Oculodentodigital dysplasia connexin43 mutations result in non-functional connexin hemichannels and gap junctions in C6 glioma cells.

Oculodentodigital dysplasia (ODDD) is a rare developmental disorder characterized by craniofacial and limb abnormalities. Over 35 separate mutations in human connexin43 (Cx43) causing ODDD have been identified. Several mutations are also associated with central nervous system involvement, including white-matter changes detected by magnetic resonance imaging. As Cx43 is abundantly expressed in astrocytes, we hypothesized that the mutant Cx43 proteins that produce neurological dysfunction have abnormal functional characteristics in astrocytes. To understand how ODDD-associated mutations affect Cx43 signaling in cells of glial origin, we conducted studies in rat C6 glioma cells, a communication-deficient glial cell line that expresses low levels of Cx43. We generated stable cell lines expressing enhanced yellow fluorescent protein (eYFP)-tagged human Cx43 constructs encoding wild-type and six eYFP-tagged mutant Cx43 mutants: Y17S, G21R, A40V, F52dup, L90V and I130T. Of these, Y17S, L90V and I130T are associated with neurological abnormalities. We found that all mutants could be detected on the cell surface. Y17S, G21R, A40V, L90V and I130T formed triton-resistant plaques representing gap junctions, although the relative ability to form plaques was decreased in these mutants compared with the wild type. F52dup formed dramatically reduced numbers of plaques. Propidium iodide uptake experiments demonstrated that all mutants were associated with reduced connexin hemichannel function compared with wild type. Scrape-loading experiments performed on the same stable cell lines showed reduced gap junctional dye transfer in all mutants compared with the wild type. These studies demonstrated that ODDD-associated Cx43 mutations result in non-functional connexin hemichannels and gap junction functions in a glial cell line regardless of whether the particular mutant is associated with neurological dysfunction.