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Structural insights into the voltage and phospholipid activation of the mammalian TPC1 channel

Abstract

The organellar two-pore channel (TPC) functions as a homodimer, in which each subunit contains two homologous Shaker-like six-transmembrane (6-TM)-domain repeats1. TPCs belong to the voltage-gated ion channel superfamily2 and are ubiquitously expressed in animals and plants3,4. Mammalian TPC1 and TPC2 are localized at the endolysosomal membrane, and have critical roles in regulating the physiological functions of these acidic organelles5,6,7. Here we present electron cryo-microscopy structures of mouse TPC1 (MmTPC1)—a voltage-dependent, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2)-activated Na+-selective channel—in both the apo closed state and ligand-bound open state. Combined with functional analysis, these structures provide comprehensive structural insights into the selectivity and gating mechanisms of mammalian TPC channels. The channel has a coin-slot-shaped ion pathway in the filter that defines the selectivity of mammalian TPCs. Only the voltage-sensing domain from the second 6-TM domain confers voltage dependence on MmTPC1. Endolysosome-specific PtdIns(3,5)P2 binds to the first 6-TM domain and activates the channel under conditions of depolarizing membrane potential. Structural comparisons between the apo and PtdIns(3,5)P2-bound structures show the interplay between voltage and ligand in channel activation. These MmTPC1 structures reveal lipid binding and regulation in a 6-TM voltage-gated channel, which is of interest in light of the emerging recognition of the importance of phosphoinositide regulation of ion channels.

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Figure 1: Overall structure of MmTPC1.
Figure 2: Ion conduction pore of MmTPC1.
Figure 3: The voltage-sensing domains.
Figure 4: PtdIns(3,5)P2 binding in MmTPC1.
Figure 5: Gating mechanism of MmTPC1.

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Acknowledgements

We thank N. Nguyen for manuscript preparation and M. X. Zhu for providing clones of animal TPC genes. Single particle cryo-EM data were collected at the University of Texas Southwestern Medical Center (UTSW) Cryo-Electron Microscopy Facility that is funded by the CPRIT Core Facility Support Award RP170644. We thank D. Nicastro and Z. Chen for facility access and data acquisition. Negatively stained sample screening was performed at UTSW Electron Microscopy core. This work was supported in part by the Howard Hughes Medical Institute (Y.J.) and by grants from the National Institute of Health (GM079179 to Y.J.) and the Welch Foundation (Grant I-1578 to Y.J.). X.B. is supported by the Cancer Prevention and Research Initiative of Texas and Virginia Murchison Linthicum Scholar in Medical Research fund.

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Contributions

J.S., J.G. and Q.C. prepared the samples; J.S., J.G., Q.C. and X.B. performed data acquisition, image processing and structure determination; W.Z. performed electrophysiology; Y.J. supervised the project and revised the manuscript; all authors participated in research design, data analysis and manuscript preparation.

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Correspondence to Youxing Jiang or Xiao-chen Bai.

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Extended data figures and tables

Extended Data Figure 1 Sequence alignment of MmTPC1, HsTPC1, AtTPC1, MmTPC2 and HsTPC2.

Secondary structure assignments are based on the structure of PtdIns(3,5)P2-bound MmTPC1. Red dots mark the ligand-binding residues; black dots mark the S4 arginine residues and residues at the gating-charge transfer centre; cyan dots mark the key S6 gating residues; green dots mark the residues predicted to participate in Ca2+ coordination in EF-hand domains of AtTPC1. MmTPC1 and AtTPC1 share about 25% sequence identity.

Extended Data Figure 2 Gating and selectivity of MmTPC1.

a, Sample traces and current density (current/capacitance) of wild-type MmTPC1 and the L11A/I12A mutant of MmTPC1, recorded in the whole-cell configuration with 100 μM PtdIns(3,5)P2 in the pipette (cytosolic). The experiments were repeated five times independently with similar results. Data points for current density are mean ± s.e.m. (n = 5 independent experiments). The L11A/I12A mutant elicited much larger whole-cell currents and was therefore used as the wild-type channel in all recordings. The extracellular side of MmTPC1 in plasma membrane is equivalent to the luminal side of MmTPC1 in lysosomes. b, Sample traces of PtdIns(3,5)P2-dependent voltage activation of MmTPC1. Whole-cell currents were recorded with varying PtdIns(3,5)P2 concentrations in the pipette (cytosolic) at pH 7.4. The experiments were repeated five times independently with similar results. c, G/GmaxV curves of MmTPC1 at various PtdIns(3,5)P2 concentrations. Boltzmann fit yields V1/2 (mV) = 21.6 ± