Association with ZO-1 correlates with plasma membrane partitioning in truncated connexin45 mutants.

Zonula occludens-1 (ZO-1), the most abundant known connexin-interacting protein in osteoblastic cells, associates with the carboxyl termini of both Cx43 and Cx45. To learn more about the role of the cormexin-ZO-1 interaction, we analyzed connexin trafficking and function in ROS 17/2.8 cells that were stably transfected either with full length Cx45 or with Cx45 lacking 34 or 37 amino acids on the carboxyl terminus (Cx45t34 or Cx45t37). All three proteins were transported to appositional membranes in the transfected cells: Cx45 and Cx45t34 displayed a punctate appositional membrane-staining pattern, while Cx45t37 staining at appositional membranes was more linear. Expression of Cx45 decreased gap junction communication as assayed by dye transfer, while expression of Cx45t34 or Cx45t37 increased the amount of dye transfer seen in these cells. We found that Cx43, Cx45 and Cx45t34 co-precipitated with ZO-1 in these cells, while Cx45t37 did not. We also found that Cx45t37 was much more soluble in 1% Triton X-100 than the other connexins examined. In addition, Cx45t37 migrated to a fraction of lighter buoyant density on sucrose flotation gradients than Cx43, Cx45, ZO-1 and Cx45t34. As ZO-1 is an actin-binding protein, this suggested that the differences in Cx45t37 solubility might be due to a difference between the interaction of gap junctions and the actin cytoskeleton in the ROS/Cx45t37 and in the other transfected ROS cells. To examine this possibility, the transfected ROS cells were stained with fluorescently labeled phalloidin and demonstrated that there was a notable loss of actin stress fibers in the ROS/Cx45t37 cells. These findings suggest that association with ZO-1 alters the plasma membrane localization of Cx45 by removing it from a lipid raft compartment and rendering it Triton-insoluble, presumably by promoting an interaction with the actin cytoskeleton; they also suggest that Cx45 has a complex binding interaction with ZO-1 that involves either an extended carboxyl terminal domain or two distinct binding sites.

Connexin45 interacts with zonula occludens-1 and connexin43 in osteoblastic cells.

The relative expression of connexin43 and connexin45 modulates gap junctional communication and production of bone matrix proteins in osteoblastic cells. It is likely that changes in gap junction permeability are determined by the interaction between these two proteins. Cx43 interacts with ZO-1, which may be involved in trafficking of Cx43 or facilitating interactions between Cx43 and other proteins. In this study we sought to identify proteins that associate with Cx45 by coprecipitation in non-denaturing conditions. Cx45 was isolated with a 220-kDa protein that we identified as ZO-1. Under the same conditions, Cx43 also was isolated with anti-Cx45 antiserum from Cx45-transfected ROS cells (ROS/Cx45 cells). Cx43 antiserum could also coprecipitate ZO-1 in the transfected and untransfected ROS cells. Double label immunofluorescence studies showed that ZO-1, Cx43, and Cx45 colocalized at appositional membranes in ROS/Cx45 cells suggesting that all three proteins are normally associated in the cells. Additionally, we found that in vitro translated ZO-1 binds to the carboxyl-terminal of Cx45 indicating that there is a direct interaction between the carboxyl-terminal of Cx45 and ZO-1. These studies demonstrate that ZO-1 interacts with Cx45 as well as with Cx43, and suggest that the interaction of connexins with ZO-1 may play a role in regulating the composition of the gap junction and may modulate connexin-connexin interactions.

Connexin45 interacts with zonula occludens-1 in osteoblastic cells.

Connexin43 (Cx43) and Cx45 are co-expressed in a number of different tissues. Studies demonstrated that Cx45 transfected ROS (ROS/Cx45) cells, were less permeable to low molecular weight dyes than untransfected ROS cells, that have gap junctions made of Cx43. This suggests that there may be a functionally important interaction between Cx43 and Cx45 in these cells. One way in which these proteins may interact is by associating with the same set of proteins. In order to isolate connexin interacting proteins, we isolated Cx45 from Cx45 transfected ROS cells (ROS/Cx45 cells) under mild detergent conditions. These studies showed that Cx45 co-purified with the tight junction protein, ZO-1. Immunofluorescence studies of ROS/Cx45 cells simultaneously stained with polyclonal Cx45 antibody and a monoclonal ZO-1 antibody showed that Cx45 and ZO-1 colocalized in ROS/Cx45 cells. Furthermore we found that ZO-1 could bind to peptides derived from the carboxyl terminal of Cx45 that had been covalently bound to an agarose resin. These data suggests that Cx45 and ZO-1 directly interact in ROS/Cx45 cells.

Dephosphorylation and intracellular redistribution of ventricular connexin43 during electrical uncoupling induced by ischemia.

Electrical uncoupling at gap junctions during acute myocardial ischemia contributes to conduction abnormalities and reentrant arrhythmias. Increased levels of intracellular Ca(2+) and H(+) and accumulation of amphipathic lipid metabolites during ischemia promote uncoupling, but other mechanisms may play a role. We tested the hypothesis that uncoupling induced by acute ischemia is associated with changes in phosphorylation of the major cardiac gap junction protein, connexin43 (Cx43). Adult rat hearts perfused on a Langendorff apparatus were subjected to ischemia or ischemia/reperfusion. Changes in coupling were monitored by measuring whole-tissue resistance. Changes in the amount and distribution of phosphorylated and nonphosphorylated isoforms of Cx43 were measured by immunoblotting and confocal immunofluorescence microscopy using isoform-specific antibodies. In control hearts, virtually all Cx43 identified immunohistochemically at apparent intercellular junctions was phosphorylated. During ischemia, however, Cx43 underwent progressive dephosphorylation with a time course similar to that of electrical uncoupling. The total amount of Cx43 did not change, but progressive reduction in total Cx43 immunofluorescent signal and concomitant accumulation of nonphosphorylated Cx43 signal occurred at sites of intercellular junctions. Functional recovery during reperfusion was associated with increased levels of phosphorylated Cx43. These observations suggest that uncoupling induced by ischemia is associated with dephosphorylation of Cx43, accumulation of nonphosphorylated Cx43 within gap junctions, and translocation of Cx43 from gap junctions into intracellular pools.

Connexin expression and turnover : implications for cardiac excitability.

Electrical activation of the heart requires current transfer from one cell to another via gap junctions, arrays of densely packed intercellular channels. The extent to which cardiac myocytes are coupled is determined by multiple mechanisms, including tissue-specific patterns of expression of diverse gap junction channel proteins (connexins), and regulatory pathways that control connexin synthesis, intracellular trafficking, assembly into channels, and degradation. Many connexins, including those expressed in the heart, have been found to turn over rapidly. Recent studies in the intact adult heart suggest that connexin43, the principal cardiac connexin, is surprisingly short-lived (half-life approximately 1.3 hours). Both the proteasome and the lysosome participate in connexin43 degradation. Other ion channel proteins, such as those forming selected voltage-gated K(+) channels, may also exhibit rapid turnover kinetics. Regulation of connexin degradation may be an important mechanism for adjusting intercellular coupling in the heart under normal and pathophysiological conditions.

Rapid turnover of connexin43 in the adult rat heart.

Remodeling of the distribution of gap junctions is an important feature of anatomic substrates of arrhythmias in patients with healed myocardial infarcts. Mechanisms underlying this process are poorly understood but probably involve changes in gap junction protein (connexin) synthesis, assembly into channels, and degradation. The half-life of the principal cardiac gap junction protein, connexin43 (Cx43), is only 1.5 to 2 hours in primary cultures of neonatal myocytes, but it is unknown whether rapid turnover of Cx43 occurs in the adult heart or is unique to disaggregated neonatal myocytes that are actively reestablishing connections in vitro. To characterize connexin turnover dynamics in the adult heart and to elucidate its potential role in remodeling of gap junctions, we measured Cx43 turnover kinetics and characterized the proteolytic pathways involved in Cx43 degradation in isolated perfused adult rat hearts. Hearts were labeled for 40 minutes with Krebs-Henseleit buffer containing [35S]methionine, and then chase perfusions were performed with nonradioactive buffer for 0, 60, 120, and 240 minutes. Quantitative immunoprecipitation assays of Cx43 radioactivity in 4 hearts at each time point yielded a monoexponential decay curve indicating a Cx43 half-life of 1.3 hours. Proteolytic pathways responsible for Cx43 degradation were elucidated by perfusing isolated rat hearts for 4 hours with specific inhibitors of either lysosomal or proteasomal proteolysis. Immunoblot analysis demonstrated significant increases ( approximately 30%) in Cx43 content in hearts perfused with either lysosomal or proteasomal pathway inhibitors. Most of the Cx43 in hearts perfused with lysosomal inhibitors consisted of phosphorylated isoforms, whereas nonphosphorylated Cx43 accumulated selectively in hearts perfused with a specific proteasomal inhibitor. These results indicate that Cx43 turns over rapidly in the adult heart and is degraded by multiple proteolytic pathways. Regulation of Cx43 degradation could play an important role in gap junction remodeling in response to cardiac injury.

Proteolysis of connexin43-containing gap junctions in normal and heat-stressed cardiac myocytes.

OBJECTIVE: The present studies were performed to examine the degradation of connexin43-containing gap junctions by the lysosome or the proteasome in normal and heat-stressed cultures of neonatal rat ventricular myocytes. METHODS: Primary cultures were prepared from neonatal rat ventricular myocytes. Connexin43 was detected by immunoblotting, immunofluorescence, or immunoprecipitation. Gap junction profiles were detected by transmission electron microscopy. RESULTS: Immunoblots of whole cell lysates demonstrated increased levels of connexin43 in cultures treated with lysosomal inhibitors (chloroquine, leupeptin, E-64, or ammonium chloride) or proteasomal inhibitors (lactacystin or ALLN). Pulse-chase experiments showed that the half-life of connexin43 was 1.4 h in control cultures, but was prolonged to 2.0 or 2.8 h in cultures treated with chloroquine or lactacystin, respectively. Immunofluorescence and electron microscopy showed a significant increase in the number of gap junction profiles in myocytes treated with either chloroquine or lactacystin. Heat treatment of cultures (43.5 degrees C for 30 min) produced a rapid loss of connexin43 as detected by immunoblotting or immunofluorescence. Heat-induced connexin43 degradation was prevented by simultaneous treatment with lactacystin, ALLN, or chloroquine. Connexin43 levels and distribution returned to normal by 3 h following a heat shock and were resistant to a subsequent repeat heat stress. The heat shock also led to production of HSP70 as detected by immunoblotting. CONCLUSIONS: These data suggest that Cx43 gap junctions in myocytes are degraded by the proteasome and the lysosome, that this proteolysis can be augmented by heat stress, and that inducible factors such as HSP70 may protect against Cx43 degradation.

Differential expression of gap junction proteins in the canine sinus node.

Electrical coupling of pacemaker cells at gap junctions appears to play an important role in sinus node function. Although the major cardiac gap junction protein, connexin43 (Cx43), is expressed abundantly in atrial and ventricular muscle, its expression in the sinus node has been a subject of controversy. The objectives of the present study were to determine whether Cx43 is expressed by sinus node myocytes, to characterize the spectrum of connexin expression phenotypes in sinus node pacemaker cells, and to define the spatial distribution of different connexin phenotypes in the intact sinus node. To fulfill these objectives, we performed high-resolution immunohistochemical analysis of disaggregated adult canine sinus node preparations. Using enhanced tissue preservation and antigen retrieval techniques, we also performed immunohistochemical studies on sections of intact canine sinus node tissue. Analysis of disaggregated sinus node preparations revealed three populations of pacemaker cells distinguished on the basis of connexin immunohistochemical phenotype: approximately 55% of cells expressed only connexin40 (Cx40); 30% to 35% of cells expressed Cx43, connexin45 (Cx45), and Cx40; and the remaining cells had no detectable connexin expression. In immunostained sections of intact sinus node, Cx43- and Cx45-positive cells were limited in their distribution and were observed in discrete bundles that appeared to abut atrial myocytes. In contrast, Cx40 immunoreactive signal was widely distributed in the sinus node region. These results indicate that subsets of pacemaker cells express distinct connexin phenotypes. Differential expression of connexins could create regions within the sinus node with different conduction properties, thereby contributing to the nonuniform conduction properties seen in this tissue.

Degradation of connexin43 gap junctions involves both the proteasome and the lysosome.

Intercellular communication may be modulated by the rather rapid turnover and degradation of gap junction proteins, since many connexins have half-lives of 1-3 h. While several morphological studies have suggested that gap junction degradation occurs after endocytosis, our recent biochemical studies have demonstrated involvement of the ubiquitin-proteasome pathway in proteolysis of the connexin43 polypeptide. The present study was designed to reconcile these observations by examining the degradation of connexin43-containing gap junctions in rat heart-derived BWEM cells. After treatment of BWEM cells with Brefeldin A to prevent transport of newly synthesized connexin43 polypeptides to the plasma membrane, quantitative confocal microscopy showed the disappearance of immunoreactive connexin43 from the cell surface with a half-life of approximately 1 h. This loss of connexin43 immunoreactivity was inhibited by cotreatment with proteasomal inhibitors (ALLN, MG132, or lactacystin) or lysosomal inhibitors (leupeptin or E-64). Similar results were seen when connexin43 export was blocked with monensin. After treatment of BWEM cells with either proteasomal or lysosomal inhibitors alone, immunoblots showed accumulation of connexin43 in both whole cell lysates and in a 1% Triton X-100-insoluble fraction. Immunofluorescence studies showed that connexin43 accumulated at the cell surface in lactacystin-treated cells, but in vesicles in BWEM cells treated with lysosomal inhibitors. These results implicate both the proteasome and the lysosome in the degradation of connexin43-containing gap junctions.

Expression of zebrafish connexin43.4 in the notochord and tail bud of wild-type and mutant no tail embryos.

Communication via gap junctions provides a mechanism for the cell-cell transfer and coordination of developmental signals. The spatial restriction of gap junctions may also serve to organize cells into domains of coordinated behavior. To investigate the role of gap junctions during embryogenesis, we have characterized the expression of a member of the gap junction gene family, zebrafish connexin43.4, a homolog of connexin45 in chicken and mammals. Expression of connexin43.4 was induced in the early gastrula, coincident with the first definitive assignments of axial cell fate and the onset of the cell movements comprising convergence and extension in zebrafish. In situ hybridization and immunohistochemistry revealed that during gastrulation connexin43.4 mRNA and protein were progressively enriched in the germ ring and in the notochord primordia on the dorsal side of the embryo. Later in development connexin43.4 expression was detected in the notochord, the paraxial mesoderm, and the tail bud but was not observed after the differentiation of these tissues. In no tail mutant embryos which are defective in tail formation and proper morphogenesis of the notochord, connexin43.4 expression was absent during gastrulation from the caudal embryonic shield and notochord primordia. During somite stages in no tail embryos, connexin43.4 expression remained absent in the notochordal precursor cells and was lost in the tail bud. Thus, the no tail gene product, a transcription factor, was required for the expression of connexin43.4 in both the notochord and tail bud during morphogenesis. By microinjection of mRNA coding for a connexin43.4/green fluorescent protein fusion in the 1-cell zebrafish embryo, we showed that connexin43.4 is capable of assembling into structures reminiscent of gap junctions. The progressively restricted, developmental expression of the zebrafish connexin43.4 gene suggests that this gap junctional protein participates in the coordination of gastrulation and the formation of the notochord and tail.

