Abstract
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.
Highlights
ClC-K channels form a subgroup of chloride channels within the ClC family of chloride channels and chloride/proton antiporters
Barttin belongs to those membrane proteins that are difficult to produce in large quantities in conventional expression systems
We confirm the usefulness of cell-free expression systems for membrane proteins that are challenging to express in bacterial systems (Katzen et al, 2009)
Summary
ClC-K channels form a subgroup of chloride channels within the ClC family of chloride channels and chloride/proton antiporters. They are expressed in the kidney and in the inner ear, and are essential for NaCl re-absorption in the loop of Henle and for potassium secretion by the stria vascularis (Fahlke and Fischer, 2010; Stolting et al, 2014). Barttin is the accessory β-subunit of ClC-K channels. It is the gene product of BSND, the disease gene of a rare human disease, called Bartter syndrome IV. Barttin has been shown to be necessary for the function of both chloride channels ClC-Ka and ClC-Kb (Estevez et al, 2001) present, respectively, in inner ears and in kidneys. Promotes its insertion into the plasma membrane, and turns the channel into a conductive state (Hayama et al, 2003; Scholl et al, 2006)
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