A dominant negative mutation of neuronal connexin 36 that blocks intercellular permeability.
Rat connexin 36 (Cx36) was mutated by substituting serine for cysteine at residue 231 (C231S) and the mutant's effect on the subcellular localization of wild-type Cx36 and the intercellular permeability that it confers was determined in human HeLa and rat PC12 cells. Cells transfected with the mutant or wild-type Cx36 cDNA expressed the expected 36 kDa protein and Cx36 immunoreactivity. Co-immunoprecipitation experiments with monkey COS-7 cells transiently transfected with both mutant and wild-type Cx36 cDNAs demonstrated that the mutant protein bound to the wild-type. Double immunofluorescence microscopy of stably transfected HeLa cells demonstrated that mutant Cx36 blocked the transport of the wild-type Cx36 to the cell membrane, primarily by trapping it in the endoplasmic reticulum around the nucleus. Coexpression of the mutant Cx36 with the wild-type protein abolished the ability of the latter to permit dye transfer in both HeLa and PC12 cells. The findings are the first demonstration of a mutation of Cx36 that inhibits wild-type Cx36 function in mammalian cells.
Placantonakis
Dimitris
D
Department of Physiology and Neuroscience, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA.
Cicirata
Federico
F
Welsh
John P
JP
D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells / Q000378:metabolism; D002450:Cell Communication / Q000235:genetics*; D002522:Chlorocebus aethiops; D017630:Connexins / Q000235:genetics* / Q000502:physiology; D018076:DNA, Complementary / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism*; D005799:Genes, Dominant; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism / Q000648:ultrastructure; D016716:PC12 Cells / Q000378:metabolism; D017354:Point Mutation; D021381:Protein Transport; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection; D000097004:Gap Junction delta-2 Protein
D017630:Connexins; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D000097004:Gap Junction delta-2 Protein