The gap junction protein connexin43 is degraded via the ubiquitin proteasome pathway.

We investigated the degradation of the gap junction protein connexin43 in E36 Chinese hamster ovary cells and rat cardiomyocyte-derived BWEM cells. Treatment of E36 cells with the lysosomotropic amine, primaquine, for 16 h doubled the amount of connexin43 detected by immunoblotting and modestly increased the half-life of connexin43 in pulse-chase studies, suggesting that the lysosome played a minor role in connexin43 proteolysis. In contrast, treatment with the proteasomal inhibitor N-acetyl-L-leucyl-L-leucinyl-norleucinal led to a 6-fold accumulation of connexin43 and increased the half-life of connexin43 to approximately 9 h. The role of ubiquitin in connexin43 degradation was examined in an E36-derived mutant, ts20, which contains a thermolabile ubiquitin-activating enzyme, E1. E36 and ts20 cells grown at the permissive temperature contained similar amounts of connexin43 detectable by immunoblotting. Heat treatment dramatically reduced the amount of connexin43 detected in E36 cells, while connexin43 levels in heat-treated ts20 cells did not change. E36 cells that were heat-treated in the presence of N-acetyl-L-leucyl-L-leucinyl-norleucinal did not lose their connexin43. Pulse-chase experiments showed the reversibility of the block to connexin43 degradation in ts20 cells that were returned to the permissive temperature. Finally, sequential immunoprecipitation using anti-connexin43 and anti-ubiquitin antibodies demonstrated polyubiquitination of connexin43. These results indicate that ubiquitin-mediated proteasomal proteolysis may be the major mechanism of degradation of connexin43.

The molecular basis of anisotropy: role of gap junctions.

Electrical activation of the heart requires transfer of current from one discrete cardiac myocyte to another, a process that occurs at gap junctions. Recent advances in knowledge have established that, like most differentiated cells, individual cardiac myocytes express multiple gap junction channel proteins that are members of a multigene family of channel proteins called connexins. These proteins form channels with unique biophysical properties. Furthermore, functionally distinct cardiac tissues such as the nodes and bundles of the conduction system and atrial and ventricular muscle express different combinations of connexins. Myocytes in these tissues are interconnected by gap junctions that differ in tissue-specific manner in terms of their number, size, and three-dimensional distribution. These observations suggest that both molecular and structural aspects of gap junctions are critical determinants of the anisotropic conduction properties of different cardiac tissues. Expression of multiple connexins also creates the possibility that "hybrid" channels composed of more than one connexin protein type can form, thus greatly increasing the potential for fine control of intercellular ion flow and communication within the heart.

Expression of multiple connexins in cultured neonatal rat ventricular myocytes.

Three gap junction proteins have been identified in mammalian cardiac myocytes: connexin43 (Cx43), connexin45 (Cx45), and connexin40 (Cx40). These proteins form channels with different electrophysiological properties and have different distributions in cardiac tissues with disparate conduction properties. We characterized the expression, phosphorylation, turnover, and subcellular distribution of these connexins in primary cultures of neonatal rat ventricular myocytes. Cx43, Cx45, and Cx40 mRNA were specifically detected in RNA blots. Immunofluorescent staining with antibodies specific for Cx43 and Cx45 revealed punctate labeling at appositional membranes, but no immunoreactive Cx40 was detected. Double-label immunofluorescence confocal microscopy of cultured myocytes revealed colocalization of Cx43 and Cx45. Cx43 and Cx45 were both identified by immunoprecipitation from [35S]methionine-labeled cultures, but anti-Cx40 antibodies did not precipitate any radiolabeled protein. Phosphorylated forms of both Cx45 and Cx43 were immunoprecipitated from cultures metabolically labeled with [32P]orthophosphate. Phosphoamino acid analysis demonstrated that Cx45 was modified on serine residues, and Cx43 was phosphorylated on serine and threonine residues. Pulse-chase labeling experiments demonstrated that the half-lives of Cx43 and Cx45 were 1.9 and 2.9 hours, respectively. Thus, both Cx43 and Cx45 turn over relatively rapidly, suggesting that myocardial gap junctions have the potential for dynamic remodeling. The results implicate multiple mechanisms of gap junction regulation that may differ for different connexins.

Properties of gap junction channels formed of connexin 45 endogenously expressed in human hepatoma (SKHep1) cells.

We have evaluated the voltage dependence and unitary conductance of gap junctional channels that were recorded in a clone isolated from the hepatoma cell line SKHep1. In this clonal population (designated SKHep1A), Northern blots, immunoprecipitation, and immunohistochemical staining demonstrated the expression of connexin (Cx) 45; no other gap junction protein was identified by these techniques, although weak hybridization with Cx40 was detected. Macroscopic junctional conductance (gj) in these cells was low, averaging 1.3 nS, and was steeply voltage dependent. Parameters of voltage sensitivity were as follows: voltage at which voltage-sensitive conductance is reduced by 50%, 13.4 mV; steepness of relation, 0.115 (corresponding to 2.7 gating charges), and voltage-insensitive fraction of residual to total conductance approximately 0.06. Unitary conductance (gamma j) of these junctional channels averaged 32 +/- 8 pS; although gamma j was independent of transjunctional voltage (Vj), at high Vj values (> 50 mV), smaller conductance values were also detected. Open probabilities of the 30-pS channels at various Vj values closely matched the predicted voltage-dependent component of macroscopic gj, the residual conductance at high Vj might be attributable to the smaller conductance events. The voltage dependence of human Cx45 gap junction channels is as steep as that seen for channels formed by Xenopus Cx38 and is much steeper than that previously reported for channels formed of the highly homologous chick Cx45 and for other mammalian connexins expressed either endogenously or exogenously.

Gap junction proteins exhibit early and specific expression during intramembranous bone formation in the developing chick mandible.

The spatial and temporal expression of three closely related members of the connexin family of gap junction proteins (connexin42, Cx42; connexin43, Cx43; and connexin45, Cx45) was evaluated during bone formation in the mandibular process of the chick embryo. Mandibles of chick embryos from Hamburger and Hamilton stage 25 (approximately 5 days) through 19 days of development were dissected, serially sectioned and processed for immunocytochemical localization, employing site-specific anti-connexin antibodies. Our data revealed that (1) Cx43 was present throughout mandibular bone formation; (2) although it appeared to be associated with all bone cell types, Cx43 was concentrated in mesenchymal cells during the earliest stages in the osteogenic lineage; (3) most importantly, the localization of Cx43 at sites of bone formation appeared to precede the overt expression of the osteogenic phenotype; (4) by contrast, Cx45 was more restricted, spatially and temporally, in its distribution; (5) Cx42 expression was not detected in osteogenic tissue during mandibular bone formation. From all of the data obtained, Cx45 appeared to be associated with stages of bone formation characterized by the elaboration of matrix and the progressive expression of the differentiated osteogenic phenotype. Cx43 appeared to be associated with condensation of mesenchyme and the earliest stages of osteogenesis. Because of these associations, we propose that connexin expression may be necessary for the initiation of bone formation and the full expression of the osteogenic phenotype.

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 Hep1, 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.

Connexin43 and connexin45 form gap junctions with different molecular permeabilities in osteoblastic cells.

