Functional Effects of Casein Kinase I-Catalyzed Phosphorylation on Lens Cell-to-Cell Coupling

The functional consequence of the casein kinase I-catalyzed phosphorylation of the lens gap junctional protein connexin49 was investigated using a sheep primary lens cell culture system. To determine whether the phosphorylation of connexin49 catalyzed by endogenous casein kinase I results in an altered junctional communication between lens cells, the effect of the casein kinase I-specific inhibitor CKI-7 on Lucifer Yellow dye transfer between cells in the lens culture was examined. Dye transfer was analyzed in cultures of different ages because we have demonstrated previously that the expression of connexin49 increases as the cultures age while that of connexin43, which is likely not a substrate for casein kinase I, has been shown to decrease [Yang & Louis (1999) Invest. Ophthalmol. Vis. Sci. 41: 2568–2564]. In 9-day old lens cultures, in which gap junctions are composed primarily of connexin43, CKI-7 had little effect on the rate of dye transfer between lens cells. In contrast, treatment of 15-day and 28-day old cultures with CKI-7 resulted in a significant increase in the rate of dye transfer. Thus, the extent of this CKI-7-dependent increase in cell-to-cell communication was positively correlated with the level of expression of connexin49, the major casein kinase I substrate in lens plasma membranes. These results suggest that the casein kinase I-catalyzed phosphorylation of connexin49 decreases cell communication between connexin49-containing gap junctions in the lens. Received: 31 July 2000/Revised: 12 January 2001     

Gap Junctional Channels Regulate Acid Secretion in the Mammalian Gastric Gland

Gap junction channels are regarded as a primary pathway for intercellular message transfer, including calcium wave propagation. Our study identified two gap junctional proteins, connexin26 and connexin32, in rat gastric glands by RT-PCR, Western blot analysis, and immunofluorescence. We demonstrated a potential physiological role of the gap junctional channels in the acid secretory process using the calcium indicator fluo-3, and microinjection of Lucifer Yellow. Application of gastrin (10⁻⁷ m) to the basolateral membrane resulted in the induction of uniphasic calcium signals in adjacent parietal cells. In addition, single parietal cell microinjections in intact glands with the cell-impermeant dye Lucifer Yellow resulted in a transfer of dye from the injected cell to the adjacent parietal cell following gastrin stimulation, demonstrating gastrin-induced cell-to-cell communication. Both calcium wave propagation and Lucifer Yellow transfer were blocked by the gap junction inhibitor 18α-glycyrrhetinic acid. Our studies demonstrate that functional gap junction channels in gastric glands provide an effective means for rapid cell-to-cell communication and allow for the rapid onset of acid secretion. Received: 4 December 2000/Revised: 5 June 2001     

Conservation of a cytoplasmic carboxy-terminal domain of connexin 43, a gap junctional protein,in mammal heart and brain

Summary According to the sequence of connexin 43, a cardiac gap junctional protein, the domain contained within residues 314–322 is located 60 amino acids away from the carboxy-terminus. Antibodies raised to a peptide corresponding to this domain label a unique 43-kD protein on immunoblots of both purified gap junctions and whole extracts from rat heart. Immunofluorescence investigations carried out on mammal heart sections reveal a pattern consistent with the known distribution of intercalated discs. Immunogold labeling performed with ultrathin frozen sections of rat heart or partially purified rat heart gap junctions demonstrate that antigenic determinants are associated exclusively with the cytoplasmic surfaces of gap junctions.The antibodies were shown to cross-react with a 43-kD protein on immunoblots of whole extracts from human, mouse and guinea pig heart. However, no labeling was seen when heart of lower vertebrates such as chicken, frog and trout, was investigated. These results, confirmed by immunofluorescence investigations, were interpreted as a loss of antigenic determinants due to sequence polymorphism of cardiac connexin 43.Proteins ofM _r 43 and 41 kD, immunologically related to cardiac connexin 43, were detected in immunoblots of mouse and rat brain whole extracts. mRNAs, homologous to those of cardiac connexin 43 and of the same size (3.0 kb), are also present in brain. Immunofluorescence investigations with primary cultures of unpermeabilized and permeabilized mouse neural cells showed that the antigenic determinants recognized by the antibodies specific for connexin 43 are cytoplasmic and that the labeling observed between clustered flat cells, is punctate, as expected for gap junctions. Double labeling experiments demonstrated that the immunoreactivity is associated with GFAP-positive cells, that is to say, astrocytes.     

