Mutation of a Conserved Threonine in the Third Transmembrane Helix of α- and β-Connexins Creates a Dominant-negative Closed Gap Junction Channel

摘要: Single site mutations in connexins have provided insights about the influence specific amino acids on gap junction synthesis, assembly, trafficking, and functionality. We discovered a single point mutation that eliminates functionality without interfering with formation. The occurs at threonine residue located near cytoplasmic end of third transmembrane helix. This is strictly conserved among members α- β-connexin subgroups but not γ-subgroup. In HeLa cells, connexin43 connexin26 mutants are synthesized, traffic to plasma membrane, make junctions same overall appearance as wild type. isolated connexin26T135A both from cells baculovirus-infected insect Sf9 cells. By using cryoelectron microscopy correlation averaging, difference images revealed small significant size change within pore region slight rearrangement subunits between mutant wild-type connexons expressed Purified, detergent-solubilized contain hexameric partially disassembled structures, although almost all hexameric, suggesting three-dimensional connexon unstable. Mammalian expressing plaques composed either connexin43T154A or showed an absence dye coupling. When Xenopus oocytes, these mutants, well cysteine substitution connexin50 (connexin50T157C), failed produce electrical coupling homotypic heteromeric pairings type dominant-negative effect. may be useful tool for knocking down out connexin function vitro vivo.