The permeability of gap junction channels to small cytosolic molecules is finely regulated by specific changes in cytosolic ionic composition. As gap junction channels close, cells uncouple from each other electrically and metabolically. Uncoupling is primarily a protective mechanism that enables healthy cells to isolate themselves from damaged neighbors, but evidence for channel gating sensitivity to nearly physiological calcium and hydrogen ion concentrations suggests that gap junction permeability modulation may play a role in normal cellular functions as well. Identifying the uncoupling agents and the molecular basis of channel gating is essential for understanding how cell communication is physiologically controlled and how cell coupling relates to specific cellular activities. Mechanism of channel gating can only be understood once a thorough knowledge of connexin domains relevant to gating and of cytosolic participants in gating processes is achieved. This chapter discusses the current knowledge of gap junction regulation by cytosolic acidification, focusing on molecular domains of connexins believed to participate in channel gating. Much of the material deals with gap junction channels made of connexin32 (Cx32), a connexin expressed widely in the mammalian tissues such as liver, pancreas, kidney, nervous system, thyroid, and mammary gland, and whose genetic mutation is involved in the pathogenesis of the X-linked Charcot-Marie-Tooth (CMTX) demyelinating disease.
Mammalian gap junction channels are formed when each cell of an adjacent pair contributes six connexins to form an oligomerized hemichannel. Any two hemichannels can form a link through extracellular loops extending from their respective membrane-spanning domains to form an aqueous intercellular pathway. Gap junction channels are therefore composed of 12 subunit proteins. These subunits are referred to as "connexins" and are named according to their predicted molecular weights. The biophysical characteristics of the gap junction family of proteins have been rigorously studied. Historically, however, most electrophysiological studies of connexins have concentrated on elucidating the macroscopic behavior and unitary conductance of homotypic gap junction channels, that is, gap junction channels formed of 12 identical connexin proteins. However, because many cell types express more than one connexin protein, distinct combinations of these intercellular channels among parenchymal cells within a given tissue are, at the very least, theoretically possible. In particular, it is quite conceivable that gap junction channels in a given tissue cell type can also be formed by the union of hemichannels that are each composed of homologous, but different connexin proteins. Recently, evidence has been advanced consistent with the supposition that the expression of more than one connexin type in a single cell results in the mixing of nonidentical connexins (i.e., heteromeric) in a given hemichannel or connexon. As such, this chapter reviews the extant evidence for heteromeric connexins formed of the two most prominent connexins found in vascular wall cells, that is, connexin43 (Cx43) and connexin37 (Cx37). In addition, the electrophysiological criteria that distinguish heteromeric connexins from their heterotypic and homotypic counterparts are reviewed, as along with the potential physiological relevance of heteromeric channels to tissue function in situ.
The mechanisms of gap junction channel gating have been studied in a variety of heterologous expression systems and cell lines. The data discussed in this chapter, however, derive from the paired Xenopus oocyte expression system, which allows more rapid analysis of connexins and their mutants than expression in the mammalian systems. The use of antisense oligonucleotides also allows the effective elimination of endogenous coupling, something that is difficult to achieve in the mammalian systems. Functional characterization can generally be accomplished within hours or days after the injection of cRNA. After microinjection of RNA encoding the gene of interest, the oocytes are stripped of their vitelline membrane and pushed together to form a pair from which intercellular currents can be recorded using the dual-cell two electrode voltage clamp technique. Virtually all connexins have been expressed in oocytes using this technique, and in almost all cases where the properties of connexins expressed in both oocytes and mammalian cells have been compared, they have proven to be identical.
This chapter focuses on the role of gap junctional intercellular communication (GJIC) in cellular growth control and neoplasia and considers the large body of evidence concerning this role. It also presents a model of how GJIC might contribute to cellular growth regulation. More than three decades ago, Loewenstein and Kanno were the first to hypothesize a role for GJIC in the control of cellular growth and defective GJIC in cancer. This hypothesis was proposed before the first ultrastructural description of a gap junction or the availability of molecular tools to study gap junctions and their protein subunits, the connexins. The hypothesis is a marvel of scientific insights. More recently, the laboratories of Yamasaki, Trosko, Mehta, Naus, Lau, and others have made major contributions to the field. They have been leaders in the validation of the Loewenstein and Kanno hypothesis and the development of this rapidly expanding area of gap junction research.
Gap junctional channels are intercellular channels that allow the passage of ions and other small molecules less than about 1 kD in size between neighbouring cells. They are composed of two oligomeric protein subunits called connexons or hemichannels which reside in the plasma membrane of closely opposed cells. Several recent studies suggest that unpaired connexons may be active on the non-junctional plasma membrane of cells. Here I present a short overview of the properties of connexon channels expressed in Xenopus oocytes.
Publisher Summary The permeability of gap junctions is rapidly (within seconds) and reversibly decreased by an experimental manipulation that decreases the cytosolic pH or increases the cytosolic concentration of free Ca2+. Large increases of the cytosolic H+ or Ca2+ concentrations impair the gap junction communication, but in many cell types these changes are slow and often irreversible, and are more akin to pathology than to a physiological regulation. Also, according to both early observations and recent data, the changes in H+ or Ca2+ levels that affect gap junctions are highly variable in different cell systems and may even differ for gap junction channels consisting of the same connexin. In this chapter, the quantitative aspects of the rises in cytosolic Ca2+ that promote uncoupling in a number of different cell types are critically considered, with particular attention to the myocytes of mammalian heart. The frequently granted hypothesis of a rapid and reversible decrease of the gap junction conductance by increases in the cytosolic Ca2+ concentration ([Ca2+]i) in the physiological range cannot be generalized and is not valid for the mammalian heart myocytes. Recent data that show an upregulation of the gap junction conductance in the mammalian heart myocytes by increases in [Ca2+]i quantitatively similar to those occurring during excitationcontraction coupling is also presented in the chapter.
Cardiac malformation in connexin43 (CX43)-disrupted mice is restricted to the junction between right ventricle and outflow tract, even though CX43 is also expressed abundantly elsewhere. We analyzed cardiac morphogenesis in immunohistochemically and hybridohistochemically stained and three-dimensionally reconstructed serial sections of CX43-deficient embryos between embryonic day (ED) 10 and birth. The establishment of the D configuration in the ascending loop of CX43-deficient hearts is markedly retarded, so that the right ventricle retains a craniomedial position and is connected with the outflow tract by a more acute bend in ED10 and ED11 embryos. Because of the subsequent growth of the right ventricle, this condition usually evolves into a D loop, but when it persists, a "crisscross" configuration develops, with the atrioventricular cushions rotated 90 degrees, a horizontal muscular ventricular septum, and a parallel course of the endocardial ridges of the outflow tract. After ED12, large intertrabecular pouches develop at the ventricular side of both shelflike myocardial structures that support the endocardial ridges of the outflow tract, ie, at the location that was earlier characterized by the acute bend between the right ventricle and the outflow tract and that subsequently develops into the anterosuperior leaflet of the tricuspid valve. Retarded development of the D configuration in the ascending loop of the embryonic heart predisposes the myocardium at the junction of the right ventricle and outflow tract to excessive development-of intertrabecular pouches during subsequent development.
The intercellular gap junction membrane channel provides a low resistance pathway for cell-cell communication in animal cells. Each of two cells contributes one hemichannel or connexon. Formation of the channel occurs when two connexons recognize each other, pair and seal at their extracellular surfaces. Here we discuss insights into how the structure of an individual connexon which we determined [1], the surface topology of the extracellular domains and the primary amino acid sequence influence the docking of two connnexons to form an intercellular channel.
