Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells.

Abstract

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.

Loss-of-function GJA12/Connexin47 mutations cause Pelizaeus–Merzbacher-like disease

“…Communication-incompetent HeLa cells (Elfgang et al 1995) were obtained from Dr. Klaus Willecke (University of Bonn, Bonn, Germany), and grown in low-glucose Dulbecco's modified Eagle's Medium (DMEM) supplemented by 10% fetal bovine serum (FBS) and antibiotics (100 units/ml penicillin & 100 μg/ml streptomycin; GIBCO Invitrogen, Carlsbad, CA) in a humidified atmosphere containing 5% CO 2 at 37°C. For transfection, 10 μl of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and 4 μg of plasmid DNA were incubated separately in Optimem (GIBCO Invitrogen, Carlsbad, CA) for 15 min at RT, then combined for another 20 min.…”

Expression of Connexin43 in mouse Leydig, Sertoli, and germinal cells at different stages of postnatal development

“…The reactivity of these antibodies was compared with that of a previously characterized anti-Cx43 antibody described elsewhere (Sá ez et al, 1997). Cx40 and Cx43-transfected HeLa cells (Elfgang et al, 1995) were kindly provided by Dr. Klaus Willecke, Institute fü r Genetik, Universitä t Bonn, Germany.…”

“…It is also indicated by the occasional detection of Cx43 between adjacent GCNA1-positive cells. Thus, homotypic, heterotypic or heteromeric channels containing Cx43 (Bukauskas et al, 1995;Elenes et al, 1999;Elfgang et al, 1995, Oh et al, 1999 may communicate between these cell types. Cx43-mediated heterocellular contacts between Sertoli and germinal cells is consistent with recent findings of Cx43 gene transcript in germinal and Sertoli cells by in situ hybridization (Batias et al, 2000).…”

Long-term connexin-mediated bystander effect in highly tumorigenic human cells in vivo in herpes simplex virus thymidine kinase/ganciclovir gene therapy

“…Their phenotype has been previously characterized. 21,32 These cell lines were transfected with an expression vector, provided by Dr G Tiraby (P Sabatier University, Toulouse, France), carrying the gene coding for the thymidine kinase of the herpes simplex 1 (HSVtk) virus under a CMV promoter, as previously described. 17 All four different phenotypes (Cx43 + -tk − , Cx43 + -tk + , Cx43 − -tk − and Cx43 − -tk + ) were cultivated in Dulbecco's minimum essential medium (Gibco, Paisley, UK) and 10% fetal calf serum (Dutscher, Brumath, France).…”