Affinity purification of a rat-brain junctional protein, connexin 43.

Immunocytochemical investigations have previously shown that antibodies specific for mammal connexins labeled in situ rat and mouse brain gap junctions. However brain gap-junction proteins have neither been identified with certainty, nor purified. By immunoblotting, anti-peptide antibodies directed against rat heart connexin 43 (CX43) detect a major protein of 41 kDa in rat brain homogenates. The specificity of these antibodies made it possible to establish an affinity-chromatography purification procedure of the 41-kDa protein. Purified antibodies specific for the sequence SAEQNRMGQ (residues 314-322) of rat heart CX43 were covalently bound to a protein-A-Sepharose-CL-4B matrix. Rat brain homogenates were recycled through the immunomatrix and the material specifically bound to the matrix was then competitively eluted with the peptide SAEQNRMGQY. Analysis by SDS/PAGE of eluates demonstrated that they contain a 41-kDa protein associated with low amounts of high-molecular-mass proteins. By immunoblotting, these proteins were shown to be specifically recognized by antibodies directed against residues 5-17, 55-56, and 314-322 of rat heart CX43. The NH2-terminal partial sequence for the 41-kDa protein was determined by microsequencing and shown to be similar to alpha 1 connexins. This is the first successful purification of a junctional protein from brain tissue and provides direct evidence that the 41-kDa protein is a CX43 gene product.     

Influence of the Molecular Structure of Steroids on Their Ability to Interrupt Gap Junctional Communication

17β-estradiol propionate was found to reduce the gap junctional communication in a concentration range similar to that of testosterone propionate, in primary cultures of rat Sertoli cells and cardiac myocytes. Uncoupling was reversible on washing out and occurred without concomitant rise in the intracellular calcium concentration. Esterification was a prerequisite for the activity of extracellularly applied steroid compounds (for example, testosterone was ineffective even at external concentrations up to 100 μm, whereas its intracellular application at 1 μm totally interrupted intercellular communication), but their uncoupling efficiency did not depend on the nature of the ester chain nor on its position on the steroid nucleus. The derivatives of two other androgen hormones (derivatives of the androstane nucleus) were also efficient as junctional uncouplers. Among five steroid molecules belonging to the pregnane family, only one (pregnanediol diacetate) interrupted the junctional communication. Neither cholic acid nor cholesteryl acetate or ouabain showed this effect. Altogether, no correlation with the presence or position of double bonds nor with the trans- or cis-fusion of the A and B rings could be recognized. These results suggest that this reversible, nondeleterious uncoupling effect of steroids is independent of the shape of the molecules and is more probably related to their size and liposolubility, that condition their insertion into the lipid bilayer. Their incorporation into the membrane could disturb the activity of the membrane proteins by a physical mechanism. Received: 10 April 1995/Revised: 27 October 1995     

Functional Characterization of Canine Connexin45

Three gap junctional proteins have been identified in canine ventricular myocytes: connexin 43 (Cx43), connexin 45 (Cx45), and connexin 40 (Cx40). We have characterized the functional properties of canine Cx45 and examined how Cx45 functionally interacts with Cx43 in Xenopus oocyte pairs. Homotypic pairs expressing Cx45 were well coupled. Heterotypic pairs composed of Cx45 paired with either Cx43 or Cx38 also developed high levels of conductance. Junctional currents in the heterotypic pairs displayed a highly asymmetrical voltage dependence. The kinetics and steady-state voltage dependence of the heterotypic channels more closely resembled those of the Cx45 channels when the Cx45 cRNA-injected cell was relatively negative suggesting that the Cx45 connexon closes for relative negativity at the cytoplasmic end of the channel. We also show that homotypic and heterotypic channels composed of Cx45 and Cx43 exhibit differences in pH _i sensitivity. Received: 18 August 1995/Revised: 21 November 1995     

Phosphorylated Carboxy Terminal Serine Residues Stabilize the Mouse Gap Junction Protein Connexin45 Against Degradation

