Properties of connexin26 gap junctional proteins derived from mutations associated with non-syndromal heriditary deafness

Three point mutations of the connexin26 (GJB2) gene associated with heredit ary 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 an d W44C is in the first extracellular loop. Wild-type and mutated connexin v ectors were constructed and transfected into communication-deficient HeLa c ells 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 coup ling. The mechanistic reasons for the functional inadequacies of the mutate d proteins were investigated, First, intracellular trafficking and targetin g of the expressed connexins were determined by immunohistochemistry. Mutat ion W77R was inefficiently targeted to the plasma membrane and retained in intracellular stores whereas the other two were targeted to the plasma memb rane, 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 pr oteins were inserted into microsomal membranes but the mutations have diffe rent effects on the post-translational properties of the expressed proteins , The results point to the conclusion that mutations in the transmembrane d omains of connexin proteins influence gap junction assembly.