T. Toyofuku et al., INTERCELLULAR CALCIUM SIGNALING VIA GAP JUNCTION IN CONNEXIN-43-TRANSFECTED CELLS, The Journal of biological chemistry, 273(3), 1998, pp. 1519-1528
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.