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, 531 (7593), 258-62

Structure, Inhibition and Regulation of Two-Pore Channel TPC1 From Arabidopsis Thaliana

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Structure, Inhibition and Regulation of Two-Pore Channel TPC1 From Arabidopsis Thaliana

Alexander F Kintzer et al. Nature.

Abstract

Two-pore channels (TPCs) comprise a subfamily (TPC1-3) of eukaryotic voltage- and ligand-gated cation channels with two non-equivalent tandem pore-forming subunits that dimerize to form quasi-tetramers. Found in vacuolar or endolysosomal membranes, they regulate the conductance of sodium and calcium ions, intravesicular pH, trafficking and excitability. TPCs are activated by a decrease in transmembrane potential and an increase in cytosolic calcium concentrations, are inhibited by low luminal pH and calcium, and are regulated by phosphorylation. Here we report the crystal structure of TPC1 from Arabidopsis thaliana at 2.87 Å resolution as a basis for understanding ion permeation, channel activation, the location of voltage-sensing domains and regulatory ion-binding sites. We determined sites of phosphorylation in the amino-terminal and carboxy-terminal domains that are positioned to allosterically modulate cytoplasmic Ca(2+) activation. One of the two voltage-sensing domains (VSD2) encodes voltage sensitivity and inhibition by luminal Ca(2+) and adopts a conformation distinct from the activated state observed in structures of other voltage-gated ion channels. 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 cytoplasm.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Extended Data Figure 1
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 orthologs with observed TPC1 (black) and secondary structure predictions by Psipred (red) and Jpred (blue). Helices are shown as cylinders, beta sheets as planks, coil as solid lines, and unstructured regions as dashed lines. Level of conservation is indicated by color (>50% yellow; >80% red). Blue dots ( formula image) mark arginines in S4. Red dots ( formula image) mark charge-transfer anions. Green dots ( formula image) mark Ca2+-binding sites in the EF-hand. Solid and dashed green lines represent observed and absent peptides from mass spectrometry experiments. Magenta stars ( formula image), orange stars ( formula image), cyan stars ( formula image), blue stars ( formula image), black stars (★), and grey stars ( formula image) mark observed phosphorylation sites using ESI-MS (Extended Data Fig. 3a), potential phosphorylation sites identified by truncation constructs (Extended Data Fig. 3e), predicted phosphorylation sites by NetPhosK, non-phosphorylated sites, unlikely sites due to solvent inaccessibility, and mark unknown or unidentified regions, respectively.
Extended Data Figure 2
Extended Data Figure 2. Crystal packing of TPC1 and experimental electron densities
a, Views of the TPC1 C2221 crystal lattice viewed along 2-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 1 σ. (left to right) Density modified phases from non-dehydrated derivatives (Extended Data Table 1), dehydrated derivatives (Extended Data Table 2), and combined phases with solvent flattening, histogram matching, phase extension to high resolution native (Native 2), and cross-crystal averaging (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
Extended Data Figure 3. Determination of phosphorylation sites in TPC1
a, Electrospray ionization mass spectrometry (ESI-MS) 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), (Middle) predicted (*) and observed (*) phosphorylation sites are shown. (Bottom) Observed phosphopeptides are listed with the sites of phosphorylation colored red. b-e, Polyacrylamide gels of purified TPC1 (10μg) stained with phosphoprotein-specific probe ProDiamond-Q, SyproRuby, or Coomassie (left to right). Molecular weights of standards are indicated in kDa. units to the left. First two lanes are PrecisionPlus protein molecular weight standards and PeppermintStick phosphoprotein molecular weight standards. b, WT TPC1 and crystallographic TPC1. c, Untreated and treated WT TPC1 with Lambda phosphatase for 1 hour at 25°C. d, Schematic of TPC1 truncations (ND1; 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
