Circulating Small Extracellular Vesicles May Contribute to Vaso-Occlusive Crises in Sickle Cell Disease.

We previously found that the plasma of patients with sickle cell disease (SCD) contains large numbers of small extracellular vesicles (EVs) and that the EVs disrupt the integrity of endothelial cell monolayers (especially if obtained during episodes of acute chest syndrome, ACS). The present study was designed to test the generality of this finding to other complications of SCD, specifically to evaluate the possibility that circulating EVs isolated during a vaso-occlusive crises (VOC) also cause damage to the intercellular connections between endothelial cells. Plasma was obtained from nine pediatric subjects at baseline and during VOC episodes. EVs isolated from these samples were added to cultures of microvascular endothelial cells. Immunofluorescence microscopy was employed to assess monolayer integrity and to localize two intercellular junction proteins (VE-cadherin and connexin43). The EVs isolated during VOC caused significantly greater monolayer disruption than those isolated at baseline. The extent of disruption varied between different episodes of VOC or ACS in the same patient. The VOC EVs disrupted the integrity of both junction proteins at appositional membranes. These results suggest that circulating EVs may be involved in modulating endothelial integrity contributing to the pathogenesis of different complications of SCD.

Circulating extracellular vesicles from patients with acute chest syndrome disrupt adherens junctions between endothelial cells.

BACKGROUND: Small cell-derived extracellular vesicles (EVs) can affect endothelial function. We previously found that patients with sickle cell disease (SCD) have greater numbers of circulating EVs than subjects without the disease, and the EVs differentially disrupt endothelial integrity in vitro. Because endothelial disruption is a critical component of acute chest syndrome (ACS), we hypothesized that EVs isolated during ACS would induce greater endothelial damage than those isolated at baseline. METHODS: Nine pediatric subjects had plasma isolated at baseline and during ACS from which EVs were isolated. Cultured microvascular endothelial cells were treated with EVs and then studied by immunofluorescence microscopy to localize VE-cadherin and F-actin. RESULTS: The EVs had a diameter of 95 nm. They contained CD63 and flotillin-1, which were increased in SCD patients (5-13-fold compared to control) and further increased between baseline and ACS (24-57%). The EVs contained hemoglobin, glycophorin A, and ferritin. Treatment with baseline EVs caused modest separation of endothelial cells, while ACS EVs caused substantial disruptions of the endothelial cell monolayers. EVs from subjects with ACS also caused a 50% decrease in protein levels of VE-cadherin. CONCLUSIONS: These results suggest that circulating EVs can modulate endothelial integrity contributing to the development of ACS in SCD patients by altering cadherin-containing intercellular junctions. IMPACT: Sickle cell disease patients have circulating extracellular vesicles (EVs) that modulate endothelial integrity by altering cadherin-containing intercellular junctions. Disruption is more severe by EVs obtained during acute chest syndrome (ACS). These results expand our knowledge of the pathophysiology of acute chest syndrome and the vasculopathies of sickle cell disease.

Gap Junctions between Endothelial Cells Are Disrupted by Circulating Extracellular Vesicles from Sickle Cell Patients with Acute Chest Syndrome.

Intercellular junctions maintain the integrity of the endothelium. We previously found that the adherens and tight junctions between endothelial cells are disrupted by plasma extracellular vesicles from patients with sickle cell disease (especially those with Acute Chest Syndrome). In the current study, we evaluated the effects of these vesicles on endothelial gap junctions. The vesicles from sickle cell patients (isolated during episodes of Acute Chest Syndrome) disrupted gap junction structures earlier and more severely than the other classes of intercellular junctions (as detected by immunofluorescence). These vesicles were much more potent than those isolated at baseline from the same subject. The treatment of endothelial cells with these vesicles led to reduced levels of connexin43 mRNA and protein. These vesicles severely reduced intercellular communication (transfer of microinjected Neurobiotin). Our data suggest a hierarchy of progressive disruption of different intercellular connections between endothelial cells by circulating extracellular vesicles that may contribute to the pathophysiology of the endothelial disturbances in sickle cell disease.

Circulating Extracellular Vesicles and Endothelial Damage in Sickle Cell Disease.

