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Fluoride-dependent interruption of the transport cycle of a CLC Cl−/H+ antiporter

Nature Chemical Biology volume 9, pages 721–725 (2013)Cite this article

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Abstract

Cl−/H+ antiporters of the CLC superfamily transport anions across biological membranes in varied physiological contexts. These proteins are weakly selective among anions commonly studied, including Cl−, Br−, I−, NO3− and SCN−, but they seem to be very selective against F−. The recent discovery of a new CLC clade of F−/H+ antiporters, which are highly selective for F− over Cl−, led us to investigate the mechanism of Cl−-over-F− selectivity by a CLC Cl−/H+ antiporter, CLC-ec1. By subjecting purified CLC-ec1 to anion transport measurements, electrophysiological recording, equilibrium ligand-binding studies and X-ray crystallography, we show that F− binds in the Cl− transport pathway with affinity similar to Cl− but stalls the transport cycle. Examination of various mutant antiporters implies a 'lock-down' mechanism of F− inhibition, in which F−, by virtue of its unique hydrogen-bonding chemistry, greatly retards a proton-linked conformational change essential for the transport cycle of CLC-ec1.

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Figure 1: F− transport and binding in wild-type CLC-ec1.
Figure 2: Crystal structures of wild-type CLC-ec1 with F−.
Figure 3: F− handling by the ungated E148A mutant.
Figure 4: H+ transport is not involved in F− inhibition.
Figure 5: Lock-down mechanism of F− inhibition.

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Acknowledgements

We are grateful to L. Tamm (University of Virginia) for providing the porin-deleted Escherichia coli strain and to the beamline scientists for their expertise and help at the Advanced Light Source, Lawrence Berkeley Labs.

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  1. Hyun-Ho Lim

    Present address: Present address: Korea Brain Research Institute, Daegu, Korea.,

Authors and Affiliations

  1. Department of Biochemistry, Brandeis University, Waltham, Massachusetts, USA

    Hyun-Ho Lim, Randy B Stockbridge & Christopher Miller

  2. Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts, USA

    Hyun-Ho Lim, Randy B Stockbridge & Christopher Miller

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H.-H.L. and R.B.S. designed and executed experiments and wrote the paper, and C.M. designed experiments and wrote the paper.

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Correspondence to Christopher Miller.

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Supplementary Results, Supplementary Figure 1 and Supplementary Tables 1–4. (PDF 639 kb)

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Lim, HH., Stockbridge, R. & Miller, C. Fluoride-dependent interruption of the transport cycle of a CLC Cl−/H+ antiporter. Nat Chem Biol 9, 721–725 (2013). https://doi.org/10.1038/nchembio.1336

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