Intercellular calcium signaling via gap junction in connexin-43-transfected cells.
In excitable cells, intracellular Ca2+ is released via the ryanodine receptor from the intracellular Ca2+ storing structure, the sarcoplasmic reticulum. To determine whether this released Ca2+ propagates through gap junctions to neighboring cells and thereby constitutes a long range signaling network, we developed a cell system in which cells expressing both connexin-43 and ryanodine receptor are surrounded by cells expressing only connexin-43. When the ryanodine receptor in cells was activated by caffeine, propagation of Ca2+ from these caffeine-responsive cells to neighboring cells was observed with a Ca2+ imaging system using fura-2/AM. Inhibitors of gap junctional communication rapidly and reversibly abolished this propagation of Ca2+. Together with the electrophysiological analysis of transfected cells, the observed intercellular Ca2+ wave was revealed to be due to the reconstituted gap junction of transfected cells. We next evaluated the functional roles of cysteine residues in the extracellular loops of connexin-43 in gap junctional communication. Mutations of Cys54, Cys187, Cys192, and Cys198 to Ser showed the failure of Ca2+ propagation to neighboring cells in accordance with the electrical uncoupling between transfected cells, whereas mutations of Cys61 and Cys68 to Ser showed the same pattern as the wild type. [14C]Iodoacetamide labeling of free thiols of cysteine residues in mutant connexin-43s showed that two pairs of intramolecular disulfide bonds are formed between Cys54 and Cys192 and between Cys187 and Cys198. These results suggest that intercellular Ca2+ signaling takes place in cultured cells expressing connexin-43, leading to their own synchronization and that the extracellular disulfide bonds of connexin-43 are crucial for this process.
Toyofuku
T
T
Department of Medicine and Pathophysiology, Osaka University Medical School, Japan. toyofuku@mr-path.med.osaka.u.ac.jp
Yabuki
M
M
Otsu
K
K
Kuzuya
T
T
Hori
M
M
Tada
M
M
D002118:Calcium / Q000502:physiology*; D015220:Calcium Channels / Q000378:metabolism; D002460:Cell Line; D018031:Connexin 43 / Q000235:genetics / Q000502:physiology*; D004963:Estrenes / Q000494:pharmacology; D017629:Gap Junctions / Q000502:physiology*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors; D016297:Mutagenesis, Site-Directed; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
D015220:Calcium Channels; D018031:Connexin 43; D004963:Estrenes; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D018160:Receptors, Cytoplasmic and Nuclear; D019837:Ryanodine Receptor Calcium Release Channel; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D002118:Calcium