INTERCELLULAR CALCIUM SIGNALING VIA GAP JUNCTION IN CONNEXIN-43-TRANSFECTED CELLS

In excitable cells, intracellular Ca2+ is released via the ryanodine r eceptor from the intracellular Ca2+ storing structure, the sarcoplasmi c reticulum, To determine whether this released Ca2+ propagates throug h gap junctions to neighboring cells and thereby constitutes a long ra nge signaling network, we developed a cell system in which cells expre ssing 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-respons ive cells to neighboring cells was observed with a Ca2+ imaging system using fura-2/AM, Inhibitors of gap junctional communication rapidly a nd reversibly abolished this propagation of Ca2+, Together with the el ectrophysiological analysis of transfected cells, the observed interce llular Ca2+ wave was revealed to be due to the reconstituted gap junct ion of transfected cells. We next evaluated the functional roles of cy steine residues in the extracellular loops of connexin-43 in gap junct ional communication. Mutations of Cys(54), Cys(187), Cys(192), and Cys (198) to Ser showed the failure of Ca2+ propagation to neighboring cel ls in accordance with the electrical uncoupling between transfected ce lls, whereas mutations of Cys(61) and Cys(68) to Ser showed the same p attern as the wild type, [C-14]Iodoacetamide labeling of free thiols o f cysteine residues in mutant connexin-43s showed that two pairs of in tramolecular disulfide bonds are formed between Cys(54) and Cys(192) a nd between Cys(187) and Cys(198). These results suggest that intercell ular Ca2+ signaling takes place in cultured cells expressing connexin- 43, leading to their own synchronization and that the extracellular di sulfide bonds of connexin-43 are crucial for this process.