Phosphoamino acid analysis of mouse connexin45 (Cx45) expressed in human HeLa cells revealed that phosphorylation occurred mainly at serine residues, but also on tyrosine and threonine residues. To characterize the role of Cx45 phosphorylation, different serine residues of the serine-rich carboxy terminal region were deleted or exchanged for other amino acids residues. Human HeLa cells deficient in gap junctional intercellular communication were stably transfected with appropriate constructs and analyzed for expression, localization, phosphorylation, formation of functional gap junction channels and degradation of mutant Cx45. After exchange or deletion of nine carboxy terminal serine residues, phosphorylation was decreased by 90%, indicating that these serine residues represented main phosphorylation sites of mouse Cx45. The various serine residues of this region contributed differently to the phosphorylation of Cx45 suggesting a cooperative mechanism for phosphorylation. Substitution of different serine residues for other amino acids did not interfere with correct intracellular trafficking and assembly of functional gap junction channels, as shown by localization of mutant Cx45 at the plasma membrane and by dye transfer to neighboring cells. Truncated Cx45 was also weakly phosphorylated but was trapped in perinuclear locations. Dye transfer of these transfectants was similar as in nontransfected HeLa cells. The half-life of mouse Cx45 protein in HeLa cells was determined as 4.2 hr. Pulse-chase experiments with the different transfectants revealed an increased turnover of Cx45, when one or both of the serine residues at positions 381 and 382 or 384 and 385 were exchanged for other amino acids. The half-life of these mutants was diminished by 50% compared to wild type Cx45. Received: 26 September 1997/Revised: 5 January 1997     

Intracellular Domains of Mouse Connexin26 and -30 Affect Diffusional and Electrical Properties of Gap Junction Channels

To evaluate the influence of intracellular domains of connexin (Cx) on channel transfer properties, we analyzed mouse connexin (Cx) Cx26 and Cx30, which show the most similar amino acid sequence identities within the family of gap junction proteins. These connexin genes are tightly linked on mouse chromosome 14. Functional studies were performed on transfected HeLa cells stably expressing both mouse connexins. When we examined homotypic intercellular transfer of microinjected neurobiotin and Lucifer yellow, we found that gap junctions in Cx30-transfected cells, in contrast to Cx26 cells, were impermeable to Lucifer yellow. Furthermore, we observed heterotypic transfer of neurobiotin between Cx30-transfectants and HeLa cells expressing mouse Cx30.3, Cx40, Cx43 or Cx45, but not between Cx26 transfectants and HeLa cells of the latter group. The main differences in amino acid sequence between Cx26 and Cx30 are located in the presumptive cytoplasmic loop and C-terminal region of these integral membrane proteins. By exchanging one or both of these domains, using PCR-based mutagenesis, we constructed Cx26/30 chimeric cDNAs, which were also expressed in HeLa cells after transfection. Homotypic intercellular transfer of injected Lucifer yellow was observed exclusively with those chimeric constructs that coded for both cytoplasmic domains of Cx26 in the Cx30 backbone polypeptide chain. In contrast, cells transfected with a construct that coded for the Cx26 backbone with the Cx30 cytoplasmic loop and C-terminal region did not show transfer of Lucifer yellow. Thus, Lucifer yellow transfer can be conferred onto chimeric Cx30 channels by exchanging the cytoplasmic loop and the C-terminal region of these connexins. In turn, the cytoplasmic loop and C-terminal domain of Cx30 prevent Lucifer yellow transfer when swapped with the corresponding domains of Cx26. In chimeric Cx30/Cx26 channels where the cytoplasmic loop and C-terminal domains had been exchanged, the unitary channel conductance was intermediate between those of the parental channels. Moreover, the voltage sensitivity was slightly reduced. This suggests that these cytoplasmic domains interfere directly or indirectly with the diffusivity, the conductance and voltage gating of the channels. Received: 26 July 2000/Revised: 15 February 2001     

