Regulation of connexin biosynthesis, assembly, gap junction formation, and removal

Gap junctions (GJs) are the only known cellular structures that allow a direct transfer of signaling molecules from cell-to-cell by forming hydrophilic channels that bridge the opposing membranes of neighboring cells. The crucial role of GJ-mediated intercellular communication (GJIC) for coordination of development, tissue function, and cell homeostasis is now well documented. In addition, recent findings have fueled the novel concepts that connexins, although redundant, have unique and specific functions, that GJIC may play a significant role in unstable, transient cell-cell contacts, and that GJ hemi-channels by themselves may function in intra-/extracellular signaling. Assembly of these channels is a complicated, highly regulated process that includes biosynthesis of the connexin subunit proteins on endoplasmic reticulum membranes, oligomerization of compatible subunits into hexameric hemi-channels (connexons), delivery of the connexons to the plasma membrane, head-on docking of compatible connexons in the extracellular space at distinct locations, arrangement of channels into dynamic, spatially and temporally organized GJ channel aggregates (so-called plaques), and coordinated removal of channels into the cytoplasm followed by their degradation. Here we review the current knowledge of the processes that lead to GJ biosynthesis and degradation, draw comparisons to other membrane proteins, highlight novel findings, point out contradictory observations, and provide some provocative suggestive solutions.     

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

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

Connexin phosphorylation as a regulatory event linked to gap junction channel assembly

Gap junctions, composed of proteins from the connexin family, allow for intercellular communication between cells and are important in development and maintenance of cell homeostasis. Phosphorylation has been implicated in the regulation of gap junctional communication at several stages of the cell cycle and the connexin “lifecycle”, such as trafficking, assembly/disassembly, degradation, as well as in the gating of “hemi” channels or intact gap junction channels. This review focuses on how phosphorylation can regulate the early stages of the connexin life cycle through assembly of functional gap junctional channels. The availability of sequences from the human genome databases has indicated that the number of connexins in the gene family is approximately 20, but we know mostly about how connexin43 (Cx43) is regulated. Recent technologies and investigations of interacting proteins have shown that activation of several kinases including protein kinase A, protein kinase C (PKC), p34^cdc2/cyclin B kinase, casein kinase 1 (CK1), mitogen-activated protein kinase (MAPK) and pp60^src kinase can lead to phosphorylation of the majority of the 21 serine and two of the tyrosine residues in the C-terminal region of Cx43. While many studies have correlated changes in kinase activity with changes in gap junctional communication, further research is needed to directly link specific phosphorylation events with changes in connexin oligomerization and gap junction assembly.     

Cytosolic stress reduces degradation of connexin43 internalized from the cell surface and enhances gap junction formation and function

The protein constituents of gap junctions, connexins, have a rapid basal rate of degradation even after transport to the cell surface. We have used cell surface biotinylation to label gap junction-unassembled plasma membrane pools of connexin43 (Cx43) and show that their degradation is inhibited by mild hyperthermia, oxidative stress, and proteasome inhibitors. Cytosolic stress does not perturb endocytosis of biotinylated Cx43, but instead it seems to interfere with its targeting and/or transport to the lysosome, possibly by increasing the level of unfolded protein in the cytosol. This allows more Cx43 molecules to recycle to the cell surface, where they are assembled into long-lived, functional gap junctions in otherwise gap junction assembly-inefficient cells. Cytosolic stress also slowed degradation of biotinylated Cx43 in gap junction assembly-efficient normal rat kidney fibroblasts, and reduced the rate at which gap junctions disappeared from cell interfaces under conditions that blocked transport of nascent connexin molecules to the plasma membrane. These data demonstrate that degradation from the cell surface can be down-regulated by physiologically relevant forms of stress. For connexins, this may serve to enhance or preserve gap junction-mediated intercellular communication even under conditions in which protein synthesis and/or intracellular transport are compromised.     

Connexin phosphorylation as a regulatory event linked to gap junction internalization and degradation

Gap junction proteins, connexins, are dynamic polytopic membrane proteins that exhibit unprecedented short half-lives of only a few hours. Consequently, it is well accepted that in addition to channel gating, gap junctional intercellular communication is regulated by connexin biosynthesis, transport and assembly as well as the formation and removal of gap junctions from the cell surface. At least nine members of the 20-member connexin family are known to be phosphorylated en route or during their assembly into gap junctions. For some connexins, notably Cx43, evidence exists that phosphorylation may trigger its internalization and degradation. In recent years it has become apparent that the mechanisms underlying the regulation of connexin turnover are quite complex with the identification of many connexin binding molecules, a multiplicity of protein kinases that phosphorylate connexins and the involvement of both lysosomal and proteasomal pathways in degrading connexins. This paper will review the evidence that connexin phosphorylation regulates, stimulates or triggers gap junction disassembly, internalization and degradation.     

Connexin phosphorylation as a regulatory event linked to gap junction internalization and degradation

Gap junction proteins, connexins, are dynamic polytopic membrane proteins that exhibit unprecedented short half-lives of only a few hours. Consequently, it is well accepted that in addition to channel gating, gap junctional intercellular communication is regulated by connexin biosynthesis, transport and assembly as well as the formation and removal of gap junctions from the cell surface. At least nine members of the 20-member connexin family are known to be phosphorylated en route or during their assembly into gap junctions. For some connexins, notably Cx43, evidence exists that phosphorylation may trigger its internalization and degradation. In recent years it has become apparent that the mechanisms underlying the regulation of connexin turnover are quite complex with the identification of many connexin binding molecules, a multiplicity of protein kinases that phosphorylate connexins and the involvement of both lysosomal and proteasomal pathways in degrading connexins. This paper will review the evidence that connexin phosphorylation regulates, stimulates or triggers gap junction disassembly, internalization and degradation.     

Ser364 of connexin43 and the upregulation of gap junction assembly by cAMP.

The assembly of gap junctions (GJs) is a process coordinated by growth factors, kinases, and other signaling molecules. GJ assembly can be enhanced via the elevation of cAMP and subsequent stimulation of connexon trafficking to the plasma membrane. To study the positive regulation of GJ assembly, fibroblasts derived from connexin (Cx)43 knockout (KO) and wild-type (WT) mice were transfected with WT Cx43 (WTCx43) or mutant Cx43. GJ assembly between untransfected WT fibroblasts or stably transfected WTCx43/KO fibroblasts was increased two- to fivefold by 8Br-cAMP, and this increase could be blocked by inhibition of cAMP-dependent protein kinase (PKA) or truncation of the Cx43 COOH terminus (CT). Although serine 364 (S364) of the Cx43 CT was determined to be a major site of phosphorylation, the molar ratio of Cx43 phosphorylation was not increased by 8Br-cAMP. Importantly, GJ assembly between either S364ECx43/KO or S364ECx43/WT fibroblasts was stimulated by 8Br-cAMP, but that between S364ACx43/KO or S364PCx43/KO fibroblasts was not stimulated, indicating that phosphorylation or a negative charge at S364 is required for enhancement of GJ assembly by cAMP. Furthermore, GJ assembly between S364ACx43/WT fibroblasts could be stimulated by 8Br-cAMP, but could not be between S364PCx43/WT fibroblasts. Thus, S364PCx43 interferes with enhanced GJ assembly when coexpressed with WTCx43.     

Myometrial connexin 43 trafficking and gap junction assembly at term and in preterm labor.

Modulation of connexin 43 (cx43) in the myometrium of timed pregnant rats was studied using enzyme-linked immunosorbent assay (ELISA), immunocytochemical localization, and immunoblot. These techniques utilized site-specific antibodies directed against a portion of the carboxyl tail of cx43. We found that cx43 is synthesized several days prior to labor but accumulates within the cytoplasm until parturition, when it is rapidly transported to the plasma membrane and assembled into gap junction plaques at the cell surface. These cx43-positive gap junctions begin to disappear from the plasma membrane within hours of delivery of the last pup and are completely absent within 24 hr following delivery. These structures are apparently internalized and degraded within the cytoplasm. ELISA documents a reduction of total cellular cx43 to baseline levels within 5 days following parturition. While the timing of synthesis, cytoplasmic storage, concentration in apparent Golgi vesicles, and transport to and assembly in the plasma membrane are accelerated in three models of preterm labor, the sequence of these events and the correlation of parturition with the formation of gap junctions are identical to those documented in normal labor. These results support the hypothesis that effective labor requires the synthesis and assembly of cx43 into functional gap junctions at the myometrial cell surface.     

Structure and closure of connexin gap junction channels

Connexin gap junctions comprise assembled channels penetrating two plasma membranes for which gating regulation is associated with a variety of factors, including voltage, pH, Ca²⁺, and phosphorylation. Functional studies have established that various parts of the connexin peptides are related to channel closure and electrophysiology studies have provided several working models for channel gating. The corresponding structural models supporting these findings, however, are not sufficient because only small numbers of closed connexin structures have been reported. To fully understand the gating mechanisms, the channels should be visualized in both the open and closed states. Electron crystallography and X-ray crystallography studies recently revealed three-dimensional structures of connexin channels in a couple of states in which the main difference is the conformation of the N-terminal domain, which have helped to clarify the structure in regard to channel closure. Here the closure models for connexin gap junction channels inferred from structural and functional studies are described in the context of each domain of the connexin protein associated with gating modulation.     

Connexin and gap junction degradation

Many of the subunit proteins (connexins) that form gap junctions are rather dynamic, with half-lives of only a few hours. Thus, alterations in connexin turnover and degradation may represent significant mechanisms for the regulation of intercellular communication. We describe a pharmacological approach to determining pathways of connexin degradation. Cell cultures are left untreated or treated with inhibitors of lysosomal or proteasomal proteolysis. Changes in connexin levels, localization, or decay curves (derived from pulse-chase experiments) are assessed by immunoblotting, immunofluorescence, and immunoprecipitation, respectively. Such experiments have provided evidence that connexin43 degradation involves both the lysosome and the proteasome.     

Coexpression of connexin 45 with connexin 43 decreases gap junction size

In the human heart, ventricular myocytes express connexin 43 (Cx43) and traces of Cx45. In congestive heart failure, Cx43 levels decrease, Cx45 levels increase and gap junction size decreases. To determine whether alterations of connexin coexpression ratio influence gap junction size, we engineered a rat liver epithelial cell line that endogenously expresses Cx43 to coexpress inducible levels of Cx45 under stimulation of the insect hormone, ponasterone A. In cells induced to express Cx45, gap junction sizes are significantly reduced (by 15% to 20%; p < 0.001), an effect that occurs despite increased levels of junctional connexons made from both connexins. In contrast, coexpression of Cx40 with Cx43 does not lead to any change in gap junction size. These results are consistent with the idea that increased Cx45 expression in the failing ventricle contributes to decreased gap junction size.     

Phosphorylation of connexin 50 by protein kinase A enhances gap junction and hemichannel function

Phosphorylation of connexins is an important mechanism regulating gap junction channels. However, the role(s) of connexin (Cx) phosphorylation in vivo are largely unknown. Here, we showed by mass spectrometry that Ser-395 in the C terminus of chicken Cx50 was phosphorylated in the lens. Ser-395 is located within a PKA consensus site. Analyses of Cx50 phosphorylation by two-dimensional thin layer chromatography tryptic phosphopeptide profiles suggested that Ser-395 was targeted by PKA in vivo. PKA activation increased both gap junction dye coupling and hemichannel dye uptake in a manner not involving increases in total Cx50 expression or relocation to the cell surface or gap junctional plaques. Single channel recordings indicated PKA enhanced transitions between the closed and ～200-pS open state while simultaneously reducing transitions between this open state and a ～65-pS subconductance state. The mutation of Ser-395 to alanine significantly attenuated PKA-induced increases in dye coupling and uptake by Cx50. However, channel records indicated that phosphorylation at this site was unnecessary for enhanced transitions between the closed and ～200-pS conductance state. Together, these results suggest that Cx50 is phosphorylated in vivo by PKA at Ser-395 and that this event, although unnecessary for PKA-induced alterations in channel conductance, promotes increased dye permeability of Cx50 channels, which plays an important role in metabolic coupling and transport in lens fibers.     

Dipyridamole-related enhancement of gap junction coupling in the GM-7373 aortic endothelial cells correlates with an increase in the amount of connexin 43 mRNA and protein as well as gap junction plaques

Previous data showed that dipyridamole enhanced gap junction coupling in vascular endothelial and smooth muscle cell lines by a cAMP-dependent mechanism. The present study investigates the level at which dipyridamole affects gap junction coupling. In the GM-7373 endothelial cell line, scrape loading/dye transfer experiments revealed a rapid increase in gap junction coupling induced during the first 6 h of dipyridamole treatment, followed by a slow increase induced by further incubation. Immunostaining analyses showed that the rapid enhancement of gap junction coupling correlated with an increased amount of Cx43 gap junction plaques and a reduced amount of Cx43 containing vesicles, while the amount of Cx43 mRNA or protein was not changed during this period, as found by semiquantitative RT-PCR and Western blot. Additionally, brefeldin A did not block this short-term-induced enhancement of gap junction coupling. Along with the dipyridamole-induced long-term enhancement of gap junction coupling, the amount of Cx43 mRNA and protein additionally to the amount of Cx43 gap junction plaques were increased. Furthermore, the anti-Cx43 antibody detected only two bands at 42 kDa and 44 kDa in control cells and cells treated with dipyridamole for 6 h, while long-term dipyridamole-treated cells showed a third band at 46 kDa. We propose that a dipyridamole-induced cAMP synthesis increased gap junction coupling in the GM-7373 endothelial cell line at different levels: the short-term effect is related to already oligomerised connexins beyond the Golgi apparatus and the long-term effect involves new expression and synthesis as well as posttranslational modification of Cx43.     

