Cytoplasmic amino acids within the membrane interface region influence connexin oligomerization.
Gap junction channels composed of connexins connect cells, allowing intercellular communication. Their cellular assembly involves a unique quality-control pathway. Some connexins [including connexin43 (Cx43) and Cx46] oligomerize in the trans-Golgi network following export of stabilized monomers from the endoplasmic reticulum (ER). In contrast, other connexins (e.g., Cx32) oligomerize early in the secretory pathway. Amino acids near the cytoplasmic aspect of the third transmembrane domain have previously been shown to determine this difference in assembly sites. Here, we characterized the oligomerization of two connexins expressed prominently in the vasculature, Cx37 and Cx40, using constructs containing a C-terminal dilysine-based ER retention/retrieval signal (HKKSL) or treatment with brefeldin A to block ER vesicle trafficking. Both methods led to intracellular retention of connexins, since the cells lacked gap junction plaques. Retention of Cx40 in the ER prevented it from oligomerizing, comparable to Cx43. By contrast, ER-retained Cx37 was partially oligomerized. Replacement of two amino acids near the third transmembrane domain of Cx43 (L152 and R153) with the corresponding amino acids from Cx37 (M152 and G153) resulted in early oligomerization in the ER. Thus, residues that allow Cx37 to oligomerize early in the secretory pathway could restrict its interactions with coexpressed Cx40 or Cx43 by favoring homomeric oligomerization, providing a structural basis for cells to produce gap junction channels with different connexin composition.
Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Emory University School of Medicine, Whitehead Biomedical Research Building, Atlanta, GA 30022, USA.
Tekla D
TD
Smith
Aditi
A
Mohankumar
Peter J
PJ
Minogue
Eric C
EC
Beyer
Viviana M
VM
Berthoud
Michael
M
Koval
D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D018031:Connexin 43 / Q000737:chemistry / Q000378:metabolism; D017630:Connexins / Q000737:chemistry / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D017629:Gap Junctions / Q000378:metabolism; D006801:Humans; D055503:Protein Multimerization; D013329:Structure-Activity Relationship; D000097006:Gap Junction alpha-4 Protein
D000596:Amino Acids; D018031:Connexin 43; D017630:Connexins; D000097006:Gap Junction alpha-4 Protein