Cardiac Gap Junction Protein Connexin43 Research Articles

Distress-Mediated Remodeling of Cardiac Connexin-43 in a Novel Cell Model for Arrhythmogenic Heart Diseases.

Gap junctions and their expression pattern are essential to robust function of intercellular communication and electrical propagation in cardiomyocytes. In healthy myocytes, the main cardiac gap junction protein connexin-43 (Cx43) is located at the intercalated disc providing a clear direction of signal spreading across the cardiac tissue. Dislocation of Cx43 to lateral membranes has been detected in numerous cardiac diseases leading to slowed conduction and high propensity for the development of arrhythmias. At the cellular level, arrhythmogenic diseases are associated with elevated levels of oxidative distress and gap junction remodeling affecting especially the amount and sarcolemmal distribution of Cx43 expression. So far, a mechanistic link between sustained oxidative distress and altered Cx43 expression has not yet been identified. Here, we propose a novel cell model based on murine induced-pluripotent stem cell-derived cardiomyocytes to investigate subcellular signaling pathways linking cardiomyocyte distress with gap junction remodeling. We tested the new hypothesis that chronic distress, induced by rapid pacing, leads to increased reactive oxygen species, which promotes expression of a micro-RNA, miR-1, specific for the control of Cx43. Our data demonstrate that Cx43 expression is highly sensitive to oxidative distress, leading to reduced expression. This effect can be efficiently prevented by the glutathione peroxidase mimetic ebselen. Moreover, Cx43 expression is tightly regulated by miR-1, which is activated by tachypacing-induced oxidative distress. In light of the high arrhythmogenic potential of altered Cx43 expression, we propose miR-1 as a novel target for pharmacological interventions to prevent the maladaptive remodeling processes during chronic distress in the heart.

Prevention of connexin-43 remodeling protects against Duchenne muscular dystrophy cardiomyopathy.

Aberrant expression of the cardiac gap junction protein connexin-43 (Cx43) has been suggested as playing a role in the development of cardiac disease in the mdx mouse model of Duchenne muscular dystrophy (DMD); however, a mechanistic understanding of this association is lacking. Here, we identified a reduction of phosphorylation of Cx43 serines S325/S328/S330 in human and mouse DMD hearts. We hypothesized that hypophosphorylation of Cx43 serine-triplet triggers pathological Cx43 redistribution to the lateral sides of cardiomyocytes (remodeling). Therefore, we generated knockin mdx mice in which the Cx43 serine-triplet was replaced with either phospho-mimicking glutamic acids (mdxS3E) or nonphosphorylatable alanines (mdxS3A). The mdxS3E, but not mdxS3A, mice were resistant to Cx43 remodeling, with a corresponding reduction of Cx43 hemichannel activity. MdxS3E cardiomyocytes displayed improved intracellular Ca2+ signaling and a reduction of NADPH oxidase 2 (NOX2)/ROS production. Furthermore, mdxS3E mice were protected against inducible arrhythmias, related lethality, and the development of cardiomyopathy. Inhibition of microtubule polymerization by colchicine reduced both NOX2/ROS and oxidized CaMKII, increased S325/S328/S330 phosphorylation, and prevented Cx43 remodeling in mdx hearts. Together, these results demonstrate a mechanism of dystrophic Cx43 remodeling and suggest that targeting Cx43 may be a therapeutic strategy for preventing heart dysfunction and arrhythmias in DMD patients.

S-nitrosylation of connexin43 hemichannels elicits cardiac stress-induced arrhythmias in Duchenne muscular dystrophy mice.

Patients with Duchenne muscular dystrophy (DMD) commonly present with severe ventricular arrhythmias that contribute to heart failure. Arrhythmias and lethality are also consistently observed in adult Dmdmdx mice, a mouse model of DMD, after acute β-adrenergic stimulation. These pathological features were previously linked to aberrant expression and remodeling of the cardiac gap junction protein connexin43 (Cx43). Here, we report that remodeled Cx43 protein forms Cx43 hemichannels in the lateral membrane of Dmdmdx cardiomyocytes and that the β-adrenergic agonist isoproterenol (Iso) aberrantly activates these hemichannels. Block of Cx43 hemichannels or a reduction in Cx43 levels (using Dmdmdx Cx43+/- mice) prevents the abnormal increase in membrane permeability, plasma membrane depolarization, and Iso-evoked electrical activity in these cells. Additionally, Iso treatment promotes nitric oxide (NO) production and S-nitrosylation of Cx43 hemichannels in Dmdmdx heart. Importantly, inhibition of NO production prevents arrhythmias evoked by Iso. We found that NO directly activates Cx43 hemichannels by S-nitrosylation of cysteine at position 271. Our results demonstrate that opening of remodeled and S-nitrosylated Cx43 hemichannels plays a key role in the development of arrhythmias in DMD mice and that these channels may serve as therapeutic targets to prevent fatal arrhythmias in patients with DMD .

On the role of the gap junction protein Cx43 (GJA1) in human cardiac malformations with Fallot-pathology. a study on paediatric cardiac specimen.

IntroductionGap junction channels are involved in growth and differentiation. Therefore, we wanted to elucidate if the main cardiac gap junction protein connexin43 (GJA1) is altered in patients with Tetralogy of Fallot or double-outlet right ventricle of Fallot-type (62 patients referred to as Fallot) compared to other cardiac anomalies (21 patients referred to as non-Fallot). Patients were divided into three age groups: 0–2years, 2–12years and >12years. Myocardial tissue samples were collected during corrective surgery and analysis of cell morphology, GJA1- and N-cadherin (CDH2)-distribution, as well as GJA1 protein- and mRNA-expression was carried out. Moreover, GJA1-gene analysis of 16 patients and 20 healthy subjects was performed.ResultsMyocardial cell length and width were significantly increased in the oldest age group compared to the younger ones. GJA1 distribution changed significantly during maturation with the ratio of polar/lateral GJA1 increasing from 2.93±0.68 to 8.52±1.41. While in 0–2years old patients ∼6% of the lateral GJA1 was co-localised with CDH2 this decreased with age. Furthermore, the changes in cell morphology and GJA1-distribution were not due to the heart defect itself but were significantly dependent on age. Total GJA1 protein expression decreased during growing-up, whereas GJA1-mRNA remained unchanged. Sequencing of the GJA1-gene revealed only few heterozygous single nucleotide polymorphisms within the Fallot and the healthy control group.ConclusionDuring maturation significant changes in gap junction remodelling occur which might be necessary for the growing and developing heart. In our study point mutations within the Cx43-gene could not be identified as a cause of the development of TOF.

211 Evaluation Of An Enhanced (sialyl LewisX) Collagen Matrix for Neovascularization And Cardiomyogenesis in a Mouse Model Of Myocardial Infarction