Regulation of gap junction coupling through the neuronal connexin Cx35 by nitric oxide and cGMP

Gap-junctional coupling among neurons is subject to regulation by a number of neurotransmitters including nitric oxide. We studied the mechanisms by which NO regulates coupling in cells expressing Cx35, a connexin expressed in neurons throughout the central nervous system. NO donors caused potent uncoupling of HeLa cells stably transfected with Cx35. This effect was mimicked by Bay 21-4272, an activator of guanylyl cyclase. A pharmacological analysis indicated that NO-induced uncoupling involved both PKG-dependent and PKG-independent pathways. PKA was involved in both pathways, suggesting that PKG-dependent uncoupling may be indirect. In vitro, PKG phosphorylated Cx35 at three sites: Ser110, Ser276, and Ser289. A mutational analysis indicated that phosphorylation on Ser110 and Ser276, sites previously shown also to be phosphorylated by PKA, had a significant influence on regulation. Ser289 phosphorylation had very limited effects. We conclude that NO can regulate coupling through Cx35 and that regulation is indirect in HeLa cells.     

The large GTPase dynamin2: a new player in connexin 43 gap junction endocytosis, recycling and degradation

Connexins (Cx) are key regulators of cell proliferation, differentiation and apoptosis. Cx trafficking and endocytosis need interactions with a large number of signaling and scaffolding proteins. We demonstrate herein that Cx43-GFP gap junction plaque endocytosis was blocked in cells transfected by the dominant-negative form of dynamin2 (Dyn2K44A) and by dynasore, an inhibitor of dynamin GTPase activity, which reduced the association between dynamin2 and Cx43. Our data also reveal that recruitment of the GTPase at the plasma membrane and its activation by c-Src are key events for Cx43 internalization. In addition they show that dynamin2 participated in internalization and degradation of the gap junction plaque but also in recycling of Cx43 to the plasma membrane through respectively Rab5/Rab7 and Rab11 pathways. These results demonstrate for the first time that dynamin2 is a new Cx partner and report an innovating mechanistic model by which dynamin2 may control Cx43 gap junction plaque invagination, endocytosis, recycling and degradation. These processes are magnified in response to carcinogen exposure underlining their potential importance during carcinogenesis.     

Spatio-temporal regulation of connexin43 phosphorylation and gap junction dynamics

Gap junctions are specialized membrane domains containing tens to thousands of intercellular channels. These channels permit exchange of small molecules (< 1000 Da) including ions, amino acids, nucleotides, metabolites and secondary messengers (e.g., calcium, glucose, cAMP, cGMP, IP₃) between cells. The common reductionist view of these structures is that they are composed entirely of integral membrane proteins encoded by the 21 member connexin human gene family. However, it is clear that the normal physiological function of this structure requires interaction and regulation by a variety of proteins, especially kinases. Phosphorylation is capable of directly modulating connexin channel function but the most dramatic effects on gap junction activity occur via the organization of the gap junction structures themselves. This is a direct result of the short half-life of the primary gap junction protein, connexin, which requires them to be constantly assembled, remodeled and turned over. The biological consequences of this remodeling are well illustrated during cardiac ischemia, a process wherein gap junctions are disassembled and remodeled resulting in arrhythmia and ultimately heart failure. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

Intracellular transport, assembly, and degradation of wild-type and disease-linked mutant gap junction proteins

