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 Cys(54), Cys(187), Cys(192), and Cys(198) to Ser showed the failure of Ca2+ propagation to neighboring cells in accordance with the electrical uncoupling between transfected cells, whereas mutations of Cys(61) and Cys(68) to Ser showed the same pattern as the wild type, [C-14]Iodoacetamide labeling of free thiols of cysteine residues in mutant connexin-43s showed that two pairs of intramolecular disulfide bonds are formed between Cys(54) and Cys(192) and between Cys(187) and Cys(198). 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.