Oculodentodigital Dysplasia-causing Connexin43 Mutants Are Non-functional and Exhibit Dominant Effects on Wild-type Connexin43

Abstract

Oculodentodigital dysplasia, a rare condition displaying congenital craniofacial deformities and limb abnormalities, has been associated with over 20 known human connexin43 (Cx43) mutations. The localization of two of these mutants, G21R and G138R, was examined in Cx43-positive normal rat kidney cells (NRK) and Cx43-negative gap junctional intercellular communication-deficient HeLa cells. Green fluorescent protein-tagged and untagged Cx43 G21R and G138R mutants were transported to the plasma membrane and formed punctate structures reminiscent of gap junction plaques in both NRK and HeLa cells. Further localization studies revealed no significant trafficking defects as subpopulations of Cx43 mutants were found in both the Golgi apparatus and lysosomes, not unlike wild-type Cx43. Dual patch clamp functional analysis of the mutants expressed in gap junctional intercellular communication-deficient N2A cells revealed that neither G21R nor G138R formed functional gap junction channels, although they successfully reached cell-cell interfaces between cell pairs. Importantly, when either mutant was expressed in NRK cells, dye coupling experiments revealed that both mutants inhibited endogenous Cx43 function. These studies suggest that, although patients suffering from oculodentodigital dysplasia possess one wild-type Cx43 allele, it is likely that Cx43-mediated gap junctional intercellular communication is reduced below 50% because of a dominant-negative effect of mutant Cx43 on wild-type Cx43.

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

“…The Cx26 (G12V, W77R, 235delC) and Cx31 (L34P, 141delI) mutants that appear to be retained intracellularly, possibly in the ER, are all associated with recessively inherited hearing loss (Martin et al 1999;Choung et al 2002;D'Andrea et al 2002;Bruzzone et al 2003;He et al 2005). Conversely, most connexin mutants that cause dominantly inherited diseases are not localized to the ER, including the Cx43 mutants that cause oculodentodigital dysplasia (ODDD, Scherer, unpublished; (Seki et al 2004;Roscoe et al 2005;Shibayama et al 2005;Lai et al 2006), a Cx46 and a Cx50 mutant that each cause cataracts (Berthoud et al 2003;Minogue et al 2005), and most of the Cx26, Cx30, and Cx31 mutants that cause hearing loss and/or skin diseases (Martin et al 1999;Rouan et al 2001;Common et al 2002;Di et al 2002;Common et al 2003;Marziano et al 2003;Oshima et al 2003;Thomas et al 2003;Essenfelder et al 2004;Thomas et al 2004;Di et al 2005;He et al 2005;Oguchi et al 2005;Piazza et al 2005). On the other hand, two of the Cx30 mutants (G11R andA88V) that cause skin diseases Essenfelder et al 2004), at least two of the Cx26 mutants (D50N, T55M), three of the Cx31 mutants (66delD, R180X, E183K) that cause dominantly inherited hearing loss Common et al 2003;Di et al 2005;He et al 2005;Melchionda et al 2005), and one Cx43 mutant that causes ODDD and skin disease (fs260, (Gong et al 2006)), appear to be localized to the ER.…”

Functional Characterization of aGJA1Frameshift Mutation Causing Oculodentodigital Dysplasia and Palmoplantar Keratoderma

“…Engineering of Mutant Cx43 cDNAs-The engineering of human Cx43-GFP construct was described previously (24). The human Cx43 mutation-fs260 was constructed with the QuikChange TM Site-directed mutagenesis kit (Stratagene, La Jolla, CA) by using forward 5Ј-GCTGAGCCCTGCCAAAGACTGGGTCTCAAAAATA-TGC-3Ј and reverse 5Ј-GCATATTTTTGAGACCCAGTCT-TTGGCAGGGCTCAGC-3Ј primers and cloned into pcDNA3.1 expression vectors.…”

“…Immunocytochemistry-Cell immunofluorescent labeling and imaging were performed as previously described (24). Briefly, cells grown on 12-mm glass coverslips were fixed with 80% methanol/20% acetone (or a 3.7% formaldehyde solution for GFP-expressing cells) at 4°C for 15 min.…”

“…All GFP-tagged variants were further cloned into the AP2 retroviral vector as described previously (25). Transient Transfection and Retroviral Infection-Cells were transfected with human Cx43-GFP, fs260-GFP, T259-GFP, or fs260-FF/AA-GFP by Lipofectamine 2000 (Invitrogen) as described previously (24). In brief, cells were allowed to reach 50% confluence in 35-mm culture dishes, the plasmids were preincubated with Lipofectamine 2000 reagent for 15 min at room temperature, the complexes were then incubated with the cells for 4 -6 h. Cells were washed and incubated in regular medium for 24 -48 h before being used for patch-clamp recording, or fixed for immunocytochemistry.…”

Cell–cell communication in the osteoblast/osteocyte lineage

“…Cell biology studies have confirmed this premise. While most ODDD mutant proteins can assemble into gap junction plaques, they do not form functional channels [37]; and some of them inhibit Cx43-mediated gap junctional intercellular communication (GJIC) [38].…”