Claudia Elfgang

Heterotypic gap junction channels (connexin26 – connexin32) violate the paradigm of unitary conductance

Human HeLa cells transfected with mouse DNA coding for connexin26 (Cx26) or connexin32 (Cx32) were used to examine the properties of heterotypic Cx26 – Cx32 gap junction channels. Intercellular current flow was examined in induced cell pairs by means of the dual voltage-clamp method. We found that Cx26 – Cx32 channels exhibit voltage-dependent conductances, γj: γj(main state) increases with increasing positivity at the cytoplasmic aspect of the Cx26 connexon and decreases with increasing negativity (slope: 32 pS/100 mV; γj γj(main state) reaches 48 pS as V j approaches 0 mV); γj(residual state) with a similar Vj-dependence is present when the cytoplasmic end of Cx26 connexon is positive, but absent when it is negative. The single channel data provide an explanation for the asymmetric relationships between the gap junction conductance, gj, and Vj. The results are consistent with the notion that docking of two connexons co-determines the biophysical properties of a gap junction channel.

Immunochemical characterization of the gap junction protein connexin45 in mouse kidney and transfected human HeLa cells

Antibodies to the gap junction protein connexin45 (Cx45) were obtained by immunizing rabbits with fusion protein consisting of glutathione S-transferase and 138 carboxy-terminal amino acids of mouse Cx45. As shown by immunoblotting and immunofluorescence, the affinity-purified antibodies recognized Cx45 protein in transfected human HeLa cells as well as in the kidney-derived human and hamster cell lines 293 and BHK21, respectively. In Cx45-transfected HeLa cells, this protein is phosphorylated as demonstrated by immunoprecipitation after metabolic labeling. The phosphate label could be removed by treatment with alkaline phosphatase. A weak phosphorylation of Cx45 protein was also detected in the cell lines 293 and BHK21. Treatment with dibutyryl cyclic adenosine or guanosine monophosphate (cAMP, cGMP) did not alter the level of Cx45 phosphorylation, in either Cx45 transfectants or in 293 or BHK21 cells. The addition of the tumor-promoting agent phorbol 12-myristate 13-acetate (TPA) led to an increased 32P phosphate incorporation into the Cx45 protein in transfected cells.The Cx45 protein was found in homogenates of embryonic brain, kidney, and skin, as well as of adult lung. In kidney of four-day-old mice, Cx45 was detected in glomeruli and distal tubules, whereas connexin32 and −26 were coexpressed in proximal tubules. No connexin43 protein was detected in renal tubules and glomeruli at this stage of development. Our results suggest that cells in proximal and distal tubules are interconnected by gap junction channels made of different connexin proteins. The Cx45 antibodies characterized in this paper should be useful for investigations of Cx45 in renal gap junctional communication.

Effect of tumor-promoting and anti-promoting chemicals on the viability and junctional coupling of human HeLa cells transfected with DNAs coding for various murine connexin proteins.

Gap-junctional intercellular communication is thought to be essential for maintaining cellular homeostasis and growth control. Its perturbation entails toxicological implications and it has been correlated with the in vivo tumor-promoting potential of chemicals. Little is known about the mechanism(s) responsible for the tumor promoters interference with the cellular coupling. Moreover, nongenotoxic carcinogens, as well as connexins (gap-junctional protein subunits), are known to be organ-/tissue-specific; this implies that the effect of different agents should be evaluated on their specific target, that is, connexin. To investigate the role of different connexins in regulating gap-junctional gating and to compare the properties of homotypic junctional channels, we evaluated the effects of tissue-specific tumor promoters and anti-promoters on the viability and intercellular coupling (dye-transfer) of HeLa cells stably transfected with cDNAs coding for connexin(cx)43, cx40, cx26 and cx32. The results demonstrate that the transfectants possess individual junctional permeabilities, differentially affected by the chemicals, they also show different sensitivities to the cytotoxic effect of the compounds. These findings confirm that connexin diversity may be responsible for the different gating properties of gap-junctional channels, being also suggestive for their separate functions and independent regulatory mechanisms.

Growth inhibition by expression of connexin 26 in HeLa cells

HeLa cells transfected with cDNAs encoding for various types of connexin (connexins 26, 40 and 43) exhibit different growth properties despite a similar dye-transfer capacity. In particular, a clone expressing connexin 26 was not tumorigenic and its growth properties in vitro were altered. These results suggest that the connexins may induce a specific cell behaviour depending on the cell type in which they are expressed.