Biochemical and immunochemical analysis of the arrangement of connexin43 in rat heart gap junction membranes.
A 43 x 10(3) Mr protein (designated connexin43 or Cx43) is a major constituent of heart gap junctions. The understanding of its arrangement in junctional membranes has been extended by means of site-directed antibodies raised against synthetic peptides of Cx43. These represent part of the first extracellular loop (EL-46), the cytoplasmic loop (CL-100), the second extracellular loop (EL-186) and carboxy-terminal sequences (CT-237 and CT-360). All of the antibodies raised reacted with their respective peptides and the Cx43 protein on Western blots. By immunoelectron microscopy two of the antibodies (CL-100 and CT-360) were shown to label the cytoplasmic surface of isolated gap junction membranes. Immunofluorescent labeling at locations of neonatal cardiac myocyte-myocyte apposition required an alkali/urea treatment when the EL-46 and EL-186 antibodies were used. Immunoblot analysis of endoproteinase Lys-C-digested gap junctions revealed that the Cx43 protein passed through the lipid bilayer four times. Alkaline phosphatase digestion of isolated junctions was used to show that the CT-360 antibody recognized many phosphorylated forms of Cx43. Our results unequivocally confirm models of the organization of Cx43 that were based on a more limited set of data and a priori considerations of the sequence.
Laird
D W
DW
Division of Biology, California Institute of Technology, Pasadena 91125.
Revel
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D000917:Antibody Formation; D015153:Blotting, Western; D002478:Cells, Cultured; D017630:Connexins; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D007365:Intercellular Junctions / Q000737:chemistry*; D008297:Male; D008565:Membrane Proteins / Q000032:analysis*; D008566:Membranes / Q000737:chemistry; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry* / Q000166:cytology; D010447:Peptide Hydrolases; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains
D017630:Connexins; D008565:Membrane Proteins; D010447:Peptide Hydrolases