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Structure, inhibition and regulation of two-pore channel TPC1 from Arabidopsis thaliana


Two-pore channels (TPCs) comprise a subfamily (TPC1–3) of eukaryotic voltage- and ligand-gated cation channels1,2 with two non-equivalent tandem pore-forming subunits that dimerize to form quasi-tetramers. Found in vacuolar3 or endolysosomal4 membranes, they regulate the conductance of sodium5 and calcium3,6 ions, intravesicular pH5, trafficking7 and excitability8,9. TPCs are activated by a decrease in transmembrane potential1,3,9,10 and an increase in cytosolic calcium concentrations1,10, are inhibited by low luminal pH and calcium11, and are regulated by phosphorylation12,13. Here we report the crystal structure of TPC1 from Arabidopsis thaliana at 2.87 Å resolution as a basis for understanding ion permeation3,4,10, channel activation1,5,10, the location of voltage-sensing domains1,9,10 and regulatory ion-binding sites11,14. We determined sites of phosphorylation3,4 in the amino-terminal and carboxy-terminal domains that are positioned to allosterically modulate cytoplasmic Ca2+ activation. One of the two voltage-sensing domains (VSD2) encodes voltage sensitivity and inhibition by luminal Ca2+ and adopts a conformation distinct from the activated state observed in structures of other voltage-gated ion channels15,16. The structure shows that potent pharmacophore trans-Ned-19 (ref. 17) acts allosterically by clamping the pore domains to VSD2. In animals, Ned-19 prevents infection by Ebola virus and other filoviruses, presumably by altering their fusion with the endolysosome and delivery of their contents into the cytoplasm7.

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Figure 1: Overview of the TPC1 Structure.
Figure 2: Structural details of the TPC1 monomer.
Figure 3: Voltage dependence and its modulation by ions.
Figure 4: Binding site for trans-Ned-19.
Figure 5: The TPC1 ion channel.
Figure 6: Cytosolic sensory domains.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors have been deposited in the Protein Data Bank under the accession number 5DQQ.


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We are grateful to J. M. Holton and G. Meigs at ALS beamline 8.3.1 and A. Gonzalez at SSRL beamline 12-2 for help with data collection and processing, B. P. Pedersen, B. H. Schmidt and J. S. Finer-Moore for critical analysis of the manuscript. This work was supported by NIH grant GM24485 to R.M.S. Beamline 8.3.1 at the Advanced Light Source is operated by the University of California Office of the President, Multicampus Research Programs and Initiatives grant MR-15-328599 and Program for Breakthrough Biomedical Research, which is partially funded by the Sandler Foundation. Figures were made with Chimera developed by the Resource for Biocomputing, Visualization and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311), and PyMOL developed by W. DeLano. Mass spectrometry was carried out by the UCSF mass spectrometry facility (supported by NIGMS P41-RR001614). 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. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.

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Authors and Affiliations



A.F.K. performed the experiments, collected and processed the data, determined, refined, and analysed the structure. R.M.S. supervised the project and aided in structure determination and analysis. A.F.K. and R.M.S. wrote the manuscript.

Corresponding author

Correspondence to Robert M. Stroud.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Sequence alignment of TPCs with TPC1 experimental and predicted secondary structure.

A sequence alignment based on seven human and plant TPC orthologues with observed TPC1 (black), and secondary structure predictions by Psipred48 (red) and Jpred49 (blue). Helices are shown as cylinders, β sheets as planks, coils as solid lines, and unstructured regions as dashed lines. Level of conservation is indicated by colour (>50% yellow; >80% red). Blue dots mark arginines in S4; red dots mark charge-transfer anions; green dots mark Ca2+-binding sites in the EF-hand. Solid and dashed green lines represent observed and absent peptides from mass spectrometry experiments. Coloured stars indicate observed phosphorylation sites using electrospray ionization mass spectrometry (magenta; Extended Data Fig. 3a), potential phosphorylation sites identified by truncation constructs (orange; Extended Data Fig. 3e), predicted phosphorylation sites by NetPhosK50 (cyan), non-phosphorylated sites (blue), unlikely sites owing to solvent inaccessibility (black), and unknown or unidentified regions (grey).

Extended Data Figure 2 Crystal packing of TPC1 and experimental electron densities.

a, Views of the TPC1 C2221 crystal lattice viewed along two-fold axes parallel to a (left) and b (right). Unit cell boundaries are shown. The asymmetric unit is shown in yellow. b, Transverse view of TPC1 is shown with overlayed FOM-weighted experimental electron density calculated using native amplitudes (native 1) and heavy-atom phases contoured at 1σ. Density-modified phases from non-dehydrated derivatives (left; Extended Data Table 1), dehydrated derivatives (middle; Extended Data Table 2), and combined phases with solvent flattening, histogram matching, phase extension to high resolution native (native 2), and cross-crystal averaging (right; see Methods, Extended Data Table 3). c, Transverse views of TPC1 with overlayed heavy-atom electron densities calculated from isomorphous differences.

