Roles of Met-34, Cys-64, and Arg-75 in the assembly of human connexin 26. Implication for key amino acid residues for channel formation and function.
Connexins form a family of membrane proteins that assemble into communication channels and directly connect the cytoplasms of adjoining cells. Malfunctioning of connexin channels often cause disease, such as the mutations M34T and R75W in human connexin 26, which are associated with hereditary deafness. Another residue known to be essential for normal channel activity in the connexin is Cys-64. To obtain structural and functional insights of connexin 26, we studied the roles of these three residues by expressing mutant connexins in insect Sf9 and HeLa cells. The M34T and M34A mutants both formed gap junction plaques, but dye transfer assays showed that the M34A mutant had a significantly reduced permeability, suggesting that for proper channel function a side chain of adequate size is required at this position. We propose that Met-34 is located in the innermost helix of the channel, where it ensures a fully open channel structure via interactions with other transmembrane helices. Gap junction channels formed by the R75W and R75D mutants dissociated upon solubilization in dodecyl maltoside, whereas the R75A mutant remained hexameric. All gap junctions formed by Arg-75 mutants also showed only negligible activity in dye transfer experiments. These results suggest that residue Arg-75 plays a role in subunit interactions needed to retain a functional and stable connexin hexamer. The C64S mutant was suggested to be defective in oligomerization and/or protein folding even in the presence of wild-type connexin.
Oshima
Atsunori
A
Department of Biophysics, Graduate School of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.
Doi
Tomoko
T
Mitsuoka
Kaoru
K
Maeda
Shoji
S
Fujiyoshi
Yoshinori
Y
D000818:Animals; D001120:Arginine / Q000502:physiology*; D002850:Chromatography, Gel; D000072259:Connexin 26; D017630:Connexins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D003545:Cysteine / Q000502:physiology*; D017629:Gap Junctions / Q000648:ultrastructure; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008715:Methionine / Q000502:physiology*; D008854:Microscopy, Electron; D016297:Mutagenesis, Site-Directed; D018411:Spodoptera
D017630:Connexins; D008164:Luminescent Proteins; D000072259:Connexin 26; D049452:Green Fluorescent Proteins; D001120:Arginine; D008715:Methionine; D003545:Cysteine