title: The cataract related mutation N188T in human connexin46 (hCx46) revealed a critical role for residue N188 in the docking process of gap junction channels.
abstract: The mutation N188T in human connexin46 (hCx46) correlates with a congenital nuclear pulverulent cataract. This mutation is in the second extracellular loop, a domain involved in docking of gap junction hemichannels. To analyze the functional consequences of this mutation, we expressed hCx46N188T and the wild type (hCx46wt) in Xenopus oocytes and HeLa cells. In Xenopus oocytes, hemichannels formed by hCx46wt and hCx46N188T had similar electrical properties. Additionally, a Ca(2+) and La(3+) sensitive current was observed in HeLa cells expressing eGFP-labeled hCx46wt or eGFP-labeled hCx46N188T. These results suggest that the N188T mutation did not alter apparent expression and the membrane targeting of the protein. Cells expressing hCx46wt-eGFP formed gap junction plaques, but plaques formed by hCx46N188T were extremely rare. A reduced plaque formation was also found in cells cotransfected with hCx46N188T-eGFP and mCherry-labeled hCx46wt as well as in cocultured cells expressing hCx46N188T-eGFP and hCx46wt-mCherry. Dye transfer experiments in cells expressing hCx46N188T revealed a lower transfer rate than cells expressing hCx46wt. We postulate that the N188T mutation affects intercellular connexon docking. This hypothesis is supported by molecular modeling of hCx46 using the crystal structure of hCx26 as a template. The model indicated that N188 is important for hemichannel docking through formation of hydrogen bonds with the residues R180, T189 and D191 of the opposing hCx46. The results suggest that the N188T mutation hinders the docking of the connexons to form gap junction channels. Moreover, the finding that a glutamine substitution (hCx46N188Q) could not rescue the docking emphasizes the specific role of N188.
authors.affiliation: Institute of Biophysics, Leibniz University Hannover, Germany.
authors.forename: Patrik
authors.initials: P
authors.lastname: Schadzek
authors.affiliation: Institute of Biophysics, Leibniz University Hannover, Germany; Division of Pulmonary, Allergy and Critical Care and Sleep Medicine, Department of Medicine and Department of Cell Biology, Emory School of Medicine, Atlanta, GA, USA.
authors.forename: Barbara
authors.initials: B
authors.lastname: Schlingmann
authors.affiliation: Institute of Biostatistics, Leibniz University Hannover, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Schaarschmidt
authors.affiliation: Institute of Biophysics, Leibniz University Hannover, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Lindner
authors.affiliation: Division of Pulmonary, Allergy and Critical Care and Sleep Medicine, Department of Medicine and Department of Cell Biology, Emory School of Medicine, Atlanta, GA, USA; Department of Cell Biology, Emory University, Atlanta, GA, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Koval
authors.affiliation: Institut für Quantenoptik, Leibniz Universität Hannover, Deutschland.
authors.forename: Alexander
authors.initials: A
authors.lastname: Heisterkamp
authors.affiliation: Institute for Biophysical Chemistry, Hannover Medical School (MHH), Hannover, Germany; Center for Structural Systems Biology, German Electron Synchrotron (DESY), Hamburg, Germany. Electronic address: preller.matthias@mh-hannover.de.
authors.forename: Matthias
authors.initials: M
authors.lastname: Preller
authors.affiliation: Institute of Biophysics, Leibniz University Hannover, Germany; Center for System Neurosciences (ZSN), Hannover, Germany. Electronic address: ngezahayo@biophysik.uni-hannover.de.
authors.forename: Anaclet
authors.initials: A
authors.lastname: Ngezahayo
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent; D017630:Connexins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017629:Gap Junctions / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006860:Hydrogen Bonding; D017136:Ion Transport; D007811:Lanthanum / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D002118:Calcium; D002413:Cations, Divalent; D017630:Connexins; D049452:Green Fluorescent Proteins; D007811:Lanthanum; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein; C082917:GJA3 protein, human; C485184:enhanced green fluorescent protein
keywords: Cataract; Connexin; Dye transfer; Hemichannel docking; Molecular dynamics; Structural modeling