Endothelial damage is central to the pathogenesis of many of the complications of sickle cell disease. Circulating extracellular vesicles (EVs) have been implicated in modulating endothelial behavior in a variety of different, diseases with vascular pathologies. As seen in other hemolytic diseases, the plasma of sickle cell patients contains EVs of different sizes and cellular sources. The medium-sized vesicles (microparticles) primarily derive from mature red blood cells and platelets; some of these EVs have procoagulant properties, while others stimulate inflammation or endothelial adhesiveness. Most of the small EVs (including exosomes) derive from erythrocytes and erythrocyte precursors, but some also originate from platelets, white blood cells, and endothelial cells. These small EVs may alter the behavior of target cells by delivering cargo including proteins and nucleic acids. Studies in model systems implicate small EVs in promoting vaso-occlusion and disruption of endothelial integrity. Thus, both medium and small EVs may contribute to the increased endothelial damage in sickle cell disease. Development of a detailed understanding of the composition and roles of circulating EVs represents a promising approach toward novel predictive diagnostics and therapeutic approaches in sickle cell disease.

ZO-1 Regulates Intercalated Disc Composition and Atrioventricular Node Conduction.

RATIONALE: ZO-1 (Zona occludens 1), encoded by the tight junction protein 1 (TJP1) gene, is a regulator of paracellular permeability in epithelia and endothelia. ZO-1 interacts with the actin cytoskeleton, gap, and adherens junction proteins and localizes to intercalated discs in cardiomyocytes. However, the contribution of ZO-1 to cardiac physiology remains poorly defined. OBJECTIVE: We aim to determine the role of ZO-1 in cardiac function. METHODS AND RESULTS: Inducible cardiomyocyte-specific Tjp1 deletion mice (Tjp1fl/fl; Myh6Cre/Esr1*) were generated by crossing the Tjp1 floxed mice and Myh6Cre/Esr1* transgenic mice. Tamoxifen-induced loss of ZO-1 led to atrioventricular (AV) block without changes in heart rate, as measured by ECG and ex vivo optical mapping. Mice with tamoxifen-induced conduction system-specific deletion of Tjp1 (Tjp1fl/fl; Hcn4CreERt2) developed AV block while tamoxifen-induced conduction system deletion of Tjp1 distal to the AV node (Tjp1fl/fl; Kcne1CreERt2) did not demonstrate conduction defects. Western blot and immunostaining analyses of AV nodes showed that ZO-1 loss decreased Cx (connexin) 40 expression and intercalated disc localization. Consistent with the mouse model study, immunohistochemical staining showed that ZO-1 is abundantly expressed in the human AV node and colocalizes with Cx40. Ventricular conduction was not altered despite decreased localization of ZO-1 and Cx43 at the ventricular intercalated disc and modestly decreased left ventricular ejection fraction, suggesting ZO-1 is differentially required for AV node and ventricular conduction. CONCLUSIONS: ZO-1 is a key protein responsible for maintaining appropriate AV node conduction through maintaining gap junction protein localization.

Connecting Exosomes and Connexins.

Intercellular communication is accomplished by passage of ions and small molecules through gap junction channels in directly contacting cells or by secretion and response to transmitters, hormones and extracellular vesicles in cells that are distant from each other. Recent studies have suggested that there may be overlap of these processes; specifically, small extracellular vesicles may contain subunit gap junction proteins, connexins. We isolated and analyzed extracellular vesicles secreted by cultured microvascular endothelial cells. These vesicles had a diameter of ~120 nm. They contained four exosomal proteins (flotillin-1, CD63, CD81 and Alix) and the gap junction protein, connexin43. They did not contain an endoplasmic reticulum protein (Grp94) or an adherens junction protein (VE-cadherin). Secretion of vesicles was increased by treatment of the cells with staurosporine. Our data confirm that the gap junction protein, connexin43, can be secreted in vesicles with the properties of exosomes. Although the role of vesicular connexin is not clearly known, we speculate that it might participate in docking/fusion of the exosomes with the recipient cell, transmission of vesicular contents, or cellular signaling.

Intermittent hypoxia causes NOX2-dependent remodeling of atrial connexins.

