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Structure of the voltage-gated two-pore channel TPC1 from Arabidopsis thaliana

Abstract

Two-pore channels (TPCs) contain two copies of a Shaker-like six-transmembrane (6-TM) domain in each subunit and are ubiquitously expressed in both animals and plants as organellar cation channels. Here we present the crystal structure of a vacuolar two-pore channel from Arabidopsis thaliana, AtTPC1, which functions as a homodimer. AtTPC1 activation requires both voltage and cytosolic Ca2+. Ca2+ binding to the cytosolic EF-hand domain triggers conformational changes coupled to the pair of pore-lining inner helices from the first 6-TM domains, whereas membrane potential only activates the second voltage-sensing domain, the conformational changes of which are coupled to the pair of inner helices from the second 6-TM domains. Luminal Ca2+ or Ba2+ can modulate voltage activation by stabilizing the second voltage-sensing domain in the resting state and shift voltage activation towards more positive potentials. Our Ba2+-bound AtTPC1 structure reveals a voltage sensor in the resting state, providing hitherto unseen structural insight into the general voltage-gating mechanism among voltage-gated channels.

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Figure 1: Voltage activation and Ca2+ modulation of AtTPC1 overexpressed in HEK cell.
Figure 2: Overall structure of AtTPC1.
Figure 3: The ion-conduction pore.
Figure 4: The calcium modulation sites.
Figure 5: The voltage-sensing domains.
Figure 6: Voltage gating mechanism.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession number 5E1J.

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Acknowledgements

We thank N. Nguyen for manuscript preparation, R. Hedrich at University of Würzburg and M. X. Zhu at University of Texas Health Science Center at Houston for providing clones of plant and animal TPC genes. 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.). The authors declare no competing financial interests.

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Authors and Affiliations

Authors

Contributions

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

Corresponding author

Correspondence to Youxing Jiang.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Sequence analysis.

a, Sequence alignment of AtTPC1, human TPC1 (HsTPC1) and TPC2 (HsTPC2). Secondary structure assignments are based on the AtTPC1 structure. Red dots indicate the residues predicted to participate in calcium coordination in EF-hand domains. b, Sequence alignment of the two 6-TM domains of AtTPC1 (AtTPC1I and AtTPC1II), NavRh (Protein Data Bank (PDB) accession: 4DXW), NavAb (PDB: 3RVY) and Kv1.2-2.1 (PDB: 2R9R). Red dots indicate the residues critical for voltage sensing. Secondary structure assignments are based on the AtTPC1 6-TM I structure.

Extended Data Figure 2 Voltage activation and Ba2+ modulation of AtTPC1 overexpressed in HEK cells.

a, Voltage-dependent activation of wild-type AtTPC1. Channel currents were recorded using patch clamp in the whole-cell configuration. The membrane was stepped from holding potential (−70 mV) to various testing potentials and then returned to the holding potential. The I/V curve was plotted using the steady peak current against the voltage. The peak tail currents were recorded to generate the G/V curves for voltage activation analysis. b, Extracellular Ba2+ inhibition of AtTPC1. The intracellular solution (pipette) contains 300 μM Ca2+ necessary for channel activation.

Extended Data Figure 3 Structure of AtTPC1 transmembrane region and its alignment with prokaryotic Nav channels.

a, Structure of the individual 6-TM domain of AtTPC1 in rainbow colour with the same pore orientation. b, Superposition of AtTPC1 (red) and NavRh (blue, PDB: 4DXW). The NavRh VSDs align well with AtTPC1 VSD1s. c, Superposition of AtTPC1 (red) and NavAb (cyan, PDB: 3RVY). The NavAb VSDs align well with AtTPC1 VSD2s. d, Pore superposition between AtTPC1 (red) and NavRh (blue). e, Pore superposition between AtTPC1 (red) and NavAb (cyan).

Extended Data Figure 4 The ion-conduction pore of AtPTC1.

a, Cross-sections of surface-rendered AtTPC1 pore along IS6 pair (left) and IIS6 pair (right). The channel is closed at the bundle crossing. b, Stereo view of the bundle crossing region from the cytosolic side. c, Partial sequence alignment of the selectivity filters from two pore channels (AtTPC1, HsTPC1 and HsTPC2), bacterial sodium channels (NavRh and NavAb) and human voltage-gated sodium channel Nav1.1. d, Stereo view of the structural alignment between AtTPC1 Filter I (carbon in yellow) and NavAb filter (carbon in cyan). e, Stereo view of structural alignment between AtTPC1 filter II and NavAb filter. f, Anomalous difference Fourier map of native crystal (green mesh, 4.5σ level) reveals the bound Ba2+ along the ion-conduction pathway. The two cavity sites are probably occupied by a single Ba2+ ion alternatively, as the two sites are only 3 Å apart, too close to accommodate two ions simultaneously.

Extended Data Figure 5 The whole cell currents and G/V curves of AtTPC1 with mutations at the luminal Ba2+ binding sites.

ad, D454N (a), D240N (b), E528Q (c) and E239Q (d). The bath solutions contained 0, 0.1, 1, or 10 mM [Ca2+]extracellular. The pipette solutions contained 300 μM [Ca2+]cytosolic. Data measured in 0.1 mM [Ca2+]extracellular are shown in the main text Fig. 4e.

Extended Data Figure 6 Functional analysis of AtTPC1 mutants.

a, The whole-cell currents of AtTPC1 containing EF-hand Ca2+-site mutations (D335A in EF1 and D376A in EF2). Currents were recorded with the presence of 300 μM [Ca2+]cytosolic. b, Whole-cell currents and G/V curves of AtTPC1 with neutralization mutations of arginines on IS4 and IIS4 of the voltage-sensing domains.

Extended Data Figure 7 Structural comparison between AtTPC1 VSD2, NavAb VSD (PDB: 3RVY) and Kv1.2-2.1 VSD (PDB: 2R9R)

All structures are aligned at the gating charge transfer centre and S1 helices are removed for clarity. The side chains of the voltage-sensing arginines in S4, residues in gating charge transfer centre and the conserved acidic residue in S2 are shown in stick model. Voltage-sensing residues in gating charge transfer centre are labelled in red. Lower panels are cross-sections of surface-rendered AtTPC1 VSD2 (left) and NavAb VSD (right) with S4 gating charge arginines in blue. NavAb VSD is rotated by 90° to visualize the external aqueous cavity.

Extended Data Figure 8 Proposed model for AtTPC1 activation.

a, The model of AtTPC1 6-TM II in voltage-activated state is generated based on the structural comparison between AtTPC1 and NavAb. Only IIS4, IIS4–S5 linker and IIS6 are considered as the moving parts, assuming IIS6 moves concurrently with IIS4–S5 linker. The moving parts are coloured red for resting state and blue for activated state. The rest of the protein is coloured in grey. Green arrows indicate the directions of the movement at the N terminus, middle part, and C terminus of IIS4, and at IIS4–S5 linker and C terminus of IIS6. Dashed arrow indicates the central axis of the channel. b, Cytosolic view of the channel-opening mechanism. Compared with the closed state (red), membrane depolarization and calcium binding to EF-hand domain lead to the opening of IIS6 and IS6 (modelled in blue), respectively.

Extended Data Figure 9 Structure determination of AtTPC1.

a, Experimental electron density maps superposed with the final refined model. Density in blue (left) is the experimental SIRAS map calculated from the native and mercury-derivative data without anisotropic truncation and B-factor sharpening. Density in magenta (right) is the experimental SIRAS map calculated from the same native and mercury-derivative data after anisotropic truncation and B-factor sharpening using ‘auto correction’ in HKL2000. This map provides much better structural features, that is, side chains. All maps are contoured at 1.5σ level. b, Anomalous difference Fourier maps of mercury-derivatized native and mutant crystals superposed on the final refined model. The blue density peaks indicate the positions of mercury bound to the native cysteine residues. The magenta density peaks indicate the positions of mercury bound to cysteine residues introduced into various part of the protein (single-cysteine mutants). The green density peaks are calculated from the wild-type crystal (no mercury soaking), indicating the barium positions in wild-type AtTPC1. All maps are contoured at 4σ. Total 20 residues in each subunit are accurately registered by the mercury sites. Arrow indicates the molecular dyad of the channel dimer.

Extended Data Table 1 Data collection and refinement statistics

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Guo, J., Zeng, W., Chen, Q. et al. Structure of the voltage-gated two-pore channel TPC1 from Arabidopsis thaliana. Nature 531, 196–201 (2016). https://doi.org/10.1038/nature16446

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