532 Research Article Introduction Connexins oligomerize to form gap junctions (Nicholson, 2003; Saez et al., 2003). Gap junctions allow direct intercellular communication between adjacent cells via transfer of ions and molecules less than 1 kDa in size. Formation of a gap junctional channel requires docking of two connexons, also known as hemichannels. The existence of connexin hemichannels has been previously viewed as a transient intermediate step in gap junction assembly. Recent studies suggest that connexin hemichannels may function independently to mediate physiological release of signaling molecules such as ATP, glutamate, PGE 2 and NAD into the extracellular space, thereby enabling paracrine stimulation of neighboring cells (Bennett et al., 2003; Goodenough and Paul, 2003; Jiang and Cherian, 2003). Cx43 is a connexin family member that is expressed throughout human tissues, including the central nervous system, heart and bone (Wei et al., 2004). Within the central nervous system, Cx43 is the primary gap junction constituent in astrocytes (Theis et al., 2004). Recently, the importance of Cx43 in promoting normal development has been emphasized by the discovery that ODDD, a rare pleiotropic developmental disorder that is usually inherited in an autosomal dominant fashion, is caused by mutations in Cx43 (Paznekas et al., 2003). Thus far, over 35 distinct missense mutations have been published that involve most domains of the protein. In addition, a mutation in the C-terminal cytoplasmic tail (C260fsX307) that results in a truncated protein with 46 incorrect amino acids has also been described (Kjaer et al., 2004; Paznekas et al., 2003; Pizzuti et al., 2004; Richardson et al., 2004; Vitiello et al., 2005). The pleiotropic phenotype can be presumably explained by the presence of Cx43 in multiple tissue types throughout the body. The mutations in Cx43 that are associated with ODDD parallel several disease-causing mutations in other connexin genes, such as Cx26 and Cx32. A large array of different 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. Key words: Connexin43, Oculodentodigital dysplasia (ODDD), Hemichannel, Gap junction, Glia Summary Oculodentodigital dysplasia connexin43 mutations result in non-functional connexin hemichannels and gap junctions in C6 glioma cells Albert Lai 1,2, *, Dung-Nghi Le 1,2 , William A. Paznekas 3 , Wes D. Gifford 1,2 , Ethylin Wang Jabs 3,4 and Andrew C. Charles 1 1 Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA 2 The Henry E Singleton Brain Cancer Research Program, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA 3 Institute of Genetic Medicine, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA 4 Departments of Medicine and Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA *Author for correspondence (email: albertlai@mednet.ucla.edu) Accepted 31 October 2005 Journal of Cell Science 119, 532-541 Published by The Company of Biologists 2006 doi:10.1242/jcs.02770 Journal of Cell Science JCS ePress online publication date 17 January 2006