The cataract related mutation N188T in human connexin46 (hCx46) revealed a critical role for residue N188 in the docking process of gap junction channels
| Autor: | Julia Lindner, Michael Koval, Frank Schaarschmidt, Alexander Heisterkamp, Patrik Schadzek, Matthias Preller, Anaclet Ngezahayo, Barbara Schlingmann |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
Hemichannel docking
0301 basic medicine Patch-Clamp Techniques Cations Divalent Green Fluorescent Proteins Molecular Sequence Data Biophysics Xenopus Gene Expression Connexin Molecular dynamics Biology Biochemistry Molecular Docking Simulation Cataract Connexins Connexon Xenopus laevis 03 medical and health sciences Genes Reporter Lanthanum Escherichia coli Animals Humans Structural modeling Amino Acid Sequence Patch clamp Ion Transport Wild type Gap junction Gap Junctions Hydrogen Bonding Cell Biology biology.organism_classification Molecular biology Recombinant Proteins Cell biology Luminescent Proteins 030104 developmental biology Amino Acid Substitution Docking (molecular) Mutation Calcium Dye transfer HeLa Cells |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - Biomembranes. 1858:57-66 |
| ISSN: | 0005-2736 |
| DOI: | 10.1016/j.bbamem.2015.10.001 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect