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Human TRPML1 channel structures in open and closed conformations

Nature volume 550, pages 366–370 (2017)Cite this article

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Abstract

Transient receptor potential mucolipin 1 (TRPML1) is a Ca2+-releasing cation channel that mediates the calcium signalling and homeostasis of lysosomes. Mutations in TRPML1 lead to mucolipidosis type IV, a severe lysosomal storage disorder. Here we report two electron cryo-microscopy structures of full-length human TRPML1: a 3.72-Å apo structure at pH 7.0 in the closed state, and a 3.49-Å agonist-bound structure at pH 6.0 in an open state. Several aromatic and hydrophobic residues in pore helix 1, helices S5 and S6, and helix S6 of a neighbouring subunit, form a hydrophobic cavity to house the agonist, suggesting a distinct agonist-binding site from that found in TRPV1, a TRP channel from a different subfamily. The opening of TRPML1 is associated with distinct dilations of its lower gate together with a slight structural movement of pore helix 1. Our work reveals the regulatory mechanism of TRPML channels, facilitates better understanding of TRP channel activation, and provides insights into the molecular basis of mucolipidosis type IV pathogenesis.

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Figure 1: Overall structure of TRPML1.
Figure 2: The structure of ML-SA1-bound TRPML1 compared with other ligand-bound channels.
Figure 3: Electrophysiological characterization of TRPML1 and its agonist-binding pocket.
Figure 4: Structural comparisons of apo and ML-SA1-bound TRPML1structures.
Figure 5: Structural rearrangements in the outer pore region and lower gate.

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Acknowledgements

We thank M. Ebrahim and J. Sotiris at the Rockefeller Cryo-EM Resource Center for assistance in data collection, Z. Zhang for help in EM data processing, D. Hilgemann, E. Coutavas and E. Debler for help in manuscript preparation, and J. Fernandez and H. Molina for mass spectrometry analyses. This work was supported by funds from the Howard Hughes Medical Institute (to G.B.) and the Endowed Scholars Program in Medical Science of University of Texas Southwestern Medical Center (to X.L.). M.F. was supported by the National Institutes of Health (T32DK007257 and HL119843). X.L. was the recipient of a Gordon and Betty Moore Foundation Fellow of the Life Sciences Research Foundation.

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Author notes

  1. Philip Schmiege and Michael Fine: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, 10065, New York, USA

    Philip Schmiege, Günter Blobel & Xiaochun Li

  2. Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, 75390, Texas, USA

    Philip Schmiege & Xiaochun Li

  3. Department of Physiology, University of Texas Southwestern Medical Center, Dallas, 75390, Texas, USA

    Michael Fine

  4. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, 75390, Texas, USA

    Xiaochun Li

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Contributions

P.S. and X.L. purified proteins and carried out cryo-EM work. M.F. carried out functional characterization by electrophysiology. All authors analysed data and contributed to manuscript preparation. X.L. conceived the project and wrote the paper.

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Correspondence to Günter Blobel or Xiaochun Li.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks S. Hansen, C. Ulens and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Sequence alignment of human TRPML1, TRPML2 and TRPML3.

Residues discussed in the paper are annotated using symbols at their positions.

Extended Data Figure 2 Biochemical properties and cryo-EM studies of human TRPML1.

a, Size-exclusion chromatogram and SDS–PAGE gel of the purified TRPML1. A degradation fragment is presented on the gel and is indicated by an asterisk. b, Density maps of structures coloured by local resolution estimation using blocres56.

Extended Data Figure 3 Data and model quality assessment.

a, Left to right, a representative electron micrograph at defocus −2.0 μm; 2D classification; and FSC curves of the apo structure. The left curve shows a plot of the FSC as a function of resolution using Frealign output, the right curve shows the FSC calculated between the refined structure and the half map used for refinement, the other half map, and the full map. b, Left to right, a representative micrograph at defocus −2.0 μm; 2D classification; and FSC curves of ML-SA1-bound TRPML1 structure. The two FSC curves represent the same as for panel a.

Extended Data Figure 4 Electron microscopy density of different portions of the structures.

a, The apo TRPML1 structure. b, The ML-SA1-bound TRPML1 structure.

Extended Data Figure 5 Comparisons of TRPML1 and PKD2.

a, Superimposition of overall structures of TRPML1 and PKD2 (PDB code: 5T4D). b, Superimposition of one subunit of TRPML1 and PKD2. The extended structural elements of pre-S1 (α1 and α2), and α4 and S2 of TRPML1, are highlighted in purple and pink, respectively.

Extended Data Figure 6 Whole-cell currents of HEK wild-type cells and TRPML1(L/A) with Y436A, F465A or Y499A mutations.

a, Whole-cell currents of HEK293T cells transfected with empty vector at pH 4.6 or pH 7.2 with or without 10 μM ML-SA1 and 50 μΜ PtdIns(3,5)P2. b, Whole-cell currents of HEK293T cells transfected with surface-expressing mutant eGFP–TRPML1(L/A) containing Y436A, F465A or Y499A under the same conditions as in panel a. c, Confocal images of patched cells. Scale bars, 10 μm.

Extended Data Figure 7 The distribution of mutations that cause mucolipidosis type IV in the TRPML1 structure.

The mutations are shown as grey balls.

Extended Data Table 1 Statistics of data collection and refinement
Full size table

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Schmiege, P., Fine, M., Blobel, G. et al. Human TRPML1 channel structures in open and closed conformations. Nature 550, 366–370 (2017). https://doi.org/10.1038/nature24036

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