In coronary ischemia and heart failure, the repair response is generally insufficient to restore blood flow and preserve cardiac function. We have previously shown that a sialyl-lewisX (sLeX)-collagen matrix could enhance endogenous vascular and myogenic repair in a model of hindlimb ischemia. In this study, we explored the use of the sLeX-collagen matrix to augment regeneration after myocardial infarction (MI). C57BL/6J mice (7-8 wk-old, n=36) underwent LAD ligation to induce MI. One week post-surgery, the mice were treated via echo-guided myocardial injections with one of the following: 1) phosphate buffered saline (PBS), 2) collagen matrix, or 3) sLeX-collagen matrix. Contractile function was assessed by echocardiography. Four weeks post-treatment, mice were sacrificed and cardiac tissue was harvested for immunohistochemistry, cytokine arrays and immunoblotting. Treatment with sLeX-collagen matrix reduced apoptosis in the heart by 33% compared to PBS (p=0.02), as measured by active caspase-3 staining. Furthermore, the infarct area of the sLeX-collagen matrix-treated group had a 1.6-fold higher number of proliferating cells, as measured by bromodeoxyuridine incorporation (p=0.008), as well as a higher number of cells per FOV that expressed the cardiac stem cell markers c-kit (2.0±0.2) and Nkx2.5 (5.2±0.4), compared to PBS (1.5±0.1 and 2.5±0.7, respectively; p≤0.02). In addition, the expression of the cardiac gap junction protein connexin43 was 1.4-fold greater in sLeX-collagen matrix-treated mice compared to the other groups (p≤0.0002). The increased expression of c-kit, Nkx2.5 and connexin43 suggests greater cardiomyogenesis in the sLeX-collagen matrix-treated hearts. Compared to baseline (1-wk post-MI), mice treated with the sLeX-collagen matrix had an improved ejection fraction (EF) of +2.5%±2% compared to the PBS group (-4.1%±1.1%; p=0.008), while the collagen-treated group had a preserved EF of +0.6%±1.9% (p=0.05). Immunostaining for arterioles showed that the vascular network was greater in both matrix-treated groups (>8.0±0.5 arterioles per FOV) compared to PBS (5.5±0.5 arterioles per FOV; p≤0.004). The sLeX-collagen matrix also reduced inflammation, as indicated by fewer CD68+ macrophages (by 23%; p=0.03) and lower levels of inflammatory cytokines (e.g. IFN-ã, TNF-α; by μ22%; p<0.05). Treatment with the sLeX-collagen matrix reduced inflammation and apoptosis and had a positive regenerative effect on the infarcted mouse heart, through improved vascular density, higher cell proliferation and possibly augmented cardiomyogenesis.

Severity of autoimmune myocarditis is influenced by Connexin43 expression (171.23)

Abstract Myocarditis, or inflammation of the heart muscle, is associated with severe morbidity and mortality, even in young, otherwise healthy individuals. The disease is initiated by exposure to viruses, parasites, and toxins, but can develop into a chronic autoimmune disease which progresses to severe fibrosis and dilated cardiomyopathy. Work in our lab using experimental autoimmune myocarditis (EAM) as a model, has demonstrated alterations in the sub-cellular localization of the cardiac gap junction protein connexin43 (Cx43) in heart tissue adjacent to myocarditis lesions, a potentially arrythmogenic effect. In the present study, we found the converse to be true; a reduction in Cx43 expression altered the course of EAM, resulting in a more pronounced disease. To generate animals with reduced Cx43, we backcrossed CX43+/- mice on the EAM-resistant C57BL/6 background to the EAM-susceptible BALB/c background. After 8 generations, we initiated EAM by two doses of α myosin heavy chain antigenic peptide at days 0 and 7. Hearts were analyzed 21 days after EAM induction for both the area and intensity of immune infiltrate. We determined that mice heterozygous for Cx43 develop more severe disease than wild type littermates. Closer analysis of infiltrating immune cells by immunofluorescence microscopy revealed differences in myeloperoxidase positive and CD3+ cells, with Cx43 heterozygotes having more of both. These results suggest that Cx43 can alter the course of EAM.

Phosphorylation of connexin43 on serine 306 regulates electrical coupling

Phosphorylation is a key regulatory event in controlling the function of the cardiac gap junction protein connexin43 (Cx43). Three new phosphorylation sites (S296, S297, S306) have been identified on Cx43; two of these sites (S297 and S306) are dephosphorylated during ischemia. The functional significance of these new sites is currently unknown. The purpose of this study was to examine the role of S296, S297, and S306 in the regulation of electrical intercellular communication. To mimic constitutive dephosphorylation, serine was mutated to alanine at the three sites and expressed in HeLa cells. Electrical coupling and single channel measurements were performed by double patch clamp. Protein expression levels were assayed by western blotting, localization of Cx43, and phosphorylation of S306 by immunolabeling. Free hemichannels were assessed by biotinylation. Macroscopic conductance in cells expressing S306A was reduced to 57% compared to wild type (WT), whereas coupling was not significantly changed in cells expressing either S296A or S297A. S306A-expressing cells displayed similar protein and free hemichannel abundance compared to WT Cx43, whereas the fractional area of plaques in cell-to-cell interfaces was increased. However, single channel measurements showed a WT Cx43 main state conductance of 119 pS, whereas the main state conductance of S306A channels was reduced to 95 pS. Furthermore, channel gating was affected in S306A channels. Lack of phosphorylation at serine 306 results in reduced coupling, which can be explained by reduced single channel conductance. We suggest that dephosphorylation of S306 partly explains the electrical uncoupling seen in myocardial ischemia.

Local effects and mechanisms of antiarrhythmic peptide AAP10 in acute regional myocardial ischemia: electrophysiological and molecular findings

Previously, it was shown that antiarrhythmic peptides and our lead substance AAP10 enhance electrical intercellular communication via gap junctions. Now, we wanted to elucidate whether AAP10 acts preferably in the ischemic area and the molecular mechanism of this peptide. Seventeen rabbit hearts were isolated, perfused according to Langendorff, and submitted to 30-min local ischemia by LAD occlusion with/without AAP10 (50 nM). Electrophysiology was assessed by 256 channel epicardial mapping. Finally, the ischemic zone, border zone, and non-ischemic zone were excised, and the cardiac gap junction protein connexin43 (Cx43), its phosphorylation state, and the distribution at the polar and lateral membrane of cardiomyocytes were determined by Western blot and immunofluorescence. Ischemia led to a decrease in activation recovery interval (ARI) homogeneity, which could be completely prevented by AAP10. Moreover, ischemia-induced activation wave slowing in the ischemic border zone was antagonized by AAP10. In ischemic center and border zone, but not in the non-ischemic area, (phospho-Cx43/dephospho-Cx43)-ratio decreased. This was also significantly antagonized by AAP10. Serine 368 was identified as one phosphorylation site for the activity of AAP10. In the non-ischemic area, AAP10 had no influence on Cx43 phosphorylation state. Interestingly, ischemia led to a loss of Cx43 from the cell poles and lateral sides in the ischemic area and border zone. AAP10 completely prevented the ischemia-induced decrease in polar Cx43 presence. In the ischemic area, AAP10 prevents from ischemia-induced Cx43 dephosphorylation and loss of Cx43 from the gap junction at cell poles and in parallel prevents the decrease in ARI homogeneity and attenuates ischemia-induced slowing of activation wave propagation. The AAP10 action seems confined to the ischemic area.

Lysosomal degradation may accelerate down-reguration of cardiac gap junction protein Connexin-43 (Cx43) via protein kinase C (PKC)-mediated hyper-phosphorylation

Lysosomal degradation may accelerate down-reguration of cardiac gap junction protein Connexin-43 (Cx43) via protein kinase C (PKC)-mediated hyper-phosphorylation

Altered connexin43 expression produces arrhythmia substrate in heart failure.

Recently, we found that repolarization heterogeneities between subepicardial and midmyocardial cells can form a substrate for reentrant ventricular arrhythmias in failing myocardium. We hypothesized that the mechanism responsible for maintaining transmural action potential duration heterogeneities in heart failure is related to intercellular uncoupling from downregulation of cardiac gap junction protein connexin43 (Cx43). With the use of the canine model of pacing-induced heart failure, left ventricles were sectioned to expose the transmural surface (n = 5). To determine whether heterogeneous Cx43 expression influenced electrophysiological function, high-resolution transmural optical mapping of the arterially perfused canine wedge preparation was used to measure conduction velocity (theta(TM)), effective transmural space constant (lambda(TM)), and transmural gradients of action potential duration (APD). Absolute Cx43 expression in failing myocardium, quantified by confocal immunofluorescence, was uniformly reduced (by 40 +/- 3%, P < 0.01) compared with control. Relative Cx43 expression was heterogeneously distributed and lower (by 32 +/- 18%, P < 0.05) in the subepicardium compared with deeper layers. Reduced Cx43 expression in heart failure was associated with significant reductions in intercellular coupling between transmural muscle layers, as evidenced by reduced theta(TM) (by 18.9 +/- 4.9%) and lambda(TM) (by 17.2 +/- 1.4%; P < 0.01) compared with control. Heterogeneous transmural distribution of Cx43 in failing myocardium was associated with lower subepicardial theta(TM) (by 12 +/- 10%) and lambda(TM) (by 13 +/- 7%), compared with deeper transmural layers (P < 0.05). APD dispersion was greatest in failing myocardium, and the largest transmural APD gradients were consistently found in regions exhibiting lowest relative Cx43 expression. These data demonstrate that reduced Cx43 expression produces uncoupling between transmural muscle layers leading to slowed conduction and marked dispersion of repolarization between epicardial and deeper myocardial layers. Therefore, Cx43 expression patterns can potentially contribute to an arrhythmic substrate in failing myocardium.

