Gating connexin 43 channels reconstituted in lipid vesicles by mitogen-activated protein kinase phosphorylation

The regulation of gap junctional permeability by phosphorylation was examin ed in a model system in which connexin 43 (Cx43) gap junction hemichannels were reconstituted in lipid vesicles. Cx43 was immunoaffinity-purified from rat brain, and Cx43 channels were reconstituted into unilamellar phospholi pid liposomes. The activities of the reconstituted channels were measured b y monitoring liposome permeability. Liposomes containing the Cx43 protein w ere fractionated on the basis of permeability to sucrose using sedimentatio n in an iso-osmolar density gradient. The gradient allowed separation of th e sucrose-permeable and -impermeable liposomes. Liposomes that were permeab le to sucrose were also permeable to the communicating dye molecule lucifer yellow. Permeability, and therefore activity of the reconstituted Cx43 cha nnels, were directly dependent on the state of Cx43 phosphorylation. The pe rmeability of liposomes containing Cx43 channels was increased by treatment of liposomes with calf intestinal phosphatase. Moreover, liposomes formed with Cx43 that had been dephosphorylated by calf intestinal phosphatase tre atment showed increased permeability to sucrose. The role of phosphorylatio n in the gating mechanism of Cx43 channels was supported further by the obs ervation that phosphorylation of Cx43 by mitogen-activated protein kinase r eversibly reduced the permeability of liposomes containing dephosphorylated Cx43. Our results show a direct correlation between gap junctional permeab ility and the phosphorylation state of Cx43.