Phosphorylation has been implicated in connexin trafficking, assembly, insertion into the plasma membrane, degradation and retrieval from the plasma membrane and in gap junctional channel gating. Chicken connexin56 (Cx56) is a phosphoprotein that is abundantly expressed in the eye lens. In immunoblots of lens homogenates, Cx56 is detected at the 4(th) embryonic day as a single band that comigrates with in vitro translated Cx56. By the 18(th) embryonic day, Cx56 is detected as multiple bands that collapse to a doublet after treatment with alkaline phosphatase. These results suggest that during development Cx56 is post-translationally modified by phosphate groups in a development-dependent process. Also, confocal immunofluorescence shows a developmental progression of immunoreactive Cx56 from large plaques to smaller dispersed punctate staining.Lens cells in culture differentiate to form lentoids that express Cx56. In lentoids, modification of Cx56 resembles that observed in the intact lens. To identify possible sites of phosphorylation of Cx56 and their functional implications, lentoid-containing cultures were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), a direct activator of protein kinase C (PKC). Treatment with TPA decreased dye coupling between lentoid cells and increased the intensity of the Cx56 bands with an apparent molecular mass of 73-77 and 83 kDa. Incorporation of P-32 into immunoprecipitated Cx56 was observed in cultures labeled with P-32-orthophosphate. The two-dimensional pattern of Cx56 tryptic phosphopeptides differed between control and TPA-treated cultures. Treatment of lens cultures with TPA increased the abundance of two constitutively phosphorylated peptides that were mapped to the intracellular loop of Cx56 corresponding to the sequence RGSVK. We also identified a constitutively phosphorylated site in the carboxy terminal domain of Cx56 in the sequence RLSR, whose phosphorylation was unaffected by activation of PKC.Pulse-chase experiments demonstrated the presence of two pools of Cx56 with half-lives of 2.4 hand 2 days. The Cx56 forms that appeared first were the ones with faster electrophoretic mobility; the forms with slower electrophoretic mobilities appeared later. These data suggest that phosphorylation may play a role in regulating Cx56 stability.
The gap junction protein connexin43 (Cx43) is a phosphoprotein that typically migrates as three bands (nonphosphorylated or NP, pi and P2) during polyacrylamide gel electrophoresis. Mitotic cells, prepared by shakeoff of either nocodazole-treated or untreated cultures, contain Cx43 that has a distinctly reduced electrophoretic mobility (P3) that migrated slower than P2. Mitotic FT210 cells, which contain a temperature sensitive mutation in the p34(cdc2) kinase, showed abundant levels of the P3 Cx43 when maintained at the permissive temperature where p34(cdc2) is active. In contrast, nocodozole-treated FT210 cells grown at the nonpermissive temperature did not contain P3 Cx43. These results indicated that generation of the P3 Cx43 was dependent upon active p34(cdc2)/cyclin B kinase. Mitotic cells exhibited a marked redistribution from cell-cell plasma membrane interfaces to multiple, distinctly-stained cytoplasmic structures. This event may be part of the dramatic structural changes observed in mitotic cells undergoing cell rounding and cytokinesis.
The aim of this study was to investigate the effects of experimental hypertension on Cx43 expression in rat aorta and heart. To this end, rats were made hypertensive by clipping one renal artery (two kidney, one-clip renal model) or by administration of deoxycorticosterone and salt (DOCA-salt model). After four weeks, all rats showed a similar increase in blood pressure, and in thickness of both aortic wall and heart. Northern blot analysis and immunolabeling for Cx43 showed that hypertensive rats expressed twice as much Cx43 than controls in aorta but not in heart. The data suggest that localized mechanical forces induced by hypertension regulate Cx43 expression in a tissue-specific manner. Since rats made similarly hypertensive by inhibiting nitric oxyde synthase with L-NAME, did not feature increased expression of Cx43 in aortic smooth muscle cells, this regulation appears related to the distensibility of conduit arteries, which differed in the 2K, 1C and the L-NAME models.
In the vascular wall, endothelial cells (ECs) are typically growth arrested; however, they proliferate in response to various pathophysiologic conditions. Whereas in the normal vessel Cxs 40, 37 and 43 are expressed in ECs, in regions of pathology and in cell culture Cx43 expression is elevated and Cx40 and 37 expression are reduced. This change in connexin expression could be a part of the proliferative response of these cells to pathophysiologic conditions, including standard cell culture. This possibility was evaluated in the current study in rat microvascular coronary endothelial cells (CECs) and in aortic endothelial cells (AECs). ECs proliferated in DMEM supplemented with 15% FBS, heparin and EC growth factor. Proliferation was blocked when the FBS content of the medium was reduced to 1%. Neither native or mildly oxidized LDL (ox-LDL) stimulated proliferation of ECs. Growth arrested AECs exhibited an similar to 5 and 16 fold reduction in Cx43 and Cx40 mRNA expression, respectively. In contrast, growth arrested CECs exhibited an similar to 2 fold increase in both Cx43 and Cx40 expression. The Cx43:Cx40 mRNA ratio in proliferating AECs and CECs was similar to 5:1 and 3.6:1, respectively; these ratios in growth arrested AECs and CECs were 17:1 and 3.6.1, respectively. Consistent with similar to 5 fold higher Cx43 levels in AECs, these cells were better coupled than the CECs. Lucifer Yellow diffused to similar to 50 neighbors in the AECs vs. 9 neighbors in the CECs. However, despite large differences in connexin levels in the growth arrested vs. proliferating AECs and CECs, differences in dye coupling could not be detected. LDL and ox-LDL also had no effect on dye coupling.