Immunolocalization of connexin 43 in the tooth germ of the neonatal rat

Summary Rabbit polyclonal antibodies to amino acids 346–360 of connexin 43, the ‘heart’ gap junction protein, were employed to immunolocalize connexin 43 gap junctions in the neonatal rat molar tooth germ. Connexin 43 appears early in the differentiation of both ectodermally derived and ectomesenchymally derived cells of the developing tooth. Connexin 43 immunoreactivity is present in the epithelial components of the enamel organ, including the area of the proximal and distal junctional complexes of the ameloblast layer, and the stratum intermedium, stellate reticulum and outer enamel epithelium. Secretory odontoblasts and developing alveolar bone also display a pattern of connexin 43 immunostaining. Both the epithelial and ectomesenchymally-derived components of the developing tooth acquire connexin 43 channels in a manner that correlates with cell differentiation. In addition, three regions can be defined by connexin 43 immunostaining: the epithelia of the enamel organ that are derived from the oral epithelium, the odontoblast layer derived from the ectomesenchyme, and the alveolar bone. The results suggest that connexin 43 may provide the mechanism for functional compartmentalization of the tissues associated with tooth formation. Compartmentalization suggested by connexin 43 expression could play important roles in the development and functions of these tissues.     

Effects of Hyperglycemia and Protein Kinase C on Connexin43 Expression in Cultured Rat Retinal Pigment Epithelial Cells

Previous results demonstrated that the intercellular communication mediated by gap junctions in retinal pigment epithelial (RPE) cells from the healthy Long Evans (LE) rat strain is higher than that from the dystrophic Royal College of Surgeons (RCS) rat strain. We examined connexin (Cx) expression in both cell types. At the mRNA level, a qualitatively similar expression pattern was found whereby Cx26, Cx32, Cx36, Cx43, Cx45 and Cx46 were all expressed. At the protein level, only Cx43 and Cx46 were detected. Expression of both isoforms was higher in LE-RPE as compared to RCS-RPE by a factor of 1.25 and 2 respectively. Phosphorylation of Cx43 was increased upon activation of protein kinase C (PKC) by 1 μM phorbol 12-myristate 13-acetate (PMA). The phosphorylation status was not changed in hyperglycemic conditions, but this treatment strongly decreased total Cx43 levels to about 75 and 40% (in LE-RPE and RCS-RPE cells respectively) of the control level in LE-RPE cells. This decrease could be overcome by PKC downregulation. These results demonstrate that PKC activation and hyperglycemic conditions have different effects on Cx43 and that PKC is involved in the metabolic pathway induced by hyperglycemic conditions. Received: 21 July 2000/Revised: 19 January 2001     

The Role of MIP in Lens Fiber Cell Membrane Transport

MIP has been hypothesized to be a gap junction protein, a membrane ion channel, a membrane water channel and a facilitator of glycerol transport and metabolism. These possible roles have been indirectly suggested by the localization of MIP in lens gap junctional plaques and the properties of MIP when reconstituted into artificial membranes or exogenously expressed in oocytes. We have examined lens fiber cells to see if these functions are present and whether they are affected by a mutation of MIP found in Cat ^Fr mouse lens. Of these five hypothesized functions, only one, the role of water channel, appears to be true of fiber cells in situ. Based on the rate of volume change of vesicles placed in a hypertonic solution, fiber cell membrane lipids have a low water permeability (p _H2O ) on the order of 1 μm/sec whereas normal fiber cell membrane p _H2O was 17 μm/sec frog, 32 μm/sec rabbit and 43 μm/sec mouse. Cat ^Fr mouse lens fiber cell p _H2O was reduced by 13 μm/sec for heterozygous and 30 μm/sec for homozygous mutants when compared to wild type. Lastly, when expressed in oocytes, the p _H2O conferred by MIP is not sensitive to Hg²⁺ whereas that of CHIP28 (AQP1) is blocked by Hg²⁺. The fiber cell membrane p _H2O was also not sensitive to Hg²⁺ whereas lens epithelial cell p _H2O (136 μm/sec in rabbit) was blocked by Hg²⁺. With regard to the other hypothesized roles, fiber cell membrane or lipid vesicles had a glycerol permeability on the order of 1 nm/sec, an order of magnitude less than that conferred by MIP when expressed in oocytes. Impedance studies were employed to determine gap junctional coupling and fiber cell membrane conductance in wild-type and heterozygous Cat ^Fr mouse lenses. There was no detectable difference in either coupling or conductance between the wild-type and the mutant lenses. Received: 17 February 1999/Revised: 16 April 1999     

