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The lysosomal potassium channel TMEM175 adopts a novel tetrameric architecture

Nature volume 547, pages 472–475 (2017)Cite this article

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Abstract

TMEM175 is a lysosomal K+ channel that is important for maintaining the membrane potential and pH stability in lysosomes1. It contains two homologous copies of a six-transmembrane-helix (6-TM) domain, which has no sequence homology to the canonical tetrameric K+ channels and lacks the TVGYG selectivity filter motif found in these channels2,3,4. The prokaryotic TMEM175 channel, which is present in a subset of bacteria and archaea, contains only a single 6-TM domain and functions as a tetramer. Here, we present the crystal structure of a prokaryotic TMEM175 channel from Chamaesiphon minutus, CmTMEM175, the architecture of which represents a completely different fold from that of canonical K+ channels. All six transmembrane helices of CmTMEM175 are tightly packed within each subunit without undergoing domain swapping. The highly conserved TM1 helix acts as the pore-lining inner helix, creating an hourglass-shaped ion permeation pathway in the channel tetramer. Three layers of hydrophobic residues on the carboxy-terminal half of the TM1 helices form a bottleneck along the ion conduction pathway and serve as the selectivity filter of the channel. Mutagenesis analysis suggests that the first layer of the highly conserved isoleucine residues in the filter is primarily responsible for channel selectivity. Thus, the structure of CmTMEM175 represents a novel architecture of a tetrameric cation channel whose ion selectivity mechanism appears to be distinct from that of the classical K+ channel family.

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Figure 1: 86Rb flux assay of CmTMEM175.
Figure 2: Overall structure of CmTMEM175.
Figure 3: CmTMEM175 ion conduction pore.
Figure 4: Selectivity analysis of hTMEM175 and CmTMEM175.

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Acknowledgements

We thank N. Nguyen for manuscript preparation. The experimental results reported in this article derive from work performed at Argonne National Laboratory, Structural Biology Center (19ID) and GM/CA (23ID) at the Advanced Photon Source, and from work performed at the Berkeley Center for Structural Biology at the Advanced Light Source (ALS). Argonne is operated by UChicago Argonne, LLC, for the US Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357. The Berkeley Center for Structural Biology is supported in part by the National Institutes of Health, National Institute of General Medical Sciences, and the Howard Hughes Medical Institute. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under Contract No. DE-AC02-05CH11231. This work was supported in part by the Howard Hughes Medical Institute and by grants from the National Institutes of Health (GM079179 to Y.J.; NS055293 and NS074257 to D.R.) and the Welch Foundation (Grant I-1578 to Y.J.).

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Authors and Affiliations

  1. Department of Physiology and Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, 75390-9040, Texas, USA

    Changkeun Lee, Jiangtao Guo, Weizhong Zeng, Sunghoon Kim, Ji She & Youxing Jiang

  2. Howard Hughes Medical Institute, Chevy Chase, 20815-6789, Maryland, USA

    Changkeun Lee, Weizhong Zeng, Sunghoon Kim & Youxing Jiang

  3. Department of Biology, University of Pennsylvania, Philadelphia, 19104, Pennsylvania, USA

    Chunlei Cang & Dejian Ren

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Contributions

C.L. and J.G. performed the structure determination; W.Z., C.C. and D.R. performed electrophysiology; S.K and J.S. participated in sample preparation; C.L., J.G., W.Z. and Y.J. designed the research, analysed data, and prepared the manuscript.

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Correspondence to Youxing Jiang.

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Extended data figures and tables

Extended Data Figure 1 Sequence comparison between prokaryotic and eukaryotic TMEM175 channels.

a, Topologies of prokaryotic (left) and eukaryotic (right) TMEM175 channel subunits. b, Sequence alignment of prokaryotic TMEM175 proteins and the first 6-TM domains of eukaryotic TMEM175 proteins. c, Sequence alignment of prokaryotic TMEM175 proteins and the second 6-TM domains of eukaryotic TMEM175 proteins. Secondary structure assignments are based on the CmTMEM175 structure. Asterisks mark the three hydrophobic filter residues. Blue triangles mark the RxxxFSD motif and the residues participating in the inter- and intra-subunit interactions with the motif.

Extended Data Figure 2 Biochemical analysis of bacterial TMEM175 channels.

a, Gel filtration profiles of three purified bacTMEM175 channels on Superdex-200 (10/30 GL column) in 20 mM HEPES, pH 7.4, 200 mM KCl and 3 mM DM. All purified bacTMEM175 channels eluted as a monodispersed peak at a position much larger than a monomer, indicating oligomerization. Arrows indicate the elution peaks of three standard proteins (29, 66 and 200 KDa) on the same column. b, Cross-linking reaction of purified bacTMEM175 channels from Streptomyces collinus (left gel) and Chryseobacterium sp. (right gel) with the cross-linking reagents DSS and DSG. Samples were analysed by SDS–PAGE and detected by coomassie blue staining. The purified proteins migrate as multiple bands on SDS–PAGE corresponding to the sizes of monomer to tetramer. The cross-linking reaction promotes the formation of a cross-linked tetramer, demonstrating that these bacterial channels form tetramers in solution. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 3 Electron density maps of CmTMEM175.

a, 2Fo−Fc electron density map of one subunit (contoured at 1.5σ). b, 2Fo−Fc electron density map of the filter region (contoured at 1.5σ). The front and back subunits have been removed for clarity. The side chains of the three layers of hydrophobic residues are coloured in magenta.

Extended Data Figure 4 Structural comparison between K+ channels.

a, CmTMEM175; b, Shaker-like K+ channel (Kv1.2-2.1 chimaera, PDB code: 2R9R). Each subunit is individually coloured. Both structures are viewed from the extracellular side.

Extended Data Figure 5 Structural comparison of the ion conduction pathways.

Ion conduction pathways are shaded grey. a, CmTMEM175; b, chicken bestrophin-1 (PDB code: 4RDQ); c, bacterial bestrophin from Klebsiella pneumoniae (KpBest, PDB code: 4WD8). Insets show zoomed-in views of the narrow filters.

Extended Data Figure 6 Ion selectivity and pharmacological properties of human TMEM175.

a, Partial sequence alignment of TM1 helices from hTMEM175 and CmTMEM175; the three layers of hydrophobic residues are boxed and the RxxxFSD motif is shaded red. b, I–V curve from a control cell. The pipette and bath solutions contained 150 mM Cs+ and 150 mM Na+, respectively. c, Extracellular Zn2+ and 4-AP blockade of human TMEM175. Currents were recorded using whole-cell patches with 150 mM extracellular Na+ (bath) and 150 mM intracellular Cs+ (pipette). d, Intracellular Zn2+ and 4-AP blockade of human TMEM175. Currents were recorded using inside-out patches with 150 mM intracellular Na+ (bath) and 150 mM extracellular Cs+ (pipette). Recordings shown in c and d indicate that human TMEM175 is sensitive to Zn2+ or 4-AP block from both sides. e, I–V curves of wild-type human TMEM175. Currents were recorded using whole-cell patches in bi-ionic conditions. The pipette solution contained 150 mM Cs+ and the bath solution contained 150 mM X+ (X = NMDG, Li, Na, K or Rb). f, I–V curves of the I46N/I271N (at layer 1) double mutant of human TMEM175 in bi-ionic conditions. Currents were recorded using the same conditions as e. g, Summary of reversal potentials of hTMEM175 and its mutants and the calculated relative permeability between Cs+ and K+ or Na+; shown are mean ± s.e.m. of ≥5 measurements. h, Summary of reversal potentials of human TMEM175 and I46N/I271N mutant with various monovalent cations in the bath solutions and the calculated relative permeability of these ions in comparison to Cs+.

Extended Data Table 1 Data collection and structure refinement statistics
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Lee, C., Guo, J., Zeng, W. et al. The lysosomal potassium channel TMEM175 adopts a novel tetrameric architecture. Nature 547, 472–475 (2017). https://doi.org/10.1038/nature23269

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