BACKGROUND: Obstructive sleep apnea has been linked to the development of heart disease and arrhythmias, including atrial fibrillation. Since altered conduction through gap junction channels can contribute to the pathogenesis of such arrhythmias, we examined the abundance and distributions of the major cardiac gap junction proteins, connexin40 (Cx40) and connexin43 (Cx43) in mice treated with sleep fragmentation or intermittent hypoxia (IH) as animal models of the components of obstructive sleep apnea. RESULTS: Wild type C57BL/6 mice or mice lacking NADPH 2 (NOX2) oxidase activity (gp91phox(-/Y)) were exposed to room air or to SF or IH for 6 weeks. Then, the mice were sacrificed, and atria and ventricles were immediately dissected. The abundances of Cx40 or Cx43 in atria and ventricles were unaffected by SF. In contrast, immunoblots showed that the abundance of atrial Cx40 and Cx43 and ventricular Cx43 were reduced in mice exposed to IH. qRT-PCR demonstrated significant reductions of atrial Cx40 and Cx43 mRNAs. Immunofluorescence microscopy revealed that the abundance and size of gap junctions containing Cx40 or Cx43 were reduced in atria by IH treatment of mice. However, no changes of connexin abundance or gap junction size/abundance were observed in IH-treated NOX2-null mice. CONCLUSIONS: These results demonstrate that intermittent hypoxia (but not sleep fragmentation) causes reductions and remodeling of atrial Cx40 and Cx43. These alterations may contribute to the substrate for atrial fibrillation that develops in response to obstructive sleep apnea. Moreover, these connexin changes are likely generated in response to reactive oxygen species generated by NOX2.

Connexin40 abnormalities and atrial fibrillation in the human heart.

Normal atrial conduction requires similar abundances and homogeneous/overlapping distributions of two connexins (Cx40 and Cx43). The remodeling of myocyte connections and altered electrical conduction associated with atrial fibrillation (AF) likely involves perturbations of these connexins. We conducted a comprehensive series of experiments to examine the abundances and distributions of Cx40 and Cx43 in the atria of AF patients. Atrial appendage tissues were obtained from patients with lone AF (paroxysmal or chronic) or normal controls. Connexins were localized by double label immunofluorescence confocal microscopy, and their overlap was quantified. Connexin proteins and mRNAs were quantified by immunoblotting and qRT-PCR. PCR amplified genomic DNA was sequenced to screen for connexin gene mutations or polymorphisms. Immunoblotting showed reductions of Cx40 protein (to 77% or 49% of control values in samples from patients with paroxysmal and chronic AF, respectively), but no significant changes of Cx43 protein levels in samples from AF patients. The extent of Cx43 immunostaining and its distribution relative to N-cadherin were preserved in the AF patient samples. Although there was variability of Cx40 staining among paroxysmal AF patients, all had some fields with substantial Cx40 heterogeneity and reduced overlap with Cx43. Cx40 immunostaining was severely reduced in all chronic AF patients. qRT-PCR showed no change in Cx43 mRNA levels, but reductions in total Cx40 mRNA (to <50%) and Cx40 transcripts A (to ~50%) and B (to <25%) as compared to controls. No Cx40 coding region mutations were identified. The frequency of promoter polymorphisms did not differ between AF patient samples and controls. Our data suggest that reduced Cx40 levels and heterogeneity of its distribution (relative to Cx43) are common in AF. Multiple mechanisms likely lead to reductions of functional Cx40 in atrial gap junctions and contribute to the pathogenesis of AF in different patients.

Degradation of a connexin40 mutant linked to atrial fibrillation is accelerated.

Several Cx40 mutants have been identified in patients with atrial fibrillation (AF). We have been working to identify physiological or cell biological abnormalities of several of these human mutants that might explain how they contribute to disease pathogenesis. Wild type (wt) Cx40 or four different mutants (P88S, G38D, V85I, and L229M) were expressed by the transfection of communication-deficient HeLa cells or HL-1 cardiomyocytes. Biophysical channel properties and the sub-cellular localization and protein levels of Cx40 were characterized. Wild type Cx40 and all mutants except P88S formed gap junction plaques and induced significant gap junctional conductances. The functional mutants showed only modest alterations of single channel conductances or gating by trans-junctional voltage as compared to wtCx40. However, immunoblotting indicated that the steady state levels of G38D, V85I, and L229M were reduced relative to wtCx40; most strikingly, G38D was only 20-31% of wild type levels. After the inhibition of protein synthesis with cycloheximide, G38D (and to a lesser extent the other mutants) disappeared much faster than wtCx40. Treatment with the proteasomal inhibitor, epoxomicin, greatly increased levels of G38D and restored the abundance of gap junctions and the extent of intercellular dye transfer. Thus, G38D, V85I, and L229M are functional mutants of Cx40 with small alterations of physiological properties, but accelerated degradation by the proteasome. These findings suggest a novel mechanism (protein instability) for the pathogenesis of AF due to a connexin mutation and a novel approach to therapy (protease inhibition).

Atrial fibrillation-associated connexin40 mutants make hemichannels and synergistically form gap junction channels with novel properties.

Mutations of Cx40 (GJA5) have been identified in people with lone chronic atrial fibrillation including G38D and M163V which were found in the same patient. We used dual whole cell patch clamp procedures to examine the transjunctional voltage (Vj) gating and channel conductance properties of these two rare mutants. Each mutant exhibited slight alterations of Vj gating properties and increased the gap junction channel conductance (γj) by 20-30 pS. While co-expression of the two mutations had similar effects on Vj gating, it synergistically increased γj by 50%. Unlike WTCx40 or M163V, G38D induced activity of a dominant 271 pS hemichannel.

c-Src kinase inhibition reduces arrhythmia inducibility and connexin43 dysregulation after myocardial infarction.

OBJECTIVES: The aim of this study was to evaluate the role of tyrosine kinase cellular-Src (c-Src) inhibition on connexin43 (Cx43) regulation in a mouse model of myocardial infarction (MI). BACKGROUND: MI is associated with decreased expression of Cx43, the principal gap junction protein responsible for propagating current in ventricles. Activated c-Src has been linked to Cx43 dysregulation. METHODS: MI was induced in 12-week-old mice by coronary artery occlusion. MI mice were treated with c-Src inhibitors (PP1 or AZD0530), PP3 (an inactive analogue of PP1), or saline. Treated hearts were compared to sham mice by echocardiography, optical mapping, telemetry electrocardiographic monitoring, and inducibility studies. Tissues were collected for immunoblotting, quantitative polymerase chain reaction, and immunohistochemistry. RESULTS: Active c-Src was elevated in PP3-treated MI mice compared to sham at the scar border (280%, p = 0.003) and distal ventricle (346%, p = 0.013). PP1 treatment restored active c-Src to sham levels at the scar border (86%, p = 0.95) and distal ventricle (94%, p = 1.0). PP1 raised Cx43 expression by 69% in the scar border (p = 0.048) and by 73% in the distal ventricle (p = 0.043) compared with PP3 mice. PP1-treated mice had restored conduction velocity at the scar border (PP3: 32 cm/s, PP1: 41 cm/s, p < 0.05) and lower arrhythmic inducibility (PP3: 71%, PP1: 35%, p < 0.05) than PP3 mice. PP1 did not change infarct size, electrocardiographic pattern, or cardiac function. AZD0530 treatment demonstrated restoration of Cx43 comparable to PP1. CONCLUSIONS: c-Src inhibition improved Cx43 levels and conduction velocity and lowered arrhythmia inducibility after MI, suggesting a new approach for arrhythmia reduction following MI.

Inducible coexpression of connexin37 or connexin40 with connexin43 selectively affects intercellular molecular transfer.

Many tissues express multiple gap junction proteins, or connexins (Cx); for example, Cx43, Cx40, and Cx37 are coexpressed in vascular cells. This study was undertaken to elucidate the consequences of coexpression of Cx40 or Cx37 with Cx43 at different ratios. EcR-293 cells (which endogenously produce Cx43) were transfected with ecdysone-inducible plasmids encoding Cx37 or Cx40. Immmunoblotting showed a ponasterone dose-dependent induction of Cx37 or Cx40 while constant levels of Cx43 were maintained. The coexpressed connexins colocalized at appositional membranes. Double whole-cell patch clamp recordings showed no significant change in total junctional conductances in cells treated with 0, 0.5, or 4 µM ponasterone; however, they did show a diversity of unitary channel sizes consistent with the induced connexin expression. In cells with induced expression of either Cx40 or Cx37, intercellular transfer of microinjected Lucifer yellow was reduced, but transfer of NBD-TMA (2-(4-nitro-2,1,3-benzoxadiol-7-yl)[aminoethyl]trimethylammonium) was not affected. In cocultures containing uninduced EcR cells together with cells induced to coexpress Cx37 or Cx40, Lucifer yellow transfer was observed only between the cells expressing Cx43 alone. These data show that induced expression of either Cx37 or Cx40 in Cx43-expressing cells can selectively alter the intercellular exchange of some molecules without affecting the transfer of others.

Atomic force microscopy of Connexin40 gap junction hemichannels reveals calcium-dependent three-dimensional molecular topography and open-closed conformations of both the extracellular and cytoplasmic faces.