Quantitative Determination of Gap Junctional Permeability in the Lens Cortex

We have developed a simple dye transfer method, which allows the gap junction permeability of lens fiber cells to be quantified. Two fixable fluorescent dyes (Lucifer yellow and rhodamine-dextran) were introduced into peripheral lens fiber cells via mechanical damage induced by removing the lens capsule. After a defined incubation period, lenses were fixed, sectioned, and the distribution of the dye recorded using confocal microscopy. Rhodamine-dextran and Lucifer yellow both labeled the extracellular space between fiber cells and the cytoplasm of fiber cells that had been damaged by capsule removal. For the gap junctional permeable dye Lucifer yellow, however, labeling was not confined to the damaged cells and exhibited intercellular diffusion away from the damaged cells. The extent of dye diffusion was quantified by collecting radial dye intensity profiles from the confocal images. Effective diffusion coefficients (D _eff ) for Lucifer yellow were then calculated by fitting the profiles to a series of model equations, which describe radial diffusion in a sphere. D _eff is the combination of dye diffusion through the cytoplasm and through gap junction channels. To calculate the gap junctional permeability (P _j ) an estimate of the cytoplasmic diffusion coefficient (D _cyt = 0.7 × 10⁻⁶ cm²/sec) was obtained by observing the time course of dye diffusion in isolated elongated fiber cells loaded with Lucifer yellow via a patch pipette. Using this approach, we have obtained a value for P _j of 31 × 10⁻⁵ cm/sec for fiber-fiber gap junctions. This value is significantly larger than the value of P _j of 4.4 × 10⁻⁶ cm/sec reported by Rae and coworkers for epithelial-fiber junctions (Rae et al., 1996. J. Membrane Biol. 150:89–103), and most likely reflects the high abundance of gap junctions between lens fiber cells. Received: 1 December 1998/Revised: 22 February 1999     

Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines

Connexin43 is a member of the highly homologous connexin family of gap junction proteins. We have studied how connexin monomers are assembled into functional gap junction plaques by examining the biosynthesis of connexin43 in cell types that differ greatly in their ability to form functional gap junctions. Using a combination of metabolic radiolabeling and immunoprecipitation, we have shown that connexin43 is synthesized in gap junctional communication-competent cells as a 42-kD protein that is efficiently converted to a approximately 46-kD species (connexin43-P2) by the posttranslational addition of phosphate. Surprisingly, certain cell lines severely deficient in gap junctional communication and known cell-cell adhesion molecules (S180 and L929 cells) also expressed 42-kD connexin43. Connexin43 in these communication-deficient cell lines was not, however, phosphorylated to the P2 form. Conversion of S180 cells to a communication-competent phenotype by transfection with a cDNA encoding the cell-cell adhesion molecule L-CAM induced phosphorylation of connexin43 to the P2 form; conversely, blocking junctional communication in ordinarily communication-competent cells inhibited connexin43-P2 formation. Immunohistochemical localization studies indicated that only communication-competent cells accumulated connexin43 in visible gap junction plaques. Together, these results establish a strong correlation between the ability of cells to process connexin43 to the P2 form and to produce functional gap junctions. Connexin43 phosphorylation may therefore play a functional role in gap junction assembly and/or activity.     

Reversible Interruption of Gap Junctional Communication by Testosterone Propionate in Cultured Sertoli Cells and Cardiac Myocytes

A direct cell-to-cell exchange of ions and molecules occurs through specialized membrane channels built by the interaction of two half channels, termed connexons, contributed by each of the two adjacent cells. The electrical and diffusional couplings have been investigated by monitoring respectively the cell-to-cell conductance and the fluorescence recovery after photobleaching, in Sertoli and cardiac cells of young rat. In both cell types, a rapid impairment of the intercellular coupling has been observed in the presence of testosterone propionate. This interruption of the cell-to-cell communication through gap junction channels was dose-dependent, observed in the concentration range 1 to 25 μm and was progressively reversed after withdrawing the testosterone ester. Pretreatment with cyproterone acetate, an antiandrogen which blocks the nuclear testosterone receptor by binding, did not prevent the uncoupling action of the androgen ester. This observation, together with the rapid time course of the uncoupling and recoupling, and the rather high effective concentration (micromolar) of the steroid compound, suggests a nongenomic mechanism of action. The uncoupling concentrations were very similar to those of other steroid compounds known to interrupt gap junctional communication. The uncoupling could result from a direct interaction of the steroid with the proteolipidic structure of the membrane, that might alter the conformation of the gap junction channels and their functional state. Received: 10 April 1995/Revised: 27 October 1995     

