Effect of antipeptide antibodies directed against three domains of connexin43 on the gap junctional permeability of cultured heart cells

J Membr Biol. 1996 Apr;150(3):243-53. doi: 10.1007/s002329900048.

Abstract

Cell-to-cell communication can be blocked by intracellular injections of antibodies raised against gap junction proteins, but the mechanism of channel obstruction is unknown. Binding to connexins could lead to a conformational change, interfere with regulatory domains or cause a steric hindrance. To address these questions, the effects on cell-to-cell communication of affinity purified polyclonal antibodies raised against peptides reproducing the intracellular sequences 5-17, 314-322 and 363-382 of rat connexin43 were investigated in cultured rat ventricular cells. The antibodies against sequence 363-382 were characterized by immunoblotting and immunocytochemistry. Characterization of antibodies 5-17 and 314-322 has been previously reported. In a first series of experiments, the effect on gap junctional communication was assessed by injecting a junction-permeant fluorescent dye into cells adjacent to one cell previously microinjected with antibodies. In a second series, junctional permeability was quantitatively determined on records of fluorescence recovery after the photobleaching of 6-carboxyfluorescein-loaded cells. Antibodies 5-17 marked a 43 kDa band on immunoblots, but did not immunolabel gap junctions and had no functional effect. Antibodies 314-322 recognized the 43 kDa protein and labeled the intercalated disks, but failed to interfere with junctional permeability. Antibodies to the nearby sequence 363-382, for which all immunospecific tests had been positive, caused a delayed diffusional uncoupling in 50% of the microinjected cells. It is suggested that the blocking of junctional communication by antibodies results from interference with a regulatory domain of the connexin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies / pharmacology*
  • Antibody Specificity / immunology
  • Cell Communication / physiology
  • Cells, Cultured
  • Connexin 43 / immunology*
  • Gap Junctions / drug effects
  • Gap Junctions / metabolism*
  • Microinjections / methods
  • Myocardium / chemistry
  • Myocardium / cytology*
  • Myocardium / metabolism
  • Peptide Fragments / immunology
  • Peptide Fragments / pharmacology*
  • Permeability / drug effects*
  • Rats

Substances

  • Antibodies
  • Connexin 43
  • Peptide Fragments