title: A dominant negative mutation of neuronal connexin 36 that blocks intercellular permeability.
abstract: 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.
authors.affiliation: Department of Physiology and Neuroscience, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA.
authors.forename: Dimitris
authors.initials: D
authors.lastname: Placantonakis
authors.forename: Federico
authors.initials: F
authors.lastname: Cicirata
authors.forename: John P
authors.initials: JP
authors.lastname: Welsh
mesh_terms: 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
chemical_list: D017630:Connexins; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D000097004:Gap Junction delta-2 Protein