More than 130 different mutations in the gap junction integral plasma membrane protein connexin32 (Cx32) have been linked to the human peripheral neuropathy X-linked Charcot-Marie-Tooth disease (CMTX). How these various mutants are processed by the cell and the mechanism(s) by which they cause CMTX are unknown. To address these issues, we have studied the intracellular transport, assembly, and degradation of three CMTX-linked Cx32 mutants stably expressed in PC12 cells. Each mutant had a distinct fate: E208K Cx32 appeared to be retained in the endoplasmic reticulum (ER), whereas both the E186K and R142W mutants were transported to perinuclear compartments from which they trafficked either to lysosomes (R142W Cx32) or back to the ER (E186K Cx32). Despite these differences, each mutant was soluble in nonionic detergent but unable to assemble into homomeric connexons. Degradation of both mutant and wild-type connexins was rapid (t(1/2) < 3 h) and took place at least in part in the ER by a process sensitive to proteasome inhibitors. The mutants studied are therefore unlikely to cause disease by accumulating in degradation-resistant aggregates but instead are efficiently cleared from the cell by quality control processes that prevent abnormal connexin molecules from traversing the secretory pathway.     

Characterization of the gap junction protein,connexin45

Connexin45 is a gap junction protein which forms channels with unique characteristics. RNA blots demonstrated that connexin45 is expressed in a number of cell lines including WB, SK Hepl, BHK, A7r5, CLEM, and BWEM cells. Connexin45 was further studied in BWEM cells using specific affinity-purified antibodies directed against a synthetic peptide representing amino acids 285–298 of its sequence. Immunofluorescence experiments demonstrated that the BWEM cells expressed both connexin43 and connexin45 and that these connexins colocalized. Connexin45 polypeptide, immunoprecipitated from BWEM cells metabolically labeled with [³⁵S]-methionine, consisted of a predominant 48 kD polypeptide. Connexin45 and connexin43 contained radioactive phosphate when immunoprecipitated from BWEM cells metabolically labeled with [³²P]-orthophosphoric acid. This phosphate label was removed from connexin45 by alkaline phosphatase digestion. Treatment of BWEM cells with the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited intercellular passage of microinjected Lucifer yellow. While TPA treatment induced phosphorylation of connexin43 in these cells, it reduced the expression of connexin45. Furthermore, the connexin45 expressed after TPA treatment was not phosphorylated. These results suggest that treatments which alter protein phosphorylation may regulate connexin43 and connexin45 in BWEM cells by different mechanisms.These studies were supported by National Institutes of Health grants HL45466 and EY08368. J.G.L. is supported by a fellowship from the Lucille P. Markey Foundation. E.C.B. is an Established Investigator of the American Heart Association.     

The connexin43 carboxyl terminus and cardiac gap junction organization

The precise spatial order of gap junctions at intercalated disks in adult ventricular myocardium is thought vital for maintaining cardiac synchrony. Breakdown or remodeling of this order is a hallmark of arrhythmic disease of the heart. The principal component of gap junction channels between ventricular cardiomyocytes is connexin43 (Cx43). Protein-protein interactions and modifications of the carboxyl-terminus of Cx43 are key determinants of gap junction function, size, distribution and organization during normal development and in disease processes. Here, we review data on the role of proteins interacting with the Cx43 carboxyl-terminus in the regulation of cardiac gap junction organization, with particular emphasis on Zonula Occludens-1. The rapid progress in this area suggests that in coming years we are likely to develop a fuller understanding of the molecular mechanisms causing pathologic remodeling of gap junctions. With these advances come the promise of novel approach to the treatment of arrhythmia and the prevention of sudden cardiac death. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Localization of a voltage gate in connexin46 gap junction hemichannels.

Cysteine replacement mutagenesis has identified positions in the first transmembrane domain of connexins as contributors to the pore lining of gap junction hemichannels (Zhou et al. 1997. Biophys. J. 72:1946-1953). Oocytes expressing a mutant cx46 with a cysteine in position 35 exhibited a membrane conductance sensitive to the thiol reagent maleimidobutyryl biocytin (MBB). MBB irreversibly reduced the single-channel conductance by 80%. This reactive cysteine was used to probe the localization of a voltage gate that closes cx46 gap junction hemichannels at negative potentials. MBB was applied to the closed channel either from outside (whole cell) or from inside (excised membrane patches). After washout of the thiol reagent the channels were tested at potentials at which the channels open. After extracellular application of MBB to intact oocytes, the membrane conductance was unaffected. In contrast, channels treated with intracellular MBB were blocked. Thus the cysteine in position 35 of cx46 is accessible from inside but not from the outside while the channel is closed. These results suggest that the voltage gate, which may be identical to the "loop gate" (Trexler et al. 1996. Proc. Natl. Acad. Sci. USA. 93:5836-5841), is located extracellular to the 35 position. The voltage gate results in regional closure of the pore rather than closure along the entire pore length.