Structure-function analysis of connexin32

Individual cell-cell channels consist of two hemichannels, located on neighboring cell membranes, that are interconnected to form an hydrophilic pathway. Each hemichannel, or connexon, is made of six protein subunits, called connexins.Connexin32 liver gap junction protein has four transmembrane segments, two extracellular regions and three cytoplasmic segments, which include the amino and carboxyl termini.The process of cell-cell channel formation was investigated by altering specific amino acids in the presumed extracellular domains of the connexin32. It is these domains that must interact when two hemichannels dock to form an open cell-cell channel. The mutant connexins were generated by site-directed in vitro mutagenesis of a connexin32 cDNA. The mutated cDNAs were then transcribed in vitro and the mRNA was injected into Xenopus oocytes for expression. Junctional conductances between paired oocytes resulting from the expression of the mRNA were measured by the double-voltage clamp technique.Every amino acid replacement in connexin32 affected its channel-forming ability. Some replacements altered the docking specificity of the hemichannel. The replacement of any one of the six cysteines in the extracellular loops by serine yielded proteins incapable of making channels, indicating that the extracellular cysteines play a crucial role in channel formation.In addition, deletion mutants in the carboxyterminal domain of connexin32 were generated in order to determine the shortest segment still capable of forming channels. The transmembrane topology of some of these deletion mutants was investigated by in vitro translation in a reticulocyte lysate supplemented with canine microsomes.Net positive charge and length of the carboxyterminal segment were found to be determinants for channel formation. Phosphorylation of Ser233 and Ser240 was found not to be necessary for channel formation when expressed in Xenopus oocytes.Finally, connexin32 hexamer formation was studied by native polyacrylamide gel electrophoresis, sucrose gradients of the in vitro translation products combined with crosslinking reagents