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The gap -junctional channels that mediate intercellular communication are formed by head -to -head docking of two gap -junctional hemichannels from adjacent cells . The hemichannels are hexamers of connexins , proteins that have four transmembrane helices . The transmembrane helices that line the gap -junctional pore have not been identified , and their identification was the main goal of my dissertation project . To accomplish this goal , I used a combination of molecular biology , biochemical and biophysical techniques that include poly -alanine helix scanning mutagenesis , the substituted cysteine accessibility method and luminescence resonance energy transfer . Using the latter methodology in particular , as well as a new method to produce purified hemichannels of controlled subunit composition , I was able to assign all helices in the available low -resolution cryoelectron microscopy structure published by others , where helices are named A through D , and generate the first model of gap -junctional channels and hemichannels based on experimental structural measurements . In this model , connexin transmembrane helices 1 through 4 correspond to helices A , C , B and D , respectively . Luminescence resonance energy transfer is a powerful method for structural studies of membrane proteins in their native bilayer environment . Taken advantage of this methodology , in combination with the generation of hemichannels of controlled subunit composition , I was also able to determine that PKC -mediated phosphorylation of Ser368 produces a partial closure of the Cx43 hemichannel pore , that this effects requires phosphorylation of all six Cx43 monomers in the hemichannel , and that the decrease in permeability is accompanied by significant conformational changes of the connexin molecules . These changes involve increases of the distances separating the C -terminal ends of the subunits and decreases in the distances separating the pore -lining helices ; both changes in inter -subunit distances are of the order of several Angstroms . These results indicate that a simple ball -and -chain mechanism cannot explain the gating of Cx43 hemichannels by PKC -mediated phosphorylation and that a significant re -arrangement of pore helices takes place instead . In summary , my results allowed me to generate an experimentally -based model gap -junctional channels and hemichannels and to gain insight into the molecular mechanism of Cx43 regulation by PKC -mediated phosphorylation . |
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