Characterization of gap junctional communication-deficient mutants of a rat liver epithelial cell line

To understand the mechanism(s) regulating gap junctional communication, we isolated gap junctional intercellular communication-deficient (GJIC-) mutant clones of a rat liver epithelial cell line, WB F-344, which is hypoxanthine-guanine phosphoribosyl transferase deficient (HGPRT-). The cells were exposed to a mutagenesis regimen and cocultured with the wild type HGPRT+ cells. Four GJIC- and one positive clones were characterized in the present study. Northern analysis of RNA isolated from both mutant and parental cells showed a single RNA species of about 3.0 kb which hybridized to connexin43 (Cx43) cDNA. Western blot analysis confirmed the expression of this junctional protein in all these clones. However, in the GJIC- clones the slowest migrating band corresponding to a hyperphosphorylated form, P2, of Cx43 protein (approximately 46 kDa) was absent suggesting that loss of this phosphorylated form of Cx43 may be involved in the failure of the mutants to establish cell-cell communication. Immunofluorescence analysis of the mutants did not reveal any differences in the distribution and localization of Cx43 between GJIC+ and GJIC- clones suggesting that the loss of phosphorylation did not affect the membrane association of this protein. Taken together, these data suggest that one mechanism for the loss of communication in these GJIC- mutants may be the consequence of a change in the intrinsic phosphorylation state of Cx43 protein.