Gap Junction Membrane Channel Research Articles

The role of pituitary gonadotropins and intraovarian regulators in follicle development: A mini-review.

BackgroundThe process of follicle development is tightly regulated by pituitary gonadotropins (follicle‐stimulating hormone [FSH] and luteinizing hormone [LH]) and intraovarian regulators (eg, steroids, growth factors, and cytokines).MethodsThis review outlines recent findings on the mechanisms of human follicle development, based on the research on animal models such as mice, rats, cows, and sheep.Main findingsPhosphatidylinositol 3‐kinase/protein kinase B signaling pathway and anti‐Müllerian hormone are involved in primordial follicle activation during the gonadotropin‐independent phase. The intraovarian regulators, such as androgen, insulin‐like growth factor system, activin, oocyte‐derived factors (growth differentiation factor‐9 and bone morphogenetic protein 15), and gap junction membrane channel protein (connexin), play a central role in the acquisition of FSH dependence in preantral follicles during the gonadotropin‐responsive phase. Antral follicle development can be divided into FSH‐dependent growth and LH‐dependent maturation. The indispensable tetralogy for follicle selection and final maturation of antral follicles involves (a) acquisition of LH dependence, (b) greater capacity for E2 production, (c) activation of the IGF system, and (d) an antiapoptotic follicular microenvironment.ConclusionWe reproductive endocrinologists should accumulate further knowledge from animal model studies to develop methods that promote early folliculogenesis and connect to subsequent gonadotropin therapy in infertile women.

Connexin40 nonsense mutation in familial atrial fibrillation

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia associated with substantial morbidity and mortality. Genetic variants play important roles in the pathogenesis of AF. However, AF is a genetically heterogeneous disorder, and the genetic determinants in most patients with AF remain to be identified. In this study, the entire coding region of the connexin40 gene, encoding the cardiac gap junction membrane channel protein alpha5, was sequenced in 126 unrelated probands with familial AF. A novel heterozygous mutation, c.145C<T, in connexin40, was identified in a proband. The mutation was predicted to introduce a premature stop codon at amino acid position 49 (p.Q49X). This nonsense mutation was present in all the living relatives of the mutation carrier, co-segregating with AF in the family with a penetrance of 100%. However, it was absent in 200 ethnically matched unrelated control individuals. The findings suggest a pathogenic link between the compromised connexin40 function and familial AF, hence providing new insight into the molecular mechanisms involved in AF.

Microarray analysis of perichondral and reserve growth plate zones identifies differential gene expressions and signal pathways

In the growth plate, the reserve and perichondral zones have been hypothesized to have similar functions, but their exact functions are poorly understood. Our hypothesis was that significant differential gene expression exists between perichondral and reserve chondrocytes that may differentiate the respective functions of these two zones. Normal Sprague-Dawley rat growth plate chondrocytes from the perichondral zone (PC) and reserve zone (RZ) were isolated by laser microdissection and then subjected to microarray analysis. In order to most comprehensively capture the unique features of the two zones, we analyzed both the most highly expressed genes and those that were most significantly different from the proliferative zone (PZ) as a single comparator. Confirmation of the differential expression of selected genes was done by quantitative real-time RT-PCR. A total of 8 transcripts showing high expression unique to the PC included translationally-controlled tumor protein (Tpt1), connective tissue growth factor (Ctgf), mortality factor 4 (Morf4l1), growth arrest specific 6 (Gas6), type V procollagen (Col5a2), frizzled-related protein (Frzb), GDP-dissociation inhibitor 2 (Gdi2) and Jun D proto-oncogene (Jund). In contrast, 8 transcripts showing unique high expression in the RZ included hyaluronan and proteoglycan link protein 1 (Hapln1), hemoglobin beta-2 subunit, type I procollagen (Col1a2), retinoblastoma binding protein 4 (LOC685491), Sparc-related modular calcium binding 2 (Smoc2), and calpastatin (Cast). Other genes were highly expressed in cells from both PC and RZ zones, including collagen II, collagen IX, catenin (cadherin associated protein) beta 1, eukaryotic translation elongation factor, high mobility group, ribosomal protein, microtubule-associated protein, reticulocalbin, thrombospondin, retinoblastoma binding protein, carboxypeptidase E, carnitine palmitoyltransferase 1, cysteine rich glycoprotein, plexin B2 (Plxnb2), and gap junction membrane channel protein. Functional classification of the most highly expressed transcripts were analyzed, and the pathway analysis indicated that ossification, bone remodeling, and cartilage development were uniquely enriched in the PC whereas both the PC and RZ showed pathway enrichment for skeletal development, extracellular matrix structural constituent, proteinaceous extracellular matrix, collagen, extracellular matrix, and extracellular matrix part pathways. We conclude that differential gene expression exists between the RZ and PC chondrocytes and these differentially expressed genes have unique roles to play corresponding to the function of their respective zones.

Stem cell protein Piwil2 modulates expression of murine spermatogonial stem cell expressed genes

The piwi family genes are highly conserved during evolution and play essential roles in stem cell self-renewal, gametogenesis, and RNA interference in diverse organisms ranging from Arabidopsis to human. Piwil2, known also as Mili gene, is one of three mouse homologues of piwi. Piwil2 was found in germ cells of adult testis, suggesting that this gene functions in spermatogonial stem cell self-renewal. In order to find molecular mechanisms underlying stem cell activity mediated by Piwil2 gene, an in vitro gain of function cell culture model was established. Messenger RNAs isolated from cells expressing Piwil2 and mRNAs isolated from cells without Piwil2 expression were compared using a stem cell array technique. It was shown that Piwil2 modulates expression of stem cell specific genes, including platelet-derived growth factor receptor, beta polypeptide (Pdgfrb), solute carrier family 2 member 1 (Slc2a1), gap junction membrane channel protein alpha 7 (Gja7), and spermatogonial cell surface markers Thy-1 (CD90), integrin alpha 6 (Itga6), CD9, and spermatogonia specific markers heat shock protein 90 alpha (Hsp90a), and stimulated by retinoic acid gene 8 (Stra8). These molecules play essential role in stem cells proliferation (Pdgfrb), energy metabolism (Slc2a1), cell adhesion, cell-cell interaction (Itga6, Gja7, Thy-1, and CD9), and germ cell differentiation (Stra8). The expression of these markers in spermatogonial stem cells and other nongerminal stem cells suggests that these cells share elements of common molecular machinery with stem cells in other tissues which are modulated by stem cell protein Piwil2.

Modulation of GJA1 Turnover and Intercellular Communication by Proinflammatory Cytokines in the Anterior Pituitary Folliculostellate Cell Line TtT/GF1

Our previous studies have advanced the idea that the folliculostellate cell GJA1 (gap junction membrane channel protein alpha1; previously known as connexin 43)-mediated gap junctions contribute to the establishment of an intercellular network that regulates the paracrine messages and the endocrine response within the anterior pituitary. The folliculostellate cells are targets for growth factors and cytokines that modulate hormone secretion. Proinflammatory cytokines modulate the cell-to-cell communication in many tissues of the body. The present study measured the effect of the proinflammatory cytokines tumor necrosis factor and interleukin-1 on the GJA1-mediated intercellular communication, specifically the expression, localization, degradation, and phosphorylation status of GJA1 in the folliculostellate cell line TtT/GF. The GJA1 localized to the plasma membrane and to minute cytoplasmic vesicles in the perinuclear area. Using different antibodies that recognize distinctly the nonphosphorylated from the phosphorylated forms of GJA1, we showed that nonphosphorylated GJA1 in Ser-368 (NP-GJA1) localized chiefly in the cytoplasm, whereas GJA1 phosphorylated in Ser-368 (P-GJA1) localized to the plasma membrane in controls. The cytokine treatment transiently increased 1) GJA1, NP-GJA1, and P-GJA1 levels; 2) NP-GJA1 and P-GJA1 degradation by both the lysosomal and proteasomal pathways; and 3) cell-to-cell communication in TtT/GF cells. The results suggest that the cytokine-evoked, transient enhancement of folliculostellate cell-mediated intercellular communication contributes to the coordination of the response among folliculostellate cells.

The Influence Of Physical Activity And Exercise On Physiological Markers And Genetic Determinants Of Blood Pressure

The interaction of genetics and environment play a major role in health and disease. Physical activity Influences the expression of specific sets of gene that regulate critical physiological functions. Modeling experiments to investigate gene-environment interaction in a laboratory setting would provide the opportunity to gain mechanistic insight under controlled conditions. In laboratory research it is unclear if the gene-environment interaction of a sedentary lifestyle predisposes laboratory animals to hypokinetic diseases such as hypertension. PURPOSE We sought to compare gene expression and physiological markers of hypertension in animals with: i. A sedentary lifestyle in a standard cage (SED); ii. access to twice-weekly physical activity in a large box outside a standard cage (PA); and iii. access to regular exercise in cages with running wheels (EX). METHODS The GeneChip® Rat Genome 230 2.0 Array was used for comprehensive screening of the transcribed rat genome on a single array. Here, we report results related to genes directly associated with hypertension. Blood pressure was measured by a tail-cuff sphygmomanometer. RESULTS Systolic blood pressure (SBP) was highest in the SED (140.6±3.1 mmHg) vs. EX (129.8±2.6 mmHg, F 1,28 =6.33, p < 0.05). Mean SBP and mean gene expressions showed the following significant correlations: SBP and farnesyl diphosphate farnesyl transferase 1 (Fdft1), r =0.95;SBP and very low-density lipoprotein receptor (Vldlr) r =0.52; and SBP and gap junction membrane channel protein alpha 1 (Gja1), r =0.36, p < 0.05. Fdft1, a regulator of lipid biosynthesis, was highest in SED followed by PA and EX (5713±101 vs. 5527±385 vs. 5097±145). Vldlr, a regular of cholesterol metabolism and lipid transport, was lower in SED vs PA (6899±144 vs. 7782±333, p < 0.05). Gja1, a regulator of cell signaling and heart rate, was lower in SED compared with EX (324±62 vs. 366.7±75, p < 0.05). CONCLUSIONS These results indicate that a sedentary environment can induce hypertension and that certain blood pressure-regulating genes are expressed differently in SED compared to animals with access to either PA or EX. Supported by NIH Grant 1 R15 AG 20526–01A1.

The Influence Of Physical Activity And Exercise On Physiological Markers And Genetic Determinants Of Blood Pressure

The interaction of genetics and environment play a major role in health and disease. Physical activity Influences the expression of specific sets of gene that regulate critical physiological functions. Modeling experiments to investigate gene-environment interaction in a laboratory setting would provide the opportunity to gain mechanistic insight under controlled conditions. In laboratory research it is unclear if the gene-environment interaction of a sedentary lifestyle predisposes laboratory animals to hypokinetic diseases such as hypertension. PURPOSE We sought to compare gene expression and physiological markers of hypertension in animals with: i. A sedentary lifestyle in a standard cage (SED); ii. access to twice-weekly physical activity in a large box outside a standard cage (PA); and iii. access to regular exercise in cages with running wheels (EX). METHODS The GeneChip® Rat Genome 230 2.0 Array was used for comprehensive screening of the transcribed rat genome on a single array. Here, we report results related to genes directly associated with hypertension. Blood pressure was measured by a tail-cuff sphygmomanometer. RESULTS Systolic blood pressure (SBP) was highest in the SED (140.6±3.1 mmHg) vs. EX (129.8±2.6 mmHg, F 1,28 =6.33, p < 0.05). Mean SBP and mean gene expressions showed the following significant correlations: SBP and farnesyl diphosphate farnesyl transferase 1 (Fdft1), r =0.95;SBP and very low-density lipoprotein receptor (Vldlr) r =0.52; and SBP and gap junction membrane channel protein alpha 1 (Gja1), r =0.36, p < 0.05. Fdft1, a regulator of lipid biosynthesis, was highest in SED followed by PA and EX (5713±101 vs. 5527±385 vs. 5097±145). Vldlr, a regular of cholesterol metabolism and lipid transport, was lower in SED vs PA (6899±144 vs. 7782±333, p < 0.05). Gja1, a regulator of cell signaling and heart rate, was lower in SED compared with EX (324±62 vs. 366.7±75, p < 0.05). CONCLUSIONS These results indicate that a sedentary environment can induce hypertension and that certain blood pressure-regulating genes are expressed differently in SED compared to animals with access to either PA or EX. Supported by NIH Grant 1 R15 AG 20526–01A1.

Gene expression profile in rat renal isografts from brain dead donors

Background Brain death (BD) and the subsequent ischemia/reperfusion (I/R) injury have cardinal implications for the pathogenesis of kidney transplantation (Tx). However, the precise mechanistic pathway of BD and the subsequent I/R injury are unknown. In this study, we performed genome-wide analysis for differential gene expression in kidney isografts from BD donors. Their gene expressions were compared with those from living sources. Methods Kidneys from BD rats were engrafted and their gene expressions were compared with those from living controls. Donors were intubated, and mechanically ventilated for 6 hours. Grafts were harvested 6 hours after BD, and 1 hour after engraftment. The expression profile of approximately 20,500 genes was analyzed. Results Gene expression of chemokines (Scya2 and Gro1), cytokines (IL-1 and -6) and adhesion molecules (E- and P-selectin and ICAM-1) were upregulated in the BD kidneys and 1 hour after engraftment. An antiapoptotic gene (Birc2), IκB-ζ, and protective gene (HO-1) were also upregulated. Other upregulated genes included oncogenes (lipocalin2, Bcl3, and CCAAT/enhancer binding protein δ), Calgranulin B, DEXRAS1, insulin-like growth factor binding protein-1, inhibin β-B-subunit gene, IgG Fc receptor, and FK 506 binding protein 5. We also observed downregulation of the genes Amphiphsin, Jagged 1, Pace 4, Slc15a2, Kcnn2, and gap junction membrane channel protein α5 only in kidneys from BD donors. Conclusions This is the first demonstration of global gene expression analysis using the rat brain-death isograft model. These results provide new insights for the detection of novel target genes for treatment and prognosis of grafts from brain-dead and extended marginal donors.

Genetic basis for chamber-specific ventricular phenotypes in the rat infarct model.

We, and others, have previously reported a strong correlation between increased inter-ventricular dispersion of repolarization and the occurrence of fatal arrhythmia in animal models of CHF. The existence of this and other such distinct electrophysiologic phenotypes in right (RV) vs. left ventricles (LV) could be explained by chamber-specific patterns of gene expression. We employed microarray gene profiling of 13824 sequence-verified, nonredundant rodent cDNAs to compare myocardial gene expression in RV vs. LV of rats with surgically induced myocardial infarction (MI: n=3) and in sham-operated animals (Sham: n=3). Significant LV infarction (32+/-4% LV) and severe CHF were observed in all MI animals at 4 weeks. In Sham animals, 937 genes exhibited significant differential expression in RV vs. LV myocardium. In MI animals, 1158 genes exhibited significant differential expression in RV vs. LV. Of those genes exhibiting significant differential expression, only 241 were common to both Sham and MI animals. Differentially expressed genes included those involved in signal transduction, cell growth and maintenance, and apoptosis. Genes with potential roles in altered dispersion of repolarization included voltage-dependent Ca(2+) channel gamma subunit (MI 8-fold increasing) and K(+) inwardly rectifying channel subfamily J, member 10 (MI 6-fold decreasing). Gap junction membrane channel protein alpha 4 (MI 6-fold decreasing) and cardiac troponin I (MI 8-fold decreasing) were also significantly differentially expressed. Inter-ventricular comparisons revealed significantly greater alterations in gene expression vs. intra-ventricular comparisons. Microarray gene profiling has revealed candidate genes, some of them novel, which may account for chamber-specific ventricular electrophysiologic phenotypes, both in physiologic as well as in arrhythmogenic states such as CHF.

