Multimeric connexin interactions prior to the trans-Golgi network.
Cells that express multiple connexins have the capacity to form heteromeric (mixed) gap junction hemichannels. We used a dominant negative connexin construct, consisting of bacterial beta-galactosidase fused to the C terminus of connexin43 (Cx43/beta-gal), to examine connexin compatibility in NIH 3T3 cells. Cx43/beta-gal is retained in a perinuclear compartment and inhibits Cx43 transport to the cell surface. The intracellular connexin pool induced by Cx43/beta-gal colocalized with a medial Golgi apparatus marker and was readily disassembled by treatment with brefeldin A. This was unexpected, since previous studies indicated that Cx43 assembly into hexameric hemichannels occurs in the trans-Golgi network (TGN) and is sensitive to brefeldin A. Further analysis by sucrose gradient fractionation showed that Cx43 and Cx43/beta-gal were assembled into a subhexameric complex. Cx43/beta-gal also specifically interacted with Cx46, but not Cx32, consistent with the ability of Cx43/beta-gal to simultaneously inhibit multiple connexins. We confirmed that interactions between Cx43/beta-gal and Cx46 reflect the ability of Cx43 and Cx46 to form heteromeric complexes, using HeLa and alveolar epithelial cells, which express both connexins. In contrast, ROS osteoblastic cells, which differentially sort Cx43 and Cx46, did not form Cx43/Cx46 heteromers. Thus, cells have the capacity to regulate whether or not compatible connexins intermix.
Das Sarma
J
J
Institute for Environmental Medicine, Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Meyer
R A
RA
Wang
F
F
Abraham
V
V
Lo
C W
CW
Koval
M
M
D016475:3T3 Cells; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D020126:Brefeldin A / Q000494:pharmacology; D002458:Cell Fractionation; D002478:Cells, Cultured; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D003902:Detergents / Q000737:chemistry; D017629:Gap Junctions / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008854:Microscopy, Electron; D017830:Octoxynol / Q000737:chemistry; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000502:physiology; D011650:Pulmonary Alveoli / Q000166:cytology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism*
D001426:Bacterial Proteins; D017630:Connexins; D003902:Detergents; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D020126:Brefeldin A; D017830:Octoxynol; D001616:beta-Galactosidase