PMID:9430691

Citation	Toyofuku, T, Yabuki, M, Otsu, K, Kuzuya, T, Hori, M and Tada, M (1998) Intercellular calcium signaling via gap junction in connexin-43-transfected cells. J. Biol. Chem. 273:1519-28
Abstract	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.
Links	PubMed
Keywords	Calcium/physiology; Calcium Channels/metabolism; Cell Line; Connexin 43/genetics; Connexin 43/physiology; Estrenes/pharmacology; Gap Junctions/physiology; Humans; Inositol 1,4,5-Trisphosphate/metabolism; Inositol 1,4,5-Trisphosphate Receptors; Mutagenesis, Site-Directed; Phosphodiesterase Inhibitors/pharmacology; Pyrrolidinones/pharmacology; Receptors, Cytoplasmic and Nuclear/metabolism; Ryanodine Receptor Calcium Release Channel/metabolism; Signal Transduction; Transfection; Type C Phospholipases/antagonists & inhibitors
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
HUMAN:CXA1		GO:0005921: gap junction	ECO:0000314:		C		Figure 1 shows northern blot analysis of RNA from transfected HEK294 cells,an immunoblot analysis of proteins from transfected HEK294 cells, and immunocytochemical localization of connexin-43 and ryanodine receptor in cultured cells.	complete CACAO 9437

References

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