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

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The regulation of gap junctional permeability by phosphorylation was examined 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 phospholipid liposomes. The activities of the reconstituted channels were measured by monitoring liposome permeability. Liposomes containing the Cx43 protein were fractionated on the basis of permeability to sucrose using sedimentation in an iso-osmolar density gradient. The gradient allowed separation of the sucrose-permeable and -impermeable liposomes. Liposomes that were permeable to sucrose were also permeable to the communicating dye molecule lucifer yellow. Permeability, and therefore activity of the reconstituted Cx43 channels, were directly dependent on the state of Cx43 phosphorylation. The permeability 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 treatment showed increased permeability to sucrose. The role of phosphorylation in the gating mechanism of Cx43 channels was supported further by the observation that phosphorylation of Cx43 by mitogen-activated protein kinase reversibly reduced the permeability of liposomes containing dephosphorylated Cx43. Our results show a direct correlation between gap junctional permeability and the phosphorylation state of Cx43.
Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Issue Date
1999-02
Language
English
Article Type
Article
Keywords

GAP-JUNCTION PROTEIN; EPIDERMAL GROWTH-FACTOR; INTERCELLULAR COMMUNICATION; SUBSTRATE RECOGNITION; EPITHELIAL-CELLS; HORIZONTAL CELLS; MEMBRANE CHANNEL; PLASMA-MEMBRANE; GLIOMA-CELLS; RAT-LIVER

Citation

JOURNAL OF BIOLOGICAL CHEMISTRY, v.274, no.9, pp.5581 - 5587

ISSN
0021-9258
URI
http://hdl.handle.net/10203/77756
Appears in Collection
BS-Journal Papers(저널논문)
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