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 III, domain IV, 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 ( formula image) mark the interaction site with CTD.
Extended Data Figure 5
Extended Data Figure 5. Comparison of VSDs between TPC1 and symmetrical ion channels
Structural alignment of S11–S12 segments of TPC1 domain II (blue) with S5–S6 of a, TPC1 domain I (red), b, NavAb (PDBID 3RVY, grey), c, Kv1.2 (PDBID 2A79, green), d, TRPV1 (PDBID 3J5P, orange), and e, TRPA1 (PDBID 3J9P, yellow). Angles between S4 segments with respect to S10 of TPC1 domain II are shown.
Extended Data Figure 6
Extended Data Figure 6. Electrostatic surfaces and Hydrophobic surfaces
(left to right) Top from the luminal side and bottom from the cytoplasmic side views of an a, electrostatic surface representation and b, surface representation colored according to Kyte-Doolittle hydropathy of TPC1. Important domains and residues are labeled. Electrostatic potential and Kyte-Doolittle hydropathy without any bound ions was generated using Chimera. 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
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 NED19, lumenal Ca2+-inhibition, and cytosolic Ca2+-activation (EF-hand), and phosphoregulation (NTD/CTD). Ca2+ binding (green) and lanthanide (red) 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.
Figure 1
Figure 1. Overview of the TPC1 Structure
a, Top view from the lumenal side onto the membrane plane and b, side view from the right side with the perpendicular to the membrane plane vertical of TPC1 structure labeled by domain and transmembrane helices. E, M, and C denote approximate endolysosomal, membrane, and cytosolic boundaries. The positions of lumenal ion binding sites, bound lipids, and NED19 are shown. Approximate geometric dimensions of TPC1 are indicated.
Figure 2
Figure 2. Structural details of the TPC1 monomer
Structure and domain boundaries in TPC1. a, A diagram describing the arrangement of structural domains, colored to match the structures below. Helices are depicted as cylinders and loops as lines. Structural details of TPC1 b, domain I and c, domain II from three perpendicular views. Phosphorylation sites are shown (*). Charges in S4 (++), S10 (++++), poly-E (----) in CTDh1, poly-R (++++) in CTDl1, and CxxCxxC (CCC) are shown. Ca2+ ions are shown in green and NED19 binding site shown as a purple hexagon.
Figure 3
Figure 3. Voltage-dependence and its modulation by ions
Side (left) and top (right) views of Ca2+-binding sites in a, VSDI and b, VSDII. Ca2+ ions are shown in green. Ion coordinating residues are labeled in VSDI and II. Voltage-sensing residues are shown in VSDII. Ba2+ (cyan) and Yb3+ (magenta) isomorphous difference density peaks and atom positions contoured at 10σ numbered according to Extended Data Fig. 2c.
Figure 4
Figure 4. Binding site for trans-NED19
Structural interactions between the TPC1 and NED19. (top) Surface rendering showing the location of the NED19 binding site. (bottom) Molecular details of the NED19 binding-pocket at the interface between PI, PII, and VSDII. Simulated-annealing omit density contoured at 1σ is shown for NED19 (red) and interacting side chains (blue).
Figure 5
Figure 5. The TPC1 ion channel
Cross-sectional views of the TPC1 ion channel separately through PI and PII. Domains removed for clarity. a, PI to PI (orange) and b, PII to PII (blue) with sharpened 2mFo-DFc electron density contoured at 1σ and Ba2+ (cyan) and Yb3+ (magenta) isomorphous difference density contoured at 10σ. Membrane boundaries are indicated by dashes. c, Pore radius calculations through separate pore domains, PI (orange) and PII (blue) using HOLE (See Methods). Approximate boundaries for the putative selectivity filter (SF), cavity (C), Gates I and II, and CTD are shown. Channel axis is vertical.
Figure 6
Figure 6. Cytosolic sensory domains
Structure of EF-hand and coupling with NTD, VSDII, and CTD. Sideviews through a, the EF-hand (green), CTD (chartreuse), and NTD (yellow), b, constriction site formed by Gate II and CTD, and c, interactions between EF-hand and CTD. Phosphorylation sites are indicated by stars (*). Transmembrane and adjacent domains removed for clarity.

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