Atomic force microscopy was used to study the three-dimensional molecular topography and calcium-sensitive conformational changes of Connexin40 hemichannels (connexons) reconstituted in 1,2-dioeloyl-sn-glycero-3-phosphatidylcholine lipid bilayers. Two classes of objects were observed that differed in their protrusion heights above the bilayer (2.6 versus 4.2 nm). Comparison to reconstituted connexons containing Connexin40 truncated to eliminate most of its C-terminal cytoplasmic domain showed that the two height classes corresponded to the shorter extracellular and taller cytoplasmic aspects of the hemichannels and that the C-terminal tail of Connexin40 contributes ∼1.6 nm in thickness. Hemichannels imaged in solutions containing < 10 µm Ca(2+) showed 3.1-3.2 nm depressions (openings) in 30% of the cytoplasmic faces and 65% of the extracellular faces, and high-resolution three-dimensional topography of extracellular or cytoplasmic aspects of some connexons was observed. After addition of 3.6 mm Ca(2+), > 75% of the connexons in either orientation adopted closed conformations. In contrast, hemichannels imaged in the presence of 0.1 mm EDTA showed large (5.6- to 5.8-nm diameter) openings in nearly all hemichannels regardless of orientation, and detailed topography was visible in many connexons. Real-time imaging following the addition of 3.6 mm Ca(2+) showed transitions of both extracellular and cytoplasmic orientations from "open" into "closed" conformations within several minutes. These studies provide the first high-resolution topographic information regarding a connexin with a large cytoplasmic domain and suggest that the extramembranous portions of Connexin40 contribute to a channel entrance that is relaxed by chelation of residual divalent cations.

Connexin40 and connexin43 determine gating properties of atrial gap junction channels.

While ventricular gap junctions contain only Cx43, atrial gap junctions contain both Cx40 and Cx43; yet the functional consequences of this co-expression remain poorly understood. We quantitated the expression of Cx40 and Cx43 and their contributions to atrial gap junctional conductance (g(j)). Neonatal murine atrial myocytes showed similar abundances of Cx40 and Cx43 proteins, while ventricular myocytes contained at least 20 times more Cx43 than Cx40. Since Cx40 gap junction channels are blocked by 2 mM spermine while Cx43 channels are unaffected, we used spermine block as a functional dual whole cell patch clamp assay to determine Cx40 contributions to cardiac g(j). Slightly more than half of atrial g(j) and

Cx30.2 can form heteromeric gap junction channels with other cardiac connexins.

Since most cells in the heart co-express multiple connexins, we studied the possible heteromeric interactions between connexin30.2 and connexin40, connexin43 or connexin45 in transfected cells. Double-label immunofluorescence microscopy showed that connexin30.2 extensively co-localized with each co-expressed connexin at appositional membranes. When Triton X-100 solubilized connexons were affinity purified from co-expressing cells, connexin30.2 was isolated together with connexin40, connexin43, or connexin45. Co-expression of connexin30.2 with connexin40, connexin43, or connexin45 did not significantly reduce total junctional conductance. Gap junction channels in cells co-expressing connexin30.2 with connexin43 or connexin45 exhibited voltage-dependent gating intermediate between that of either connexin alone. In contrast, connexin30.2 dominated the voltage-dependence when co-expressed with connexin40. Our data suggest that connexin30.2 can form heteromers with the other cardiac connexins and that mixed channel formation will influence the gating properties of gap junctions in cardiac regions that co-express these connexins.

N-terminal residues in Cx43 and Cx40 determine physiological properties of gap junction channels, but do not influence heteromeric assembly with each other or with Cx26.

The cytoplasmic N-terminal domain in the connexins (Cx) has been implicated in determining several properties including connexin hetero-oligomerization, channel gating and regulation by polyamines. To elucidate the roles of potentially crucial amino acids, we produced site-directed mutants of connexins Cx40 and Cx43 (Cx40E12S,E13G and Cx43D12S,K13G) in which the charged amino acids at positions 12 and 13 were replaced with serine and glycine as found in Cx32. HeLa, N2a and HEK293 cells were transfected and studied by immunochemistry and double whole-cell patch clamping. Immunoblotting confirmed production of the mutant proteins, and immuno-fluorescence localized them to punctuate distributions along appositional membranes. Cx40E12S,E13G and Cx43D12S,K13G formed homotypic gap junction channels that allowed intercellular passage of Lucifer Yellow and electrical current, but these channels exhibited negligible voltage-dependent gating properties. Unlike wild-type Cx40, Cx40E12S,E13G channels were insensitive to block by 2 mM spermine. Affinity purification of material solubilized by Triton X-100 from cells co-expressing mutant Cx43 or mutant Cx40 with wild-type Cx40, Cx43 or Cx26 showed that introducing the mutations did not affect the compatibility or incompatibility of these proteins for heteromeric mixing. Co-expression of Cx40E12S,E13G with wild-type Cx40 or Cx43 dramatically reduced voltage-dependent gating. Thus, whereas the charged amino acids at positions 12 and 13 of Cx40 or Cx43 are not required for gap junction assembly or the compatibility of oligomerization with each other or with Cx26, they strongly influence several physiological properties including those of heteromeric channels.

