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A voltage-gated proton-selective channel lacking the pore domain

Abstract

Voltage changes across the cell membrane control the gating of many cation-selective ion channels. Conserved from bacteria to humans1, the voltage-gated-ligand superfamily of ion channels are encoded as polypeptide chains of six transmembrane-spanning segments (S1–S6). S1–S4 functions as a self-contained voltage-sensing domain (VSD), in essence a positively charged lever that moves in response to voltage changes. The VSD ‘ligand’ transmits force via a linker to the S5–S6 pore domain ‘receptor’2, thereby opening or closing the channel. The ascidian VSD protein Ci-VSP gates a phosphatase activity rather than a channel pore, indicating that VSDs function independently of ion channels3. Here we describe a mammalian VSD protein (HV1) that lacks a discernible pore domain but is sufficient for expression of a voltage-sensitive proton-selective ion channel activity. Hv1 currents are activated at depolarizing voltages, sensitive to the transmembrane pH gradient, H+-selective, and Zn2+-sensitive. Mutagenesis of Hv1 identified three arginine residues in S4 that regulate channel gating and two histidine residues that are required for extracellular inhibition of Hv1 by Zn2+. Hv1 is expressed in immune tissues and manifests the characteristic properties of native proton conductances ( G vH + ). In phagocytic leukocytes4, G vH + are required to support the oxidative burst that underlies microbial killing by the innate immune system4,5. The data presented here identify Hv1 as a long-sought voltage-gated H+ channel and establish Hv1 as the founding member of a family of mammalian VSD proteins.

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Figure 1: Biophysical properties of expressed H v 1 currents.
Figure 2: H v 1 voltage-dependent gating.
Figure 3: Mutations in H v 1 reveal residues required for Zn 2+ inhibition.

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Acknowledgements

We thank T. DeCoursey, C. Miller, R. MacKinnon and P. Bezanilla for comments on the manuscript, and K.-H. Lee for technical assistance. This work was supported by the Sandler Program for Asthma Research and the Howard Hughes Medical Institute.

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Correspondence to David E. Clapham.

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Supplementary information

Supplementary Figure Legends

This file contains text to accompany the Supplementary Figures. (DOC 56 kb)

Supplementary Figure 1

Hv1 amino acid sequence and expression profile. (PDF 1523 kb)

Supplementary Figure 2

a, Human tissue Western blot probed with 4234 antibody (5 µg/ml) demonstrates expression of native Hv1 protein (~32 kDa) in immune tissues. b, Western blot of total cell lysates prepared from Jurkat (lane 1) or HEK-293T cells transfected with the indicated cDNA (lanes 3-5). c, in HL-60 cells, Hv1 protein appeared to be increased when cells were cultured in the presence of 1.3% DMSO (lane 2). (PDF 1536 kb)

Supplementary Figure 3

a, Hv1-like currents were not detectable in HM1 cells. b, Native GvH+ in a DMSO-differentiated HL-60 cell. c, Temperature dependence of Hv1 currents. d, Monoexponential fits of τACT from the same cell as shown in panel c illustrate strong temperature-dependence of Hv1 kinetics. e, R205A currents. f, R208A currents. g, R211A currents. (PDF 2674 kb)

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Ramsey, I., Moran, M., Chong, J. et al. A voltage-gated proton-selective channel lacking the pore domain. Nature 440, 1213–1216 (2006). https://doi.org/10.1038/nature04700

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