Analysis of Connexin43 phosphorylated at S325, S328 and S330 in normoxic and ischemic heart.
The functional consequences of Connexin43 (Cx43) phosphorylation remain largely unexplored. Using an antibody that specifically recognizes Cx43 phosphorylated at serine residues 325, 328 and/or 330 (pS325/328/330-Cx43), we show that labeling of this form of Cx43 as well as of total Cx43 is restricted to the intercalated disk region of normal ventricular tissue. In ischemic heart, significant relocalization of total Cx43 to the lateral edges of myocytes was evident; however pS325/328/330-Cx43 remained predominately at the intercalated disk. Western blots indicated a eightfold decrease in pS325/328/330-Cx43 in ischemic tissue. Peptide-binding- and competition-experiments indicated that our antibody mainly detected Cx43 phosphorylated at S328 and/or S330 in heart tissue. To evaluate how this change in Cx43 phosphorylation contributes to ischemia-induced downregulation of intercellular communication, we stably transfected Cx43(-/-) cells with a Cx43 construct in which serine residues 325, 328 and 330 had been mutated to alanine (Cx43-TM). Cx43-TM was not efficiently processed to isoforms that have been correlated with gap junction assembly. Nevertheless, Cx43-TM cells were electrically coupled, although development of coupling was delayed. Fully opened channels were only rarely observed in Cx43-TM cells, and Lucifer-Yellow-dye-coupling was significantly reduced compared with wild-type cells. These data suggest that phosphorylation of Cx43 at serine residues 325, 328 and/or 330 influences channel permselectivity and regulates the efficiency of gap junction assembly.
Lampe
Paul D
PD
Molecular Diagnostics Program, Fred Hutchinson Cancer Research Center, Department of Pathobiology, University of Washington, 1100 Fairview Avenue N., M5C800, PO Box 19024, Seattle, WA 98109, USA. plampe@fhcrc.org
Cooper
Cynthia D
CD
King
Timothy J
TJ
Burt
Janis M
JM
D000818:Animals; D002450:Cell Communication / Q000502:physiology*; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism*; D006352:Heart Ventricles / Q000378:metabolism; D007074:Immunoglobulin G / Q000276:immunology; D015640:Ion Channel Gating; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D017202:Myocardial Ischemia / Q000378:metabolism* / Q000473:pathology; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D051381:Rats; D012694:Serine / Q000378:metabolism
D018031:Connexin 43; D007074:Immunoglobulin G; D012694:Serine