Connexin43 with a cytoplasmic loop deletion inhibits the function of several connexins.

Connexins (Cx) form gap junction channels mediating direct intercellular communication. To study the role of amino acids within the cytoplasmic loop, we produced a recombinant adenovirus containing Cx43 with a deletion of amino acids 130-136 (Cx43del(130-136)). Cx43del(130-136) expressed alone in HeLa cells localized within the cytoplasm and did not allow transfer of ions, neurobiotin or Lucifer yellow. When co-expressed with wild type Cx43, Cx43del(130-136) blocked electrical coupling and transfer of neurobiotin or Lucifer yellow. Cx43del(130-136) and Cx43 co-localized by immunofluorescence and were co-purified from Triton X-100-solubilized cell extracts. Intercellular transfer mediated by Cx37 and Cx45 (but not Cx26 or Cx40) was inhibited when co-expressed with Cx43del(130-136). Cx43del(130-136) co-localized with Cx37, Cx40, or Cx45, but not Cx26. These data suggest that Cx43del(130-136) produces connexin-specific inhibition of intercellular communication through formation of heteromeric connexons that are non-functional and/or retained in the cytoplasm.

Dynamic model for ventricular junctional conductance during the cardiac action potential.

The ventricular action potential was applied to paired neonatal murine ventricular myocytes in the dual whole cell configuration. During peak action potential voltages >100 mV, junctional conductance (g(j)) declined by 50%. This transjunctional voltage (V(j))-dependent inactivation exhibited two time constants that became progressively faster with increasing V(j). G(j) returned to initial peak values during action potential repolarization and even exceeded peak g(j) values during the final 5% of repolarization. This facilitation of g(j) was observed <30 mV during linearly decreasing V(j) ramps. The same behavior was observed in ensemble averages of individual gap junction channels with unitary conductances of 100 pS or lower. Immunohistochemical fluorescent micrographs and immunoblots detect prominent amounts of connexin (Cx)43 and lesser amounts of Cx40 and Cx45 proteins in cultured ventricular myocytes. The time dependence of the g(j) curves and channel conductances are consistent with the properties of predominantly homomeric Cx43 gap junction channels. A mathematical model depicting two inactivation and two recovery phases accurately predicts the ventricular g(j) curves at different rates of stimulation and repolarization. Functional differences are apparent between ventricular myocytes and Cx43-transfected N2a cell gap junctions that may result from posttranslational modification. These observations suggest that gap junctions may play a role in the development of conduction block and the genesis and propagation of triggered arrhythmias under conditions of slowed conduction (<10 cm/s).

Connexin43 and connexin26 form gap junctions, but not heteromeric channels in co-expressing cells.

Many cells contain two (or more) gap junction proteins that are able to oligomerize with each other to form heteromeric gap junction channels and influence the properties of intercellular communication. Cx26 and Cx43 are found together in a number of cell types, but previous data have suggested that they might not form heteromeric connexons. We studied the possible interactions of these connexins by co-expression in three different cell lines. Analysis of N2aCx26/Cx43 cell pairs by double whole-cell patch-clamp methods showed that these cells were coupled, but contained only a small number of sizes of single channels consistent with those formed by homomeric Cx26 or Cx43 channels. Immunofluorescence studies showed that both connexins localized to appositional membranes, but in largely distinct domains. Analysis of Triton X-100-solubilized connexons from co-expressing cells by centrifugation through sucrose gradients or by affinity purification using a Ni-NTA column showed no evidence of mixing of Cx26 and Cx43. These results contrast with our observations in cells co-expressing other connexins with Cx43 and suggest that Cx26 and Cx43 do not form heteromeric hemichannels. Moreover, the incorporation of Cx26 and Cx43 into oligomers and into the membrane were similarly affected by treatment of co-expressing cells with brefeldin A or nocodazole, suggesting that the lack of mixing is due to incompatibility of these connexins, not to differences in biosynthetic trafficking.