DNAs coding for seven murine connexins (Cx) (Cx26, Cx31, Cx32, Cx37, Cx40, Cx43, and Cx45) are functionally expressed in human HeLa cells that were deficient in gap junctional communication. We compare the permeabilities of gap junctions comprised of different connexins to iontophoretically injected tracer molecules. Our results show that Lucifer yellow can pass through all connexin channels analyzed. On the other hand, propidium iodide and ethidium bromide penetrate very poorly or not at all through Cx31 and Cx32 channels, respectively, but pass through channels of other connexins. 4,6 Diamidino-2-phenylindole (DAPI) dihydrochloride shows less transfer among Cx31 or Cx43 transfectants. Neurobiotin is weakly transferred among Cx31 transfectants. Total junctional conductance in Cx31 or Cx45 transfected cells is only about half as high as in other connexin transfectants analyzed and does not correlate exactly with any of the tracer permeabilities. Permeability through different connexin channels appears to be dependent on the molecular structure of each tracer, i.e. size, charge and possibly rigidity. This supports the hypothesis that different connexin channels show different permeabilities to second messenger molecules as well as metabolites and may fulfill in this way their specific role in growth control and differentiation of cell types. In addition, we have investigated the function of heterotypic gap junctions after co-cultivation of two different connexin transfectants, one of which had been prelabeled with fluorescent dextran beads. Analysis of Lucifer yellow transfer reveals that HeLa cells expressing Cx31 (beta-type connexin) do not communicate with any other connexin transfectant tested but only with themselves. Two other beta-type connexin transfectants, HeLa-Cx26 and -Cx32, do not transmit Lucifer yellow to any of the alpha-type connexins analyzed. Among alpha- type connexins, Cx40 does not communicate with Cx43. Thus, connexins differ in their ability to form functional heterotypic gap junctions among mammalian cells.

By using antibodies directed against gapjunction proteins of liver (connexins 26 and 32) and heart (connexin 43), we have localized immunoreactivity to specific cell types in frozen sections of adult rodent brains. Connexin 32 reactivity was found in oligodendrocytes and also in a few neurons, whereas reactivity to connexins 26 and 43 was localized to leptomeningeal cells, ependymal cells, and pineal gland. Immunoreactivity with antibodies to connexin 43 also occurred in astrocytes. Furthermore, during embryonic and postnatal maturation of brain tissues, gap junction proteins were differentially expressed. Connexins 43 and 26 predominated in the neuroepithelium of embryonic brains, whereas connexin 32 was virtually absent. Between 3 and 6 weeks after birth, connexin 26 largely disappeared from immature brain; this time course corresponded to the increased expression of connexin 32. Expression of connexin 43 remained high throughout embryonic and postnatal development. These findings demonstrate that gap junction expression in the brain is diverse, with specific cell types expressing different connexins; this cell-specific distribution may imply differences in the function of these intercellular channels in different loci and developmental stages.Gap junctions are the structural domains through which electrical transmission and metabolic and ionic cooperation between contiguous cells are thought to be mediated. Recent progress in the understanding of the molecular composition of at least four types of gapjunctions found in liver, heart, and lens fibers has been derived from immunochemical (1-5) and molecular biological studies (6-10). The message emerging from these discoveries is that gap junctions are composed of a family of homologous proteins that are expressed in various amounts in different cell types (11,12). We have shown (11, 12) that at least two gap junction proteins are present in liver.Nervous tissue was among the first organs where gap junction membrane contacts were structurally and physiologically characterized (for review, see ref. 13). Recent studies using antibodies against multiple determinants have demonstrated that gap junctions are abundant in brain (14,15), but the composition of these gap junction proteins was not unambiguously determined.By using affinity-purified polyclonal and monoclonal anticonnexin-32 antibodies, an antibody to the liver protein connexin 26, and a polyclonal antibody directed to the carboxylterminal domain of the heart protein connexin 43, we have evaluated the patterns of expression of these proteins in adult and embryonic brains. Our data provide evidence that specific sets of gap junction proteins are expressed by specific brain cell populations. In addition, we found that during development relative amounts of connexin 26 and connexin 32 shift. Connexin 26, which is abundant in embryonic brain, becomes confined to leptomeningeal and ependymal cells and to pinealocytes. Connexin 32, which is not expressed to a large extent in embryonic brain, is expressed ...

The gap junctional protein connexin32 is expressed in hepatocytes, exocrine pancreatic cells, Schwann cells, and other cell types. We have inactivated the connexin32 gene by homologous recombination in the mouse genome and have generated homozygous connexin32-deficient mice that were viable and fertile but weighed on the average -17% less than wild-type controls. Electrical stimulation of sympathetic nerves in connexin32-deficient liver triggered a 78% lower amount of glucose mobilization from glycogen stores, when compared with wild-type liver. Thus, connexin32-containing gap junctions are essential in mouse liver for maximal intercellular propagation of the noradrenaline signal from the periportal (upstream) area, where it is received from sympathetic nerve endings, to perivenous (downstream) hepatocytes. In connexin32-defective liver, the amount of connexin26 protein expressed was found to be lower than in wild-type liver, and the total area of gap junction plaques was -1000-fold smaller than in wild-type liver. In contrast to patients with connexin32 defects suffering from X chromosome-linked Charcot-Marie-Tooth disease (CMTX) due to demyelination in Schwann cells of peripheral nerves, connexin32-deficient mice did not show neurological abnormalities when analyzed at 3 months of age. It is possible, however, that they may develop neurodegenerative symptoms at older age.

Intercellular channels of gap junctions are formed in vertebrates by the protein family of connexins and allow direct exchange of ions, metabolites and second messenger molecules between apposed cells (reviewed in [1-3]). In the mouse, connexin40 (Cx40) protein has been detected in endothelial cells of lung and heart and in certain heart muscle cells: atrial myocytes, cells of the atrial ventricular (AV) node and cells of the conductive myocardium, which conducts impulses from the AV node to ventricular myocyctes [3]. We have generated mice homozygous for targeted disruption of the Cx40 gene (Cx40-/-mice). The electrocardiograph (ECG) parameters of Cx40-/- mice were very prolonged compared to those of wild type (Cx40+/+) mice, indicating that Cx40-/- mice have lower atrial and ventricular conduction velocities. For 6 out of 31 Cx40-/- animals, different types of atrium-derived abnormalities in cardiac rhythm were recorded, whereas continuous sinus rhythm was observed for the 26 Cx40+/+ and 30 Cx40+/- mice tested. The expression levels of other connexins expressed in heart (Cx37, Cx43 and Cx45) were the same in Cx40-/- and Cx40+/+ mice. Our results demonstrate the function of Cx40 in the regulation and coordination of heart contraction and show that cardiac arrhythmogenesis can not only be caused by defects in the ion channels primarily involved in cellular excitation but also by defects in intercellular communication through gap junction channels. As the distribution of Cx40 protein is similar in mouse and human hearts, further functional analysis of Cx40 should yield relevant insights into arrhythmogenesis in human patients.

Gap junction channels which are responsible for direct intercellular communication are composed of connexin proteins. Different connexins are distributed in a tissue-specific manner. Up to now only connexin26 has been identified to be widely expressed in the inner ear. In order to investigate the role of additional gap junction proteins, the expression of connexin30 and 43 was investigated in the rat cochlea. Connexin26 and connexin30 were both expressed in the spiral limbus, the spiral ligament, the stria vascularis and between supporting cells of the organ of Corti. Double-labeling experiments suggest that both connexins are partly colocalized between cells. Weak staining of connexin43 could only be detected in the stria vascularis, the spiral ligament and between organ of Corti supporting cells. The corresponding transcripts for connexin26, 30 and 43 could be detected by Northern blot analysis. The expression of different gap junction channels in the cochlea suggests functional diversity. Gap junctions in the inner ear may control ion concentrations of cochlear fluids or act as conduits through which glucose and other metabolites diffuse.