Analysis of gap junction assembly using mutated connexins detected in Charcot-Marie-Tooth X-linked disease

The assembly of gap junction intercellular communication channels was studi ed by analysis of the molecular basis of the dysfunction of connexin 32 mut ations associated with the X-linked form of Charcot-Marie-Tooth disease in which peripheral nervous transmission is impaired. A cell-free translation system showed that six recombinant connexin 32 mutated proteins-four point mutations at the cytoplasmic amino terminus, one at the membrane aspect of the cytoplasmic carboxyl terminus, and a deletion in the intracellular loop -were inserted into microsomal membranes and oligomerised into connexon hem ichannels with varying efficiencies. The functionality of the connexons was determined by the ability of HeLa cells expressing the respective connexin cDNAs to transfer Lucifer yellow, The intracellular trafficking properties of the mutated connexins were determined by immunocytochemistry. The resul ts show a relationship between intracellular interruption of connexin traff icking, the efficiency of intercellular communication, and the severity of the disease phenotype, Intracellular retention was explained either by defi ciencies in the ability of connexins to oligomerise or by mutational change s at two targeting motifs, The results point to dominance of two specific t argeting motifs: one at the amino terminus and one at the membrane aspect o f the cytoplasmically located carboxyl tail. An intracellular loop deletion of six amino acids, associated with a mild phenotype, showed partial oligo merisation and low intercellular dye transfer compared with wild-type conne xin 32, The results show that modifications in trafficking and assembly of gap junction channels emerge as a major feature of Charcot-Marie-Tooth X-li nked disease.