We examined the expression and function of gap junctions in two rat osteoblastic cell lines, ROS 17/2.8 and UMR 106-01. The pattern of expression of gap junction proteins in these two cell lines was distinct: ROS cells expressed only connexin43 on their cell surface, while UMR expressed predominantly connexin45. Immunoprecipitation and RNA blot analysis confirmed the relative quantitation of these connexins. Microinjected ROS cells passed Lucifer yellow to many neighboring cells, but UMR cells were poorly coupled by this criterion. Nevertheless, both UMR and ROS cells were electrically coupled, as characterized by the double whole cell patch-clamp technique. These studies suggested that Cx43 in ROS cells mediated cell-cell coupling for both small ions and larger molecules, but Cx45 in UMR cells allowed passage only of small ions. To demonstrate that the expression of different connexins alone accounted for the lack of dye coupling in UMR cells, we assessed dye coupling in UMR cells transfected with either Cx43 or Cx45. The UMR/Cx43 transfectants were highly dye coupled compared with the untransfected UMR cells, but the UMR/Cx45 transfectants demonstrated no increase in dye transfer. These data demonstrate that different gap junction proteins create channels with different molecular permeabilities; they suggest that different connexins permit different types of signalling between cells.

Multiple connexins colocalize in canine ventricular myocyte gap junctions.

We have recently shown that adult canine ventricular myocytes express three distinct gap junction channel proteins, connexin40 (Cx40), connexin43 (Cx43), and connexin45 (Cx45). These proteins have unique cytoplasmic domains that likely confer connexin-specific physiological properties. To determine whether the three distinct channel proteins are distributed in identical or different populations of gap junctions, we performed double-label immunofluorescence on disaggregated canine ventricular myocytes incubated simultaneously with a mouse monoclonal anti-Cx43 and affinity-purified polyclonal rabbit antibodies against Cx40 or Cx45. Analysis of double-labeled cardiac myocytes using laser scanning confocal microscopy revealed virtually identical patterns of immunoreactivity for both the Cx43/Cx40 and Cx43/Cx45 pairs. Double-label immunoelectron microscopy confirmed that ultrastructurally identified cardiac myocyte gap junctions contain multiple channel proteins. Thus, three channel proteins colocalize in canine cardiac myocyte gap junctions. The presence of multiple functionally distinct connexins suggests complex possibilities regarding the composition of individual channels and the regulation of intercellular coupling.

Distinct patterns of connexin expression in canine Purkinje fibers and ventricular muscle.

Electrical conduction is more rapid in Purkinje fibers than in ventricular muscle, which are distinct cardiac tissues that have different active and passive electrophysiological properties. We have recently demonstrated that canine myocardium contains multiple gap junction proteins or connexins that form channels with unique electrophysiological properties. To determine whether differences in connexin expression may account, in part, for the characteristic conduction properties of Purkinje fibers and ventricular muscle, we assessed the amounts of mRNA for two connexins, Cx40 and Cx43, in these tissues obtained from canine hearts by Northern blot analysis and in situ hybridization. We also characterized the distribution and relative abundance of these two connexins in gap junctions with immunocytochemistry. A significantly greater amount of Cx40 mRNA was observed in Purkinje fibers compared with ventricular muscle, a difference that was at least threefold according to quantitative in situ hybridization (p < 0.001) and densitometric analysis of Northern blots. Purkinje fibers also demonstrated greater immunostaining intensity when incubated with anti-Cx40 antibodies than did ventricular muscle. In contrast, Cx43 mRNA and protein appeared to be abundant in both tissues. Quantitative in situ hybridization demonstrated a modest but not statistically significant increase in Cx43 mRNA in Purkinje fibers compared with ventricular myocardium. These results indicate that Purkinje fibers and ventricular muscle express distinct patterns of connexins. This tissue-specific pattern of connexin expression could contribute to differences in the conduction properties of Purkinje fibers and ventricular muscle.