Biochemical and immunochemical analysis of the arrangement of connexin43 in rat heart gap junction membranes
A 43 × 10^3 M_r 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.
Additional Information© 1990 The Company of Biologists Limited. Received 23 February 1989 - Accepted, in revised form, 6 June 1990. The authors thank Dr S. B. Yancey, Dr R. Lai and Dr K. Puranam and Mr J. Hoh for their advice and critical comments in preparing this manuscript. A special thanks to Dr S. A. John for his expert help in constructing the Cx43 model. We also extend our gratitude to J. Edens, P. Koen and B. Austin for their excellent technical assistance. This research was supported by National Institutes of Health grant HL 37109, a Canadian Heart Foundation research fellowship, the A. B. Ruddock Fund and the Markey Foundation, as well as BRSG grant RR07003.
Published - LAIjcs90.pdf