Reconstitution and NMR Characterization of the Ion-Channel Accessory Subunit Barttin in Detergents and Lipid-Bilayer Nanodiscs
Barttin is an accessory subunit of ClC-K chloride channels expressed in the kidney and the inner ear. Main functions of ClC-K/barttin channels are the generation of the cortico-medullary osmotic gradients in the kidney and the endocochlear potential in the inner ear. Mutations in the gene encoding b...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2019-03-01
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| Series: | Frontiers in Molecular Biosciences |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fmolb.2019.00013/full |
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| author | Thibault Viennet Thibault Viennet Stefanie Bungert-Plümke Shantha Elter Aldino Viegas Christoph Fahlke Manuel Etzkorn Manuel Etzkorn |
| author_facet | Thibault Viennet Thibault Viennet Stefanie Bungert-Plümke Shantha Elter Aldino Viegas Christoph Fahlke Manuel Etzkorn Manuel Etzkorn |
| author_sort | Thibault Viennet |
| collection | DOAJ |
| description | Barttin is an accessory subunit of ClC-K chloride channels expressed in the kidney and the inner ear. Main functions of ClC-K/barttin channels are the generation of the cortico-medullary osmotic gradients in the kidney and the endocochlear potential in the inner ear. Mutations in the gene encoding barttin, BSND, result in impaired urinary concentration and sensory deafness. Barttin is predicted to be a two helical integral membrane protein that directly interacts with its ion channel in the membrane bilayer where it stabilizes the channel complex, promotes its incorporation into the surface membrane and leads to channel activation. It therefore is an attractive target to address fundamental questions of intermolecular communication within the membrane. However, so far inherent challenges in protein expression and stabilization prevented comprehensive in vitro studies and structural characterization. Here we demonstrate that cell-free expression enables production of sufficient quantities of an isotope-labeled barttin variant (I72X Barttin, capable to promote surface membrane insertion and channel activation) for NMR-based structural studies. Additionally, we established purification protocols as well as reconstitution strategies in detergent micelles and phospholipid bilayer nanodiscs. Stability, folding, and NMR data quality are reported as well as a suitable assignment strategy, paving the way to its structural characterization. |
| first_indexed | 2024-12-21T03:53:04Z |
| format | Article |
| id | doaj.art-906940fdf8b44d32a92c8751e623d547 |
| institution | Directory Open Access Journal |
| issn | 2296-889X |
| language | English |
| last_indexed | 2024-12-21T03:53:04Z |
| publishDate | 2019-03-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Molecular Biosciences |
| spelling | doaj.art-906940fdf8b44d32a92c8751e623d5472022-12-21T19:16:55ZengFrontiers Media S.A.Frontiers in Molecular Biosciences2296-889X2019-03-01610.3389/fmolb.2019.00013446884Reconstitution and NMR Characterization of the Ion-Channel Accessory Subunit Barttin in Detergents and Lipid-Bilayer NanodiscsThibault Viennet0Thibault Viennet1Stefanie Bungert-Plümke2Shantha Elter3Aldino Viegas4Christoph Fahlke5Manuel Etzkorn6Manuel Etzkorn7Institute of Physical Biology, Heinrich-Heine-University Düsseldorf, Düsseldorf, GermanyInstitute of Complex Systems 6, Forschungszentrum Jülich, Wilhelm-Johnen-Strasse, Jülich, GermanyInstitute of Complex Systems 4, Forschungszentrum Jülich, Wilhelm-Johnen-Strasse, Jülich, GermanyInstitute of Physical Biology, Heinrich-Heine-University Düsseldorf, Düsseldorf, GermanyInstitute of Physical Biology, Heinrich-Heine-University Düsseldorf, Düsseldorf, GermanyInstitute of Complex Systems 4, Forschungszentrum Jülich, Wilhelm-Johnen-Strasse, Jülich, GermanyInstitute of Physical Biology, Heinrich-Heine-University Düsseldorf, Düsseldorf, GermanyInstitute of Complex Systems 6, Forschungszentrum Jülich, Wilhelm-Johnen-Strasse, Jülich, GermanyBarttin is an accessory subunit of ClC-K chloride channels expressed in the kidney and the inner ear. Main functions of ClC-K/barttin channels are the generation of the cortico-medullary osmotic gradients in the kidney and the endocochlear potential in the inner ear. Mutations in the gene encoding barttin, BSND, result in impaired urinary concentration and sensory deafness. Barttin is predicted to be a two helical integral membrane protein that directly interacts with its ion channel in the membrane bilayer where it stabilizes the channel complex, promotes its incorporation into the surface membrane and leads to channel activation. It therefore is an attractive target to address fundamental questions of intermolecular communication within the membrane. However, so far inherent challenges in protein expression and stabilization prevented comprehensive in vitro studies and structural characterization. Here we demonstrate that cell-free expression enables production of sufficient quantities of an isotope-labeled barttin variant (I72X Barttin, capable to promote surface membrane insertion and channel activation) for NMR-based structural studies. Additionally, we established purification protocols as well as reconstitution strategies in detergent micelles and phospholipid bilayer nanodiscs. Stability, folding, and NMR data quality are reported as well as a suitable assignment strategy, paving the way to its structural characterization.https://www.frontiersin.org/article/10.3389/fmolb.2019.00013/fullbarttinion channellipid bilayer nanodiscdetergent micellenuclear magnetic resonance |
| spellingShingle | Thibault Viennet Thibault Viennet Stefanie Bungert-Plümke Shantha Elter Aldino Viegas Christoph Fahlke Manuel Etzkorn Manuel Etzkorn Reconstitution and NMR Characterization of the Ion-Channel Accessory Subunit Barttin in Detergents and Lipid-Bilayer Nanodiscs Frontiers in Molecular Biosciences barttin ion channel lipid bilayer nanodisc detergent micelle nuclear magnetic resonance |
| title | Reconstitution and NMR Characterization of the Ion-Channel Accessory Subunit Barttin in Detergents and Lipid-Bilayer Nanodiscs |
| title_full | Reconstitution and NMR Characterization of the Ion-Channel Accessory Subunit Barttin in Detergents and Lipid-Bilayer Nanodiscs |
| title_fullStr | Reconstitution and NMR Characterization of the Ion-Channel Accessory Subunit Barttin in Detergents and Lipid-Bilayer Nanodiscs |
| title_full_unstemmed | Reconstitution and NMR Characterization of the Ion-Channel Accessory Subunit Barttin in Detergents and Lipid-Bilayer Nanodiscs |
| title_short | Reconstitution and NMR Characterization of the Ion-Channel Accessory Subunit Barttin in Detergents and Lipid-Bilayer Nanodiscs |
| title_sort | reconstitution and nmr characterization of the ion channel accessory subunit barttin in detergents and lipid bilayer nanodiscs |
| topic | barttin ion channel lipid bilayer nanodisc detergent micelle nuclear magnetic resonance |
| url | https://www.frontiersin.org/article/10.3389/fmolb.2019.00013/full |
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