properties of the pore-lining residues and their contribution to the electric field in the pore are important factors for the rate of ion permeation of Cx50 and possibly other GJ channels.