Evidence for gap junctional communication of second messengers

Abstract

Gap junction channels reconstituted in liposomes show direct evidence for the transfer of the second messengers of cyclic AMP (cAMP) and inositol 1,4,5-trisphosphate ($IP_3$) through the channels. Two types of connexin proteins, Connexin 43 and connexin 32, were purified by using immunoaffinity gel chromatography under nondenaturing conditions from rat brain and liver respectively. It appeared that the purified samples contained both connexin monomers and the hexamers, connexon. The purified connexin 43 and connexin 32 were then reconstituted into artificial unilamella liposomes. The liposomes were applied on the isoosmolar sucrose density gradient and separated into two populations of sucrose-permeable and impermeable liposomes based on the permeability. It was found that the sucrose-permeability was due to the presence of the open gap junction channels in the liposome bilayer and the reconstituted channels appeared to be pH-sensitive and permeable to Lucifer Yellow, a well-known communicating dye molecule. To assess the transfer of second messenger molecules through the channels, the liposomes were formed to entrap the tritium labeled second messengers and the distribution of the second messengers in the gradient was examined. Both cAMP and $IP_3$ were retained only in sucrose-impermeable liposomes which do not contain open channel of connexon while they were leaked out from liposomes containing open channels of Cx43. Liposomes containing open channels of Cx32 also failed to retain the second messengers. These results provide a direct evidence of the transfer of the second messengers through gap junction channels.