Extended Data Figure 3 Determination of phosphorylation sites in TPC1.

a, Electrospray ionization mass spectrometry peptide sequence coverage from an in-gel digest of wild-type (WT) TPC1 by four enzyme combinations (Trypsin/Asp-N, Trypsin/Glu-C, Lys-C or Chymotrypsin). Measured peptides (top, red highlight), predicted (middle, black star) and observed (middle, red star) phosphorylation sites are shown. Observed phosphopeptides are listed with the sites of phosphorylation coloured red. be, Polyacrylamide gels of purified TPC1 (10 μg) stained with phosphoprotein-specific probe ProDiamond-Q, SyproRuby or Coomassie (left to right). Molecular weights of standards are indicated in kilodaltons. The first two lanes are PrecisionPlus protein molecular weight standards and PeppermintStick phosphoprotein molecular weight standards. b, Wild-type TPC1 and crystallographic TPC1. c, Untreated and treated wild-type TPC1 with lambda phosphatase for 1 h at 25 °C. d, Schematic of TPC1 truncations (NΔ1; 2–11, NΔ2; 2–21, NΔ3; 2–30, CΔ1; 682–733, CΔ2; 693–733, CΔ3; 709–733, CΔ4; 724–733). e, Analysis of TPC1 N- and C-terminal truncations for binding to ProDiamond-Q. NΔ1CΔ1 was unstable during purification.

Extended Data Figure 4 Sequence alignment of TPC1 subdomains with hTPC1, hTPC2, human Cav 1.1–1.4, Nav 1.1, NavAb and hCaM.

a, S4 voltage-sensing segments. Conserved arginines are highlighted in cyan. Stars mark potential voltage-sensing residues. b, S2 segments. Stars mark conserved charge-transfer residues. c, S6 segments, pore gate and poly-E motif. Conserved hydrophobic residues are highlighted in cyan. Dihydropyridine-binding residues in the Cav S6 domain 3, domain 4, and corresponding residues in TPC1 are highlighted in red and green, respectively. Stars mark the position of phenylalkylamine drug-binding residues in Cavs. Conserved residues in the gate and poly-E motif are highlighted in magenta and purple, respectively. d, Pore loops. Conserved residues are highlighted in red. e, Alignment of hCaM EF-hand domains (EF-hand 1, residues 1–71; EF-hand 2, residues 72–149) and AtTPC1 (residues 322–398). Stars mark calcium binding motifs and orange dots mark the interaction site with CTD.

Extended Data Figure 5 Comparison of VSDs between TPC1 and symmetrical ion channels.

ae, Structural alignment of S11–S12 segments of TPC1 domain 2 (blue) with S5–S6 of TPC1 domain 1 (a; red), NavAb16 (b; PDB ID 3RVY, grey); Kv1.2 (c; PDB ID 2A79, green)15, TRPV1 (d; PDB ID 3J5P, orange)51, and TRPA1 (e; PDB ID 3J9P, yellow)52. Angles between S4 segments with respect to S10 of TPC1 domain 2 are shown.

Extended Data Figure 6 Electrostatic surfaces and hydrophobic surfaces.

a, b, Top from the luminal side (left) and bottom from the cytoplasmic side (right) views of an electrostatic surface representation (a) and surface representation, coloured according to Kyte–Doolittle hydropathy of TPC1 (b). Notable domains and residues are labelled. Electrostatic potential and Kyte–Doolittle hydropathy without any bound ions were generated using Chimera46. The EF-hand and NTD domains are negatively charged and bind cations. The poly-R motif accounts for the positively charged region in the CTD.

Extended Data Figure 7 Mechanism for TPC gating.

A schematic summarizing structural features of TPC1 that suggest mechanisms for voltage-sensing, ion permeation, inhibition by Ned-19, luminal Ca2+ inhibition, cytosolic Ca2+-activation (EF-hand), and phosphoregulation (NTD/CTD). Ca2+ (green) and lanthanide (red) ion binding sites are shown. An ion permeation pathway through the putative selectivity filter, gate, and poly-E and poly-R motifs are summarized. M+ represents a general cation (Na+, K+, Ca2+) and ‘+’ signs are gating charges.

Extended Data Table 1 Data collection and phasing statistics for non-dehydrated derivatives
Extended Data Table 2 Data collection and phasing statistics for partially dehydrated derivatives
Extended Data Table 3 Data collection and refinement statistics

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Kintzer, A., Stroud, R. Structure, inhibition and regulation of two-pore channel TPC1 from Arabidopsis thaliana. Nature 531, 258–264 (2016).

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