Connexin43 (Cx43) is the most abundantly expressed gap junction protein in ventricular myocytes of the heart and in astrocytes of the brain. In order to determine the functional importance of Cx43 in coupling between these cell types, we have compared coupling strength and junctional channel properties in cardiac myocytes and cortical astrocytes obtained from wildtype (WT) mice and littermates in which Cx43 has been deleted through homologous recombination. Because the Cx43 (-/-) mice die at birth, cultures were established perinatally from individual animals and genotypes subsequently determined from PCR of tail DNA. For cardiac myocytes from Cx43 (-/-) mice, spontaneous contractions were less frequent and more arrhythmic than in wildtype littermates; dye coupling was virtually absent in Cx43(-/-) cardiocytes, although junctional conductance (g(j)) was 36% that of WT. Voltage sensitivity of g(j) and unitary junctional conductances (gamma(j)) were primarily Cx43-like in WT cardiocytes and Cx40-like in Cx43 (-/-) cells; although smaller channel sizes, perhaps reflecting Cx45 activity, were also prominent in event histograms prepared from recordings from cardiac myocytes of both genotypes. Conduction velocity and extent of spread of mechanically elicited Ca2+ waves were reduced in Cx43 (-/-) cardiocytes compared to WT littermates. For astrocytes from Cx43 (-/-) mice, dye coupling was present, though less extensive than in WT glia; g(j) in Cx43 (-/-) astrocytes was only 5% that measured in WT littermates. Voltage sensitivity of g(j) was markedly stronger in Cx43 (-/-) astrocytes than in WT, in many cases exhibiting no voltage-induced substate, suggesting the presence of Cx46. Cx43 (-/-) gamma(j) values were substantially lower than in WT astrocyes, although larger Cx40-like channels were also present in some cell pairs. Comparing results from individual littermates, Ca2+ wave spread among Cx43 (-/-) and WT astrocytes was remarkably similar with regard to velocity of propagation, amplitude, and extent of spread, although significant differences in each parameter were obtained when all animals from each genotype were compared. Ca2+ wave spread was similarly but only partially reduced in Cx43 (-/-) and WT cultures exposed to the ATP receptor blocker suramin and almost totally eliminated by heptanol treatment, indicating that under the conditions of these experiments the primary route of Ca2+ wave spread is through junctional channels No differences were detected in coupling strength or Cx43 mRNA expression between neonatal heterozygous and WT cardiac or glial cells, and no compensatory changes in mRNA expression of other connexins were seen in brain or cardiac tissue. We conclude from these studies that the impact of Cx43 deficiency on intercellular coupling differs markedly in cardiac myocytes and CNS astrocytes. The substantially higher g(j) values in cardiocytes than in astrocytes from Cx43 (-/-) mice contrasts with the lower strength of dye coupling and Ca2+ wave spread in cardiocytes than in astrocytes from these animals and presumably reflects the different properties of channels formed by the connexins responsible for the residual coupling.
The purpose of this study was to screen the entire Cx50 (MP70) protein coding region of the No2 (nuclear opacity #2) mouse mutant for a mutation that was linked to and consistent with the observed cataractous phenotype.Genomic DNA's were prepared from both mutant and normal, parental spleens. DNA oligonucleotides were designed and used to amplify the entire Cx50 coding region, including the upstream splice acceptor site from these DNA's via PCR. PCR products were gel purified and then directly sequenced.A single A-->C transversion was observed in the coding strand of the mutant DNA. This DNA substitution results in the nonconservative amino acid substitution of Ala for the normally encoded Asp in a conserved domain of the encoded polypeptide. The DNA alteration also creates a restriction site not present in the wild type DNA sequence. This was used to corroborate the sequence data.A mutation has been identified in the Cx50 gene of the No2 mouse mutant. This mutation is both linked to and consistent with the observed cataractous phenotype, as it is only present in mutant DNA and is predicted to alter a conserved domain of the ocular-specific membrane protein encoded by the gene. These results indicate that Cx50 plays an important role in normal lens development and maintenance and heralds its gene as a likely candidate in human congenital, hereditary cataract.
