title: Functional characterization of a GJA1 frameshift mutation causing oculodentodigital dysplasia and palmoplantar keratoderma.
abstract: A frameshift mutation generated from a dinucleotide deletion (780-781del) in the GJA1 gene encoding Cx43 results in a frameshift yielding 46 aberrant amino acids after residue 259 and a shortened protein of 305 residues compared with the 382 in wild-type Cx43. This frameshift mutant (fs260) causes oculodentodigital dysplasia (ODDD) that includes the added condition of palmoplantar keratoderma. When expressed in a variety of cell lines, the fs260 mutant was typically localized to the endoplasmic reticulum and other intracellular compartments. The fs260 mutant, but not the G138R ODDD-linked Cx43 mutant or a Cx43 mutant truncated at residue 259 (T259), reduced the number of apparent gap junction plaques formed from endogenous Cx43 in normal rat kidney cells or keratinocytes. Interestingly, mutation of a putative FF endoplasmic reticulum retention motif encoded within the 46 aberrant amino acid domain failed to restore efficient assembly of the fs260 mutant into gap junctions. Dual whole cell patch-clamp recording revealed that fs260-expressing N2A cells exerted severely reduced electrical coupling in comparison to wild-type Cx43 or the T259 mutant, whereas single patch capacitance recordings showed that fs260 could also dominantly inhibit the function of wild-type Cx43. Co-expression studies further revealed that the dominant negative effect of fs260 on wild-type Cx43 was dose-dependent, and at a predicted 1:1 expression ratio the fs260 mutant reduced wild-type Cx43-mediated gap junctional conductance by over 60%. These results suggest that the 46 aberrant amino acid residues associated with the frameshift mutant are, at least in part, responsible for the manifestation of palmoplantar keratoderma symptoms.
authors.affiliation: Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario N6A 5C1, Canada.
authors.forename: Xiang-Qun
authors.initials: XQ
authors.lastname: Gong
authors.forename: Qing
authors.initials: Q
authors.lastname: Shao
authors.forename: Crystal S
authors.initials: CS
authors.lastname: Lounsbury
authors.forename: Donglin
authors.initials: D
authors.lastname: Bai
authors.forename: Dale W
authors.initials: DW
authors.lastname: Laird
mesh_terms: D000015:Abnormalities, Multiple / Q000235:genetics; D000818:Animals; D018031:Connexin 43 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005124:Eye Abnormalities / Q000235:genetics*; D016368:Frameshift Mutation; D006367:HeLa Cells; D006801:Humans; D007645:Keratoderma, Palmoplantar / Q000235:genetics*; D007668:Kidney / Q000378:metabolism; D017880:Limb Deformities, Congenital / Q000235:genetics*; D051379:Mice; D009154:Mutation; D051381:Rats; D014071:Tooth Abnormalities / Q000235:genetics*
chemical_list: D018031:Connexin 43