TY - JOUR
T1 - Reconstitution and NMR Characterization of the Ion-Channel Accessory Subunit Barttin in Detergents and Lipid-Bilayer Nanodiscs
AU - Viennet, Thibault
AU - Bungert-Plümke, Stefanie
AU - Elter, Shantha
AU - Viegas, Aldino
AU - Fahlke, Christoph
AU - Etzkorn, Manuel
N1 - Publisher Copyright:
© 2019 Viennet, Bungert-Plümke, Elter, Viegas, Fahlke and Etzkorn.
PY - 2019
Y1 - 2019
N2 - 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.
AB - 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.
KW - Barttin
KW - Detergent micelle
KW - Ion channel
KW - Lipid bilayer nanodisc
KW - Nuclear magnetic resonance
UR - http://www.scopus.com/inward/record.url?scp=85065418304&partnerID=8YFLogxK
U2 - 10.3389/fmolb.2019.00013
DO - 10.3389/fmolb.2019.00013
M3 - Journal article
AN - SCOPUS:85065418304
SN - 2296-889X
VL - 6
JO - Frontiers in Molecular Biosciences
JF - Frontiers in Molecular Biosciences
IS - 13
M1 - 13
ER -