title: Properties of connexin26 gap junctional proteins derived from mutations associated with non-syndromal heriditary deafness.
abstract: Three point mutations of the connexin26 (GJB2) gene associated with hereditary deafness were studied using in vitro expression systems. Mutation M34T results in an amino acid substitution in the first transmembrane domain of the connexin protein, W77R is located in the second transmembrane domain and W44C is in the first extracellular loop. Wild-type and mutated connexin vectors were constructed and transfected into communication-deficient HeLa cells to obtain transient expression of the connexin proteins. Intercellular coupling was subsequently assessed by examining transfer of Lucifer yellow between cells. All three mutations resulted in impaired intercellular coupling. The mechanistic reasons for the functional inadequacies of the mutated proteins were investigated. First, intracellular trafficking and targeting of the expressed connexins were determined by immunohistochemistry. Mutation W77R was inefficiently targeted to the plasma membrane and retained in intracellular stores whereas the other two were targeted to the plasma membrane. Oligomerization assays showed that connexins M34T and W77R failed to assemble efficiently into hexameric gap junction hemichannels, but the W44C mutation did so. A cell-free translation system showed that the mutated proteins were inserted into microsomal membranes but the mutations have different effects on the post-translational properties of the expressed proteins. The results point to the conclusion that mutations in the transmembrane domains of connexin proteins influence gap junction assembly.
authors.affiliation: Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, UK. wmbpem@cardiff.ac.uk
authors.forename: P E
authors.initials: PE
authors.lastname: Martin
authors.forename: S L
authors.initials: SL
authors.lastname: Coleman
authors.forename: S O
authors.initials: SO
authors.lastname: Casalotti
authors.forename: A
authors.initials: A
authors.lastname: Forge
authors.forename: W H
authors.initials: WH
authors.lastname: Evans
mesh_terms: D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D000072259:Connexin 26; D017630:Connexins / Q000235:genetics* / Q000378:metabolism; D003638:Deafness / Q000235:genetics*; D017629:Gap Junctions / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D017354:Point Mutation; D011233:Precipitin Tests
chemical_list: D017630:Connexins; C000607042:GJB2 protein, human; D000072259:Connexin 26