title: Gap junction assembly: PTX-sensitive G proteins regulate the distribution of connexin43 within cells.
abstract: Cells expressing connexin43 are able to upregulate gap junction (GJ) communication by enhancing the assembly of new GJs, apparently through increased connexin trafficking. Because G proteins are known to regulate different aspects of protein trafficking, we examined the effects of pertussis toxin (PTX; a specific inhibitor of certain G proteins) on GJ assembly. Dissociated Novikoff hepatoma cells were reaggregated for 60 min to form nascent junctions. PTX inhibited GJ assembly, as indicated by a reduction in dye transfer. Electron microscopy also revealed a 60% decrease in the number of GJ channels per cell interface. Importantly, PTX blocked the twofold enhancement in GJ assembly found in the presence of low-density lipoprotein. Two G(i alpha) proteins (G(i alpha 2) and G(i alpha 3)), which have been implicated in the control of membrane trafficking, reacted with PTX in ADP-ribosylation studies. PTX and/or the trafficking inhibitors, brefeldin A and monensin, inhibited GJ assembly to comparable degrees. In addition, assays for GJ hemichannels demonstrated reduced plasma membrane levels of connexin43 following PTX treatment. These results suggest that PTX-sensitive G proteins regulate connexin43 trafficking, and, as a result of inhibition with PTX, the number of plasma membrane hemichannels available for GJ assembly is reduced.
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA. plampe@fhcrc.org
authors.forename: P D
authors.initials: PD
authors.lastname: Lampe
authors.forename: Q
authors.initials: Q
authors.lastname: Qiu
authors.forename: R A
authors.initials: RA
authors.lastname: Meyer
authors.forename: E M
authors.initials: EM
authors.lastname: TenBroek
authors.forename: T F
authors.initials: TF
authors.lastname: Walseth
authors.forename: T A
authors.initials: TA
authors.lastname: Starich
authors.forename: H L
authors.initials: HL
authors.lastname: Grunenwald
authors.forename: R G
authors.initials: RG
authors.lastname: Johnson
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology; D008078:Cholesterol, LDL / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D005614:Freeze Fracturing; D019204:GTP-Binding Proteins / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism* / Q000648:ultrastructure; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D007476:Ionophores / Q000494:pharmacology; D008985:Monensin / Q000494:pharmacology; D037342:Pertussis Toxin; D010766:Phosphorylation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
chemical_list: D008078:Cholesterol, LDL; D018031:Connexin 43; D007476:Ionophores; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D010566:Virulence Factors, Bordetella; D005576:Colforsin; D020126:Brefeldin A; D000246:Adenosine Diphosphate Ribose; D008985:Monensin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins