Abstract
Renal salt loss in Bartter's syndrome is caused by impaired transepithelial transport in the loop of Henle. Sodium chloride is taken up apically by the combined activity of NKCC2 (Na+-K--2Cl- cotransporters) and ROMK potassium channels. Chloride ions exit from the cell through basolateral ClC-Kb chloride channels. Mutations in the three corresponding genes have been identified1,2,3 that correspond to Bartter's syndrome types 1–3. The gene4 encoding the integral membrane protein barttin is mutated in a form of Bartter's syndrome that is associated with congenital deafness and renal failure. Here we show that barttin acts as an essential β-subunit for ClC-Ka and ClC-Kb chloride channels, with which it colocalizes in basolateral membranes of renal tubules and of potassium-secreting epithelia of the inner ear. Disease-causing mutations in either ClC-Kb or barttin compromise currents through heteromeric channels. Currents can be stimulated further by mutating a proline-tyrosine (PY) motif on barttin. This work describes the first known β-subunit for CLC chloride channels and reveals that heteromers formed by ClC-K and barttin are crucial for renal salt reabsorption and potassium recycling in the inner ear5.
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Acknowledgements
We thank S. Alper for the AE1 antibody, M. Knepper for the aquaporin-2 antibody, M. Knipper for advice on inner ear immunohistochemistry, and J. Enderich and M. Kolster for technical assistance. R.E. is a recipient of a Marie Curie Human Potential Fellowship of the European Union, and F.H. is a Heisenberg scholar of the Deutsche Forschungsgemeinschaft (DFG). This work was supported by grants from the DFG, the Fonds der Chemischen Industrie, and the Prix Louis Jeantet de Médecine to T.J.J., and from the Federal State of Baden-Württemberg to F.H.
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Supplementary information
Figure A. Functional characterisation of CIC-K/barttin channels expressed in transfected tsA201 cells
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Figure B. Topology of barttin
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The first hydrophobic region of barttin may span the membrane or may act as a cleavable signal peptide. The latter possibility is suggested by the program SMART (http://smart.embl-heidelberg.de). To distinguish between these possibilities, we epitope-tagged barttin at either end. This did not affect its ability to elicit currents with ClC-Ka. When these constructs were expressed in COS cells with or without ClC-Ka (Ka) and analysed by Western blotting using an antibody against the epitope, bands corresponding in size to barttin were stained with comparable intensities irrespective of the position of the epitope (Fig. B). Mock-transfected COS cells (Ctrl) and ClC-Ka transfected COS cells were used as a controls (left two lanes). This argues against an amino-terminal cleavage, and supports a model in which barttin has two transmembrane spanning segments in its amino-terminal end.
Figure C. RT-PCR detection of ClC-K1 and ClC-K2 in the cochlea
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To examine whether ClC-K1 and ClC-K2 is expressed in the cochlea, we performed RT-PCR experiments on RNA obtained from mouse cochlea. The primers used could differentiate between the highly homologous (~90% identity) ClC-K1 and ClC-K2 mRNAs. Using PCR conditions and primers described in reference 24, we observed bands of the correct sizes in samples containing cochlear RNA, but not in controls (Fig. C). Thus, both ClC-K1 and ClC-K2 are expressed in the cochlea. Because immunofluorescence indicates that ClC-K channels are only present in the stria vascularis, this strongly suggests that these cells express both isoforms.
Figure D. Antibodies against barttin
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Estévez, R., Boettger, T., Stein, V. et al. Barttin is a Cl- channel β-subunit crucial for renal Cl- reabsorption and inner ear K+ secretion. Nature 414, 558–561 (2001). https://doi.org/10.1038/35107099
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DOI: https://doi.org/10.1038/35107099
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