To explore the hypothesis that intercellular Ca2+ waves are a mechanism of heterotypic cell communication and convey information across biological interfaces, such as the blood-brain barrier, intercellular Ca2+ signaling was investigated in cocultures of glial and endothelial cells. Mechanically-initiated, intercellular Ca2+ waves were found to propagate from glial to endothelial cells or vice versa. To determine if glial-endothelial Ca2+ waves propagate by the intercellular diffusion of inositol trisphosphate (IP3,) through gap junctions, the multicellular Ca2+ responses to increases in intracellular concentrations of IP3 and Ca2+, generated by the photolysis of caged compounds, were examined. Caged-IP3 was microinjected into a single cell but was free to diffuse to adjacent cells. Photolytic release of IP3, in the injected cell or in a distal, non-injected cell, initiated an intercellular Ca2+ wave. By contrast, photolysis of caged-Ca2+ induced an increase in [Ca2+](i) in the illuminated cell but did not initiate an intercellular Ca2+ wave. These results indicate that 1) intercellular Ca2+ waves propagate over short distances by the intercellular diffusion of IP3, via gap junctions, 2) Ca2+ itself does not serve as an intercellular messenger in these cells and 3) Ca2+ waves may contribute to the regulation of blood-brain barrier function.
Short term exposures to 17 beta-estradiol propionate (1 to 25 mu M) completely abolish cell-to-cell communication between cardiac myocytes, but the mechanism(s) of this action remain(s) to be elucidated. As several 17 beta-estradiol effects have been found affected by protein kinases inhibitors, we have examined the influence of this compound, in combination with several protein kinase activators and inhibitors, on junctional permeability and on macroscopic and single channel conductances. Protein kinases A or C as well as protein tyrosine kinase do not seem involved in this 17 beta-estradiol effect. This compound, known able to reduce membrane fluidity, might, like several other lipophilic junctional uncouplers (e.g. heptanol, gossypol, tamoxifen), interfere with the plasma membrane structure, leading to perturbations of junctional channel conformation, with accompanying effects on their functions.
Evidence is accumulating that connexin genes form a family of tumor-suppressor genes. Thus, overexpression of Cx43 in rat C6 glioma cells, and of Cx26 in HeLa cells, suppresses growth both in vitro and in vivo. Previously, we showed that the mutant Cx32 proteins, found in X-linked Charcot-Marie-Tooth disease patients, abolish the GJIC restored in HeLa cells by the wild-type Cx32 gene in a dominant-negative fashion. However, it was not clear whether such a dominant-negative effect of mutant connexins could lead to biological phenomena such as aberrant growth control. We have. therefore, examined the dominant-negative effects of mutant Cx43 constructs in C6 cells, and of mutant Cx26 constructs in HeLa cells, on cell growth control exerted by the wild types. When two mutant Cx43 constructs -L160M (Leu 160 to Met) and A253V (Ala 253 to Val) - were transfected into Cx43-transfected C6 cells (clone C6-13), they restored anchorage-independent growth capacity and reinforced the tumorigenicity of C6-13, meaning that these two mutants can inhibit growth-suppressive function of wild-type Cx43 in a dominant-negative manner. Neither of the mutants seemed to affect subcellular localization of Cx43 proteins. In HeLa cells transfected with wild-type Cx26, the mutant constructs P87L (Pro 87 to Leu) and R143W (Arg 143 to Trp) restored tumorigenicity in nude mice after their second transfection, without showing any effect on subcellular localization of Cx26 proteins. However, there was no clear correlation between the level of GJIC and the tumorigenicity of these double transfectants. These results show that some mutant forms of Cx43 and Cx26 can inhibit the tumor-suppressive function of their wild-type counterparts, and suggest that the level of GJIC, as revealed by Lucifer Yellow transfer, is not necessarily the determinant of growth control.
Cancer preventive retinoids and carotenoids are antiproliferative and upregulate connexin43 expression and gap junctional communication (GJC). To determine if effects on GJC are central to actions of these compounds we have created HeLa cell clones containing a tet-inducible Cx43 gene. Clones respond rapidly to induction by doxycycline with Cx43 synthesis and incorporation into junctional plaques. In dense cultures, induction results in decreased proliferation. Clonal heterogeneity among HeLa cells with regard to Cx43 expression and neoplastic phenotype was also discovered. These results support the hypothesis of growth control via GJC.