Isolation and characterization of gap junctions from tissue culture cells

The purification of membrane proteins in a form and amount suitable for structural or biochemical studies still remains a great challenge. Gap junctions have long been studied using electron microscopy and X-ray diffraction. However, only a limited number of proteins in the connexin family have been amenable to protein or membrane purification techniques. Molecular biology techniques for expressing large gap junctions in tissue culture cells combined with improvements in electron crystallography have shown great promise for determining the channel structure to better than 10 Å resolution. Here, we have isolated two-dimensional (2D) gap junction crystals from HeLa Cx26 transfectants. This isoform has never been isolated in large fractions from tissues. We characterize these preparations by SDS-PAGE, Western blotting, negative stain electron microscopy and atomic force microscopy. In our preparations, the Cx26 is easily detected in the Western blots and we have increased expression levels so that connexin bands are visible on SDS-PAGE gels. Preliminary assessment of the samples by electron cryo-microscopy shows that these 2D crystals diffract to at least ∼22 Å. Atomic force microscopy of these Cx26 gap junctions show exquisite surface modulation at the extracellular surface in force dissected gap junctions. We also applied our protocol to cell lines such as NRK cells that express endogenous Cx43 and NRK and HeLa cell lines transfected with exogenous connexins. While the gap junction membrane channels are recognizable in negatively stained electron micrographs, these lattices are disordered and the gap junction plaques are smaller. SDS-PAGE and Western blotting revealed expression of connexins, but at a lower level than with our HeLa Cx26 transfectants. Therefore, the purity and morphology of the gap junction plaques depends the size and abundance of the gap junctions in the cell line itself.

Transmembrane α‐helices in the gap junction membrane channel: Systematic search of packing models based on the pair potential function

Transmembrane α‐helices in the gap junction membrane channel: Systematic search of packing models based on the pair potential function

Transmembrane alpha-helices in the gap junction membrane channel: systematic search of packing models based on the pair potential function.

Recent progress in the field of electron cryo-microscopy and image analysis has shown that there is an overwhelming need to interpret medium resolution (5 to 10 A) three-dimensional maps. Traditional methods of fitting amino acid residues into electron density using molecular modeling programs must be supplemented with further analysis. We have used a potential of mean force (PMF) method, derived from Boltzmann statistics in protein structure, to generate models for the packing of alpha-helices, using pairwise potentials between amino acid residues. The approach was tested using the three-dimensional map of a recombinant cardiac gap junction membrane channel provided by electron cryo-crystallography (Unger et al., 1997; 1999a, 1999b) which had a resolution of 7.5 A in the membrane plane and 21 A in the vertical direction. The dodecameric channel was formed by the end-to-end docking of two hexamers, each of which displayed 24 rods of density in the membrane interior, which was consistent with an alpha-helical conformation for the four transmembrane domains of each connexin subunit. Based on the three-dimensional map and the amino acid sequence for the 4 transmembrane domains determined by hydropathy analysis, we used the modeling utility SymServ (Macke et al., 1998) to build hexameric connexons with 24 transmembrane alpha-helices. Canonical alpha-helices were aligned to the axes of the rods of density and translated along the density so that the center of masses coincided. The PMF function was used to evaluate 162,000 conformations for each of the 24 possible alpha-helical packing models. Since the different packing models yielded different energy distributions, the pair potential function appears to be a promising tool for evaluating the packing of alpha-helices in membrane proteins. The analysis will be refined by energy calculations based on the expectations that the outer boundary of the channel will be formed by hydrophobic residues in contact with the lipids.

Purification of gap junctions.

The descriptive term gap junction originally arose from the electron micrograph studies of Revel and Karnovsky in 1967 (). This morphological name referred to a cell-cell contact area where two cell membranes were bridged by a specialized membrane protein complex. Gap junctions are found in almost all tissues where cells abut each other. The “gap” is a distinct morphological feature whereby heavy metal stains are able to intercalate into a space between the two joined cell membranes, as opposed to the situation in tight junctions, where the membranes fuse and the stain cannot penetrate in between the cells. Subsequent electron microscopy revealed that gap junctions consist of tens to thousands of membrane channels (also called intercellular channels) containing proteins generally called connexins. Each membrane channel was shown to contain two connexons, the hexamer of connexins, which dock at their extracellular surfaces. In essence, the gap junction membrane channel is a dimer of two hexamers joined together in the gap region. In this way, the membrane channel extends across both cell membranes. Both freeze-fracture and thin section electron micrographs showed that these gap junctional plaques contained quasicrystalline areas of the membrane channels. During the next decade, intensive efforts by many researchers were directed toward purifying the gap junction plaques first seen in these micrographs for both biochemical and structural analysis. Many man-years were spent developing and refining isolation protocols that reliably gave pure preparations of gap junctions, which were used for structure determination by electron crystallography () and X-ray diffraction (). The specimens that are considered most desirable for electron crystallography are thin, uniform two-dimensional crystals. Early on, gap junctions were looked on as a tantalizing specimen, not only because of their biological importance, but also because they form quasicrystals in the cell and with limited detergent treatments could be induced into well-ordered twodimensional crystals (). It is clear from the structural work that has been done to date that the critical step in the structure determination is the isolation of crystalline material in sufficient and pure quantities (a problem that structural biologists also refer to as “no crystal, no grant”). In liver, the gap junction arrays are estimated to cover only ~0.1% of the surface area of a hepatocyte, therefore, one generally begins with large amounts of starting material to end with a small quantity of pure gap junctions (, , ). Liver, lens and heart tissue (, , ) have classically been the organs of choice for isolating gap junctions because the majority of the tissue contains only one or two types of connexins and starting material can be obtained in large quantities. The situation has improved with the advent of molecular biology techniques for expression of the connexins in tissue culture systems such as the baby hamster kidney (BHK) cell line () and the insect cell line derived from Spodoptera (,). In these cell lines, the gap junctions can be overexpressed, isolated and purified with larger yields than from native tissues. Recent advances in the three-dimensional structure determination at ~7Å have occurred because of the development of overexpression systems and generation of recombinant connexin material (see ref. and Chapter 4 in this volume) as well as improved electron crystallographic structure determination methods. These EM crystallographic methods have been greatly enhanced by both improvements in electron microscopes as well as in computer reconstruction techniques (see Chapter 4 and also ref. ).

Expression, Two-Dimensional Crystallization, and Electron Cryo-crystallography of Recombinant Gap Junction Membrane Channels

We used electron cryo-microscopy and image analysis to examine frozen-hydrated, two-dimensional (2D) crystals of a recombinant, 30-kDa C-terminal truncation mutant of the cardiac gap junction channel formed by 43-kDa α 1 connexin. To our knowledge this is the first example of a structural analysis of a membrane protein that has been accomplished using microgram amounts of starting material. The recombinant α 1 connexin was expressed in a stably transfected line of baby hamster kidney cells and spontaneously assembled gap junction plaques. Detergent treatment with Tween 20 and 1,2-diheptanoyl- sn-phosphocholine resulted in well-ordered 2D crystals. A three-dimensional density (3D) map with an in-plane resolution of ∼7.5 Å revealed that each hexameric connexon was formed by 24 closely packed rods of density, consistent with an α-helical conformation for the four transmembrane domains of each connexin subunit. In the extracellular gap the aqueous channel was bounded by a continuous wall of protein that formed a tight electrical and chemical seal to exclude exchange of substances with the extracellular milieu.

Three-dimensional structure of a recombinant gap junction membrane channel.

Gap junction membrane channels mediate electrical and metabolic coupling between adjacent cells. The structure of a recombinant cardiac gap junction channel was determined by electron crystallography at resolutions of 7.5 angstroms in the membrane plane and 21 angstroms in the vertical direction. The dodecameric channel was formed by the end-to-end docking of two hexamers, each of which displayed 24 rods of density in the membrane interior, which is consistent with an alpha-helical conformation for the four transmembrane domains of each connexin subunit. The transmembrane alpha-helical rods contrasted with the double-layered appearance of the extracellular domains. Although not indicative for a particular type of secondary structure, the protein density that formed the extracellular vestibule provided a tight seal to exclude the exchange of substances with the extracellular milieu.

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

Synthesis, assembly and structure of gap junction intercellular channels

Gap junction membrane channels assemble as dodecameric complexes, in which a hexameric hemichannel (connexon) in one plasma membrane docks end to end with a connexon in the membrane of a closely apposed cell. Steps in the synthesis, assembly and turnover of gap junction channels appear to follow the general secretory pathway for membrane proteins. In addition to homo-oligomeric connexons, different connexin polypeptide subunits can also assemble as hetero-oligomers. The ability to form homotypic and heterotypic channels that consist of two identical or two different connexons, respectively, adds even greater versatility to the functional modulation of gap junction channels. Electron cryocrystallography of recombinant gap junction channels has recently provided direct evidence for α-helical folding of at least two of the transmembrane domains within each connexin subunit. The potential to correlate the structure and biochemistry of gap junction channels with recently identified human diseases involving connexin mutations makes this a particularly exciting area of research.

Structure of a Recombinant Gap Junction Channel at 7Å Resolution

Gap junction membrane channels mediate the electrical and metabolic coupling between cells. The channels are formed by the end-to-end docking of two hemichannels (connexons), each of which is formed by a hexameric cluster of protein subunits (connexins). The principal gap junction protein in the heart, α1 connexin (also designated Cx43), mediates action potential propagation between cells in order to synchronize cardiac contraction.We recently utilized electron cryo-microscopy and image analysis to examine frozen-hydrated two-dimensional (2D) crystals of a recombinant, truncated α1- connexin (α1Cx263T). The projection map at 7Å resolution revealed that each 30kD connexin subunit has a transmembrane α-helix that lines the aqueous pore and a second α-helix in close contact with the membrane lipids [Nature Struct. Biol. 4: 39-43 (1997)]. The distribution of densities allowed us to propose a model in which the two apposing connexons that form the channel are staggered by ∼30°. Furthermore, apparent non-crystallographic twofold axes predicted that the two apposing connexons adopt identical conformations.

Formation of the gap junction intercellular channel requires a 30° rotation for interdigitating two apposing connexons

Intercellular communication via gap junction membrane channels cannot occur until two apposing hemichannels (connexons) meet and dock to form a sealed cell-cell conduit. In particular, an important question is how does the structure at the extracellular surface influence the molecular recognition of the two connexons. In this study, cryoelectron microscopy and computer modeling provide evidence that the formation of the gap junction intercellular channel requires a 30° rotation between hemichannels for proper docking. With this amount of rotation, the peaks (protrusions) on one connexon fit into the valleys of the apposed connexon in the 3-D model, which would make for an ionically tight interface necessary for a functional cell-cell channel. Docking appears to be governed by a “lock and key” mechanism via a simple interdigitation of the six protrusions from each connexon. This interdigitation increases significantly the contact surface area and potential number of hydrogen bonds or hydrophobic interactions and/or other attractive interactions. Having a larger surface area than if the surfaces were flat would explain the biochemical requirements for conditions characterized previously for splitting of channels into hemichannels. The docked connexons were computationally fitted into two gap junction structures, which further confirmed the interdigitated manner of docking.

Three-Dimensional Structure of the Gap Junction Connexon and Intercellular Channel

Gap junctions are specialized cell-cell contact areas by which cells communication with each other. Within these contact areas are tens to thousands of membrane channels. A gap junction membrane channel (also referred to as an intercellular channel) is unique among membrane channels in that it is composed of two oligomers with each of two adjacent tissue cells contributing one oligomer (called a connexon or hemichannel). The pore of the intercellular channel controls the passage of small molecules and ions from one cell to another.We are interested in how the structure and surface topology of the gap junction connexon at its extracellular surface influences the docking and formation of an intercellular communicating channel. It has been demonstrated that connexons made from some connexins will dock and form functional channels with some but not all connexons made from other isoforms. This selectivity is surprising considering that the primary sequences of the docking domains are highly conserved.