Effect of antipeptide antibodies directed against three domains of connexin43 on the gap junctional permeability of cultured heart cells.
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.
Bastide
B
B
Laboratoire de Physiologie Cellulaire, Unité de Recherche Associée au CNRS no 1869, Faculté des Sciences, Poitiers, France.
Jarry-Guichard
T
T
Briand
J P
JP
Délèze
J
J
Gros
D
D
D000818:Animals; D000906:Antibodies / Q000494:pharmacology*; D000918:Antibody Specificity / Q000276:immunology; D002450:Cell Communication / Q000502:physiology; D002478:Cells, Cultured; D018031:Connexin 43 / Q000276:immunology*; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism*; D008845:Microinjections / Q000379:methods; D009206:Myocardium / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology / Q000494:pharmacology*; D010539:Permeability / Q000187:drug effects*; D051381:Rats
D000906:Antibodies; D018031:Connexin 43; D010446:Peptide Fragments