Structural bases for the chemical regulation of Connexin43 channels

Connexins proteins associate with a variety of catalytic and non-catalytic molecules. Also, different domains of connexin can bind to each other, providing a mechanism for channel regulation. Here, we review some of these associations, placing particular emphasis on the intramolecular interactions that regulate Connexin43 (Cx43). We also describe some novel methods that allow for the characterization of protein-protein interactions such as those observed in the cardiac gap junction protein Connexin43. Overall, intra- and inter-molecular interactions may regulate gap junctions to filter the passage of molecular messages between cells at the appropriate time and between the appropriate cells. As a potential area for future investigations, we also speculate as to whether some of the inter-molecular interactions involving connexins lead to modifications in the function of the associated protein, rather than on the function of connexin itself.

The organization of adherens junctions and desmosomes at the cardiac intercalated disc is independent of gap junctions.

Adherens junctions and desmosomes are responsible for mechanically coupling myocytes in the heart and are found closely apposed to gap junction plaques at the intercalated discs of cardiomyocytes. It is not known whether loss of cardiac gap junctions, such as described in cardiac disease states, may influence the expression patterns of other intercalated disc-associated proteins. We investigated whether the major cardiac gap junction protein connexin43 (Cx43) may be responsible for regulating adherens junctions, desmosomes and their associated catenins, in terms of abundance and localization at the intercalated discs of cardiomyocytes. In order to study the effect of loss of cardiac gap junctions on the intercalated disc-associated proteins, we used a combination of immunoblotting, immunofluorescence with confocal microscopy and electron microscopy to evaluate heart tissue from mice with cardiac-specific conditional knockout of Cx43. We found that the cardiac adherens junctions, desmosomes and their associated catenins, as well as vinculin and ZO-1, maintain their normal abundance, structural appearance and localization in the absence of Cx43. We conclude from these data that Cx43 is not required for the organization of the cell adhesion junctions and their associated catenins at the intercalated disc in the adult cardiac myocyte.

Discontinuous conduction in the heart

Preface Section I: Normal Cardiac Conduction: Introduction 1. Discontinuous Cardiac Conduction: Its Origin in Cellular Connectivity With Long-Term Adaptive Changes That Cause Arrhythmias 2. Gap Junction Channels and Cardiac Conduction 3. Nonjunctional Channels of Cardiac Cells and Metabolic Oscillations as Contributors to Discontinuous Conduction Section II: Discontinuous Conduction and Arrhythmias: Introduction: 4. Gap Junction Structure, Conduction, and Arrhythmogenesis: Directions for Future Research 5. Role of Discontinuous Electrical Propagation in Clinical Atrial Arrhythmias: Current Evidence and Future Directions 6. Role of Discontinuous Conduction/Nonuniform Anisotropy in Clinical Arrhythmias Section III: Gating and Modulation of Gap Junctions: Introduction: 7. Structure of Cardiac Gap Junction Membrane Channels: Progress Toward a Higher-Resolution Model 8. Major Cell Biological Issues and Strategies in Discontinuous Conduction 9. Molecular Analysis of the pH Regulation of the Cardiac Gap Junction Protein Connexin43 10. Triggered Propagated Contractions and Arrhythmias Caused by Acute Damage to Cardiac Muscle 11. Microscopic Conduction in Cell Cultures Assessed by High-Resolution Optical Mapping and Computer Simulation Section IV: Waves and Arrhythmias: Introduction: 12. Scale of Geometric Structures Responsible for Discontinuous Propagation in Myocardial Tissue 13. Wave-Front Curvature Leads to Slow Conduction and Block in Two-Dimensional Cardiac Muscle 14. Antiarrhythmic and Proarrhythmic Mechanisms in Cardiac Tissue: Linking Spiral Waves, Reentrant Arrhythmias, and Electrocardiographic Patterns Section V: Junctional Channels and Molecular Genetics: Introduction: 15. Molecular and Biochemical Regulation of Connexins: Potential Targets for Modulation of Cardiac Intercellular Communication 16. Can We Learn About Conduction by Use of Genetic Approaches? Section VI: Discontinuous Conduction in Myocardial Ischemia: Introduction: 17. Characteristics and Causes of Conduction Changes Associated With 1a and 1b Arrhythmias in Acute Ischemia 18. Discontinuous Conduction in the Epicardial Border Zone of Infarcted Hearts and Its Role in Anisotropic Reentry 19. Is There a Pharmacology for Discontinuous Conduction? Section VII: Contemporary Model Studies of Discontinuous Conduction: Introduction: 20. Discontinous Conduction in the AV Junctional Area 21. Experimental Simulations of Variations in Junctional Conductance 22. Membrane Factors and Gap Junction Factors as Determinants of Ventricular Conduction and Reentry Perspectives and Future Directions: Arrhythmias and Discontinuous Conduction Index

Effects of angiotensin II on expression of the gap junction channel protein connexin43 in neonatal rat ventricular myocytes.

To elucidate signal transduction pathways regulating expression of myocardial gap junction channel proteins (connexins) and to determine whether mediators of cardiac hypertrophy might promote remodeling of gap junctions, we characterized the effects of angiotensin II on expression of the major cardiac gap junction protein connexin43 (Cx43) in cultured neonatal rat ventricular myocytes. Remodeling of the distribution of myocardial gap junctions appears to be an important feature of anatomic substrates of ventricular arrhythmias in patients with heart disease. Remodeling of intercellular connections may be initiated by changes in connexin expression caused by chemical mediators of the hypertrophic response. Cultures were exposed to 0.1 micromol/liter angiotensin II for 6 or 24 h, and Cx43 expression was characterized by immunoblotting, confocal microscopy and electron microscopy. Immunoblot analysis revealed a twofold increase in Cx43 content in cells treated for 24 h with angiotensin II (n=4, p < 0.05). This response was inhibited by the presence of 1.0 micromol/liter losartan, an AT1-receptor blocker. Confocal and electron microscopy demonstrated enhanced Cx43 immunoreactivity and increases in the number and size of gap junction profiles in cells exposed to angiotensin II for 24 h. These effects were also blocked by losartan. Immunoprecipitation of Cx43 from cells metabolically labeled with [35S]methionine demonstrated 2.4- and 2.9-fold increases in Cx43 radioactivity after 6 and 24 h exposure to angiotensin II, respectively (p < 0.03 at each time point). Angiotensin II up-regulates gap junctions in cultured neonatal rat ventricular myocytes by increasing Cx43 synthesis. Signal transduction pathways activated by angiotensin II under pathophysiologic conditions could initiate remodeling of conduction pathways, leading to the development of anatomic substrates of arrhythmias.

Intramolecular interactions mediate pH regulation of connexin43 channels

We have previously proposed that acidification-induced regulation of the cardiac gap junction protein connexin43 (Cx43) may be modeled as a particle-receptor interaction between two separate domains of Cx43: the carboxyl terminal (acting as a particle), and a region including histidine 95 (acting as a receptor). Accordingly, intracellular acidification would lead to particle-receptor binding, thus closing the channel. A premise of the model is that the particle can bind its receptor, even if the particle is not covalently bound to the rest of the protein. The latter hypothesis was tested in antisense-injected Xenopus oocyte pairs coexpressing mRNA for a pH-insensitive Cx43 mutant truncated at amino acid 257 (i.e., M257) and mRNA coding for the carboxyl terminal region (residues 259-382). Intracellular pH (pHo) was recorded using the dextran form of the proton-sensitive dye seminaphthorhodafluor (SNARF). Junctional conductance (Gj) was measured with the dual voltage clamp technique. Wild-type Cx43 channels showed their characteristic pH sensitivity. M257 channels were not pH sensitive (pHo tested: 7.2 to 6.4). However, pH sensitivity was restored when the pH-insensitive channel (M257) was coexpressed with mRNA coding for the carboxyl terminal. Furthermore, coexpression of the carboxyl terminal of Cx43 enhanced the pH sensitivity of an otherwise less pH-sensitive connexin (Cx32). These data are consistent with a model of intramolecular interactions in which the carboxyl terminal acts as an independent domain that, under the appropriate conditions, binds to a separate region of the protein and closes the channel. These interactions may be direct (as in the ball-and-chain mechanism of voltage-dependent gating of potassium channels) or mediated through an intermediary molecule. The data further suggest that the region of Cx43 that acts as a receptor for the particle is conserved among connexins. A similar molecular mechanism may mediate chemical regulation of other channel proteins.

Role of histidine 95 on pH gating of the cardiac gap junction protein connexin43.

We have studied the role of histidine 95 (H95) on the pH gating of the cardiac gap junction protein connexin43 (Cx43). Wild-type and mutant rat cardiac Cx43 channels were expressed in antisense-injected Xenopus oocytes. Junctional conductance was measured using the dual voltage-clamp technique, and intracellular acidification was induced by superfusion with a sodium acetate-containing solution balanced at a pH of 6.2. H95 was substituted by other amino acids by use of oligonucleotide-directed site-specific mutagenesis. Replacing H95 for the hydrophobic residues methionine or phenylalanine, for the charged basic residue arginine, or for the noncharged residue glutamine (H95Q) yielded nonfunctional channels. Functional expression of H95Q was rescued by placing a histidine residue in position 93 (H95Q-L93H), 94 (H95Q-A94H), or 97 (H95Q-F97H) but not in position 96. Further experiments showed that replacing H95 with either aspartate (an acidic residue) or tyrosine (a polar uncharged residue) led to the expression of functional channels with a reduced susceptibility to acidification-induced uncoupling, whereas lysine (a basic residue) was more susceptible to uncoupling than the wild-type protein. The susceptibility to acidification-induced uncoupling was enhanced for the H95Q-A94H mutant when compared with the wild-type mutant, but it was significantly reduced when histidine was placed at position 93 (H95Q-L93H). Our data indicate that a properly placed histidine residue is an important structural element for functional expression as well as for pH regulation of Cx43. The results suggest that the importance of H95 on pH gating may be associated with a possible protonation of this residue on acidification of the intracellular environment.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative analysis of intercellular connections by immunohistochemistry of the cardiac gap junction protein connexin43.

Polyclonal antisera directed against epitopes in the cytoplasmic domain of rat connexin43, the predominant cardiac gap junction protein, were used to delineate immunohistochemically the distribution of gap junctions in sections of canine left ventricle. Antigen-antibody binding and tissue structure were preserved after paraformaldehyde fixation and paraffin embedment of canine myocardium. Specific binding of antibody to the cytoplasmic surfaces of ultrastructurally identified gap junctions was confirmed with electron microscopy. Light microscopic morphometric analysis of immunostained sections in five separate experiments revealed a mean gap junction surface density of 0.0052 micron2/micron3 myocyte volume, which is consistent with previously reported values determined by use of quantitative electron microscopy. This new method permits quantitative determinations of gap junction surface density and distribution in relatively large heterogeneous areas of myocardium in which ultrastructural morphometry would be impractical. This approach should facilitate analysis of the relation between potential alterations in electrical coupling of myocytes and abnormalities of myocardial conduction occurring at the macroscopic scale in regions such as structurally heterogeneous infarct border zones.