Assembly of gap junction channels: mechanism, effects of calmodulin antagonists and identification of connexin oligomerization determinants.
The assembly of connexins (Cxs) into gap junction intercellular communication channels was studied. An in vitro cell-free synthesis system showed that formation of the hexameric connexon hemichannels involved dimeric and tetrameric connexin intermediates. Cx32 contains two putative cytoplasmic calmodulin-binding sites, and their role in gap junction channel assembly was investigated. The oligomerization of Cx32 into connexons was reversibly inhibited by a calmodulin-binding synthetic peptide, and by W7, a naphthalene sulfonamide calmodulin antagonist. Removing the calmodulin-binding site located at the carboxyl tail of Cx32 limited connexon formation and resulted in an accumulation of intermediate connexin oligomers. This truncation mutant, Cx32Delta215, when transiently expressed in COS-7 cells, accumulated intracellularly and had failed to target to gap junctions. Immunoprecipitation studies suggested that a C-terminal sequence of Cx32 incorporating the calmodulin-binding site was required for the formation of hetero-oligomers of Cx26 and Cx32 but not for Cx32 homomeric association. A chimera, Cx32TM3CFTR, in which the third transmembrane and proposed channel lining sequence of Cx32 was substituted by a transmembrane sequence of the cystic fibrosis transmembrane conductance regulator, did not oligomerize in vitro and it accumulated intracellularly when expressed in COS-7 cells. The results indicate that amino-acid sequences in the third transmembrane domain and a calmodulin-binding domain in the cytoplasmic tail of Cx32 are likely candidates for regulating connexin oligomerization.
Ahmad
S
S
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, Wales, UK.
Martin
P E
PE
Evans
W H
WH
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017630:Connexins / Q000737:chemistry*; D006224:Cricetinae; D013449:Sulfonamides / Q000494:pharmacology
D002147:Calmodulin; D017630:Connexins; D013449:Sulfonamides; C017967:W 7