Barttin modulates trafficking and function of ClC-K channels

Ute Scholl

Simon Hebeisen

Audrey Janssen

Gerhard Müller-Newen

Alexi Alekov

Christoph Fahlke

*Abteilung Neurophysiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany;

^{Abteilung Physiologie and}

^{Institut für Biochemie, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen, 52074 Aachen, Germany;}

^{Centro de Estudios Cientificos (CECS), Valdivia 509000, Chile; and}

^{Zentrum für Systemische Neurowissenschaften Hannover (ZSN), 30559 Hannover, Germany}

Edited by Steven C. Hebert, Yale University School of Medicine, New Haven, CT, and approved June 9, 2006

^{U.S. and S.H. contributed equally to this work.}

**To whom correspondence should be addressed. E-mail: ed.revonnah-hm@hpotsirhc.eklhaf

Author contributions: U.S., S.H., A.G.H.J., G.M.-N., A.A., and C.F. designed research; U.S., S.H., A.G.H.J., and A.A. performed research; U.S., S.H., A.G.H.J., G.M.-N., A.A., and C.F. analyzed data; and U.S., A.A., and C.F. wrote the paper.

Edited by Steven C. Hebert, Yale University School of Medicine, New Haven, CT, and approved June 9, 2006

Received 2006 Feb 27

Abstract

Barttin is an accessory subunit of a subgroup of ClC-type chloride channels expressed in renal and inner ear epithelia. In this study, we examined the effects of barttin on two ClC-K channel isoforms, rat ClC-K1 and human ClC-Kb, using heterologous expression, patch clamping, confocal imaging, and flow cytometry. In the absence of barttin, only a small percentage of rClC-K1 and hClC-Kb channels are inserted into the plasma membrane. Coexpression of barttin enhances surface membrane insertion and furthermore modifies permeation and gating of ClC-K channels. hClC-Kb channels are nonfunctional without barttin and require the coexpressed accessory subunit to become anion conducting. In contrast, rClC-K1 channels are active without barttin, but at the cost of reduced unitary conductance as well as altered voltage dependence of activation. We mapped the separate functions of barttin to structural domains by a deletion analysis. Whereas the transmembrane core is necessary and sufficient to promote ClC-K channel exit from the endoplasmic reticulum, a short cytoplasmic segment following the second transmembrane helix modifies the unitary conductance. The entire cytoplasmic carboxyl terminus affects the open probability of ClC-K channels. The multiple functions of barttin might be necessary for a tight adjustment of epithelial Cl^{conductances to ensure a precise regulation of body salt content and endocochlear potential.}

Keywords: chloride channels, gating, kidney, ClC family, accessory subunit

Abstract

Barttin was identified as a protein encoded by the disease gene of a variant of Bartter’s syndrome, BSND, that combines a salt-losing nephropathy with sensorineural deafness and renal failure (1, 2), and later shown to be an accessory subunit of a subclass of ClC channels, namely the ClC-K channels (3, 4). ClC-K channels are necessary for chloride absorption in various sections of the nephron (5, 6) as well as for potassium secretion in the stria vascularis and the vestibular labyrinth. For the majority of these channels, heterologous expression results in measurable anion currents only when expressed together with barttin (3, 4, 7, 8). So far, this effect of barttin on ClC channels has been interpreted in terms of trafficking alone, i.e., that ClC-K channels are retained in intracellular membrane systems and reach the cell surface after association with barttin (3, 4, 9).

We examined the functional and molecular basis of the regulation of two different ClC-K channels, rat ClC-K1 (10) and human ClC-Kb (11), by barttin using a combination of heterologous expression, site-directed mutagenesis, confocal imaging, flow cytometry, and cellular electrophysiology. Our study demonstrates that this accessory subunit modifies not only the surface membrane density, but also permeation and gating of ClC-K channels. By controlling these diverse parameters, barttin effectively and tightly controls the macroscopic chloride conductance in renal and inner ear epithelia.

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Acknowledgments

We thank Drs. A. L. George (Vanderbilt University, Nashville, TN) and S. Uchida (Tokyo Medical and Dental University) for providing the expression constructs for hClC-Kb, rClC-K1, and barttin. We thank Drs. Tania Alekova, Patricia Hidalgo, Heider Linder, Günther Schmalzing, and Saba Sile for helpful discussions; Dr. S. Radtke for help with the flow cytometry; Drs. A. Lückhoff and P. Heinrich for support; Barbara Poser for excellent technical assistance; and Dr. Stefan Wüllner (RWTH, Aachen, Germany) for providing the MDCK cells. These studies were supported by Deutsche Forschungsgemeinschaft Grants FOR450, TP10 (to C.F.), and SFB 542, Zentralprojekt Z1 (to G.M.-N.) and by the Studienstiftung des deutschen Volkes (U.S.).

Acknowledgments

Footnotes

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Footnotes

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