Modulation of connexin43 alters expression of osteoblastic differentiation markers

Gap junctional channels between cells provide a pathway for exchange of regulatory ions and small molecules. We previously demonstrated that expression of connexins and cell-to-cell communication parallel osteoblastic differentiation and that nonspecific pharmacological inhibitors of gap junctional communication inhibit alkaline phosphatase activity. In this study, we stably transfected connexin (Cx)43 antisense cDNA into the immortalized human fetal osteoblastic cell line hFOB 1.19 (hFOB/Cx43^–). hFOB/Cx43^– cells express lower levels of Cx43 protein and mRNA and display a 50% decrease in gap junctional intercellular communication relative to control [hFOB/plasmid vector control (pvc)]. This suggests that other connexins, such as Cx45, which is expressed to a similar degree in hFOB/Cx43^– cells and hFOB/pvc cells, contribute to cell-to-cell communication in hFOB 1.19 cells. We observed almost total inhibition of alkaline phosphatase activity in hFOB/Cx43^– cells despite only a 50% decrease in cell-to-cell communication. This suggests the intriguing possibility that Cx43 expression per se, independent of cell-to-cell communication, influences alkaline phosphatase activity and perhaps bone cell differentiation. Quantitative real-time RT-PCR revealed that mRNA levels for osteocalcin and core binding factor 1 (Cbfa1) increased as a function of time in hFOB/pvc but were inhibited in hFOB/Cx43^–. Osteopontin mRNA levels were increased in hFOB/Cx43^– relative to hFOB/pvc and decreased as a function of time in both hFOB/Cx43^– and hFOB/pvc. Transfection with Cx43 antisense did not affect expression of type I collagen in hFOB 1.19 cells. These results suggest that gap junctional intercellular communication and expression of Cx43 contribute to alkaline phosphatase activity, as well as osteocalcin, osteopontin, and Cbfa1 expression in osteoblastic cells. gap junction communication; alkaline phosphatase activity; osteopontin; osteocalcin; hFOB 1.19     

Transfection with Different Connexin Genes Alters Growth and Differentiation of Human Choriocarcinoma Cells

To examine the role of cell–cell communication via gap junctions in controlling proliferation and differentiation we transfected the malignant trophoblast cell line Jeg-3, which exhibits extremely low cell–cell communication mediated by endogenously expressed connexin40, with connexin26, connexin40, and connexin43, respectively.In vitrogrowth of all cell clones transfected with connexin genes was significantly reduced compared to controls. This effect corresponded to a significant increase in total junctional conductance of all clones. Single-channel conductances for channels formed by the transfected connexins were in the range of the values published previously. Though total junctional conductance varied highly among clones and even within one clone, differentiation of the cells indicated by β-hCG secretion was most prominent in the clones that revealed the largest amount of well-coupled cell pairs. Connexin26 channels enable cells of one clone to reduce drastically growth rate and produce significantly higher secretion of β-hCG. Connexin43 had only moderate effects on the differentiation properties of Jeg-3 cells. These findings suggest that restoration of cell–cell communication plays a role in growth reduction and in differentiation of tumor cells and that different channel proteins might have different effects.     

Identification of Isoforms of G Proteins and PKC that Colocalize with Tight Junctions

Recent evidence suggests that the formation and permeability of tight junctions are actively regulated by second-messenger-generating systems involving G proteins and protein kinase C (PKC). A possible specific target for these regulatory proteins is the tight junction protein ZO-1. An extensive immunocytochemical study was performed in cultured epithelial monolayers of MDCK and Caco-2 cells to identify which isoforms of G proteins and PKC are present at or near the zonula occludens complex. Antibodies against α-subunits of each one of the four major subfamilies were used for the localization of the G proteins. For the PKC localization, antibodies against eight different isoforms were used. In confluent monolayers, Gα₁₂ and PKC ζ, were the only isoforms of these proteins present at the cell borders. In subconfluent monolayers, Gα₁₂ and PKC ζ were found at the plasma membrane only along the areas of lateral cell-cell contact. These isoforms formed a pattern of distribution very similar to the ZO-1 protein. The present findings indicate that Gα₁₂ and PKC ζ may be part of the zonula occludens complex and may locally regulate formation and permeability of tight junctions. Received: 29 July 1995/Revised: 13 October 1995     

The stage-specific function of gap junctions during tumourigenesis

Tumour development is a process resulting from the disturbance of various cellular functions including cell proliferation, adhesion and motility. While the role of these cell parameters in tumour promotion and progression has been widely recognized, the mechanisms that influence gap junctional coupling during tumorigenesis remain elusive. Neoplastic cells usually display decreased levels of connexin expression and/or gap junctional coupling. Thus, impaired intercellular communication via gap junctions may facilitate the release of a potentially neoplastic cell from the controlling regime of the surrounding tissue, leading to tumour promotion. However, recent data indicates that metastatic tumour cell lines are often characterized by relatively high levels of connexin expression and gap junctional coupling. This review outlines current knowledge on the role of connexins in tumorigenesis and the possible mechanisms of the interference of gap junctional coupling with the processes of tumour invasion and metastasis. Paper authored by participants of the international conference: XXXIV Winter School of the Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University, Zakopane, March 7–11, 2007, “The Cell and Its Environment”. Publication costs were covered by the organisers of this meeting.     

Voltage Gating of Gap Junctions in Cochlear Supporting Cells: Evidence for Nonhomotypic Channels

The organ of Corti has been found to have multiple gap junction subunits, connexins, which are localized solely in nonsensory supporting cells. Connexin mutations can induce sensorineural deafness. However, the characteristics and functions of inner ear gap junctions are not well known. In the present study, the voltage-dependence of gap junctional conductance (G _j ) in cochlear supporting cells was examined by the double voltage clamp technique. Multiple types of asymmetric voltage dependencies were found for both nonjunctional membrane voltage (V _m ) and transjunctional (V _j ) voltage. Responses for each type of voltage dependence were categorized into four groups. The first two groups showed rectification that was polarity dependent. The third group exhibited rectification with either voltage polarity, i.e., these cells possessed a bell-shaped G _j -V _j or G _j -V _m function. The rectification due to V _j had fast and slow components. On the other hand, V _m -dependent gating was fast (<5 msec), but stable. Finally, a group was found that evidenced no voltage dependence, although the absence of V _j dependence did not preclude V _m dependence and vice versa. In fact, for all groups V _j sensitivity could be independent of V _m sensitivity. The data show that most gap junctional channels in the inner ear have asymmetric voltage gating, which is indicative of heterogeneous coupling and may result from heterotypic channels or possibly heteromeric configurations. This heterogeneous coupling implies that single connexin gene mutations may affect the normal physiological function of gap junctions that are not limited to homotypic configurations. Received: 17 September 1999/Revised: 12 January 2000     

Definition of Epitopes for Monoclonal Antibodies Developed Against Purified Sodium Channel Protein: Implications for Channel Structure

To test sodium channel structural models, we defined the epitopes for nineteen independently cloned monoclonal antibodies previously generated against purified, detergent-solubilized, adult rat skeletal muscle sodium channel protein using channel proteolysis, synthetic peptides, and fusion proteins. All identified epitopes were continuous and unique to the skeletal muscle subtype α-subunit. Of the nineteen independent clones, seventeen had epitopes located either in the origin of the amino-terminus or in the interdomain 2–3 region while only two antibodies had epitopes located in the mid-portion of the interdomain 1–2 region. No immunogenic regions were identified on the α-subunit's extracellular regions, interdomain 3–4 segment, or carboxyl-terminus or on channel β-subunits. While immune tolerance may explain the lack of immunogenicity of extracellular regions, the lack of immunogenicity of most of the channel's cytoplasmic mass may be due to segment inaccessibility from organization of these regions as globular domains, to insertion of parts of these regions into the membrane phase, or to interaction with other protein elements. The definition of monoclonal antibody epitopes allows us to reinterpret previously reported monoclonal antibody competition studies, providing independent support for our model of sodium channel cytoplasmic domain structure. In addition, these data suggest additional testable hypotheses concerning the interactions of the sodium channel amino- and carboxyl-termini with each other as well as with other protein elements. Received: 4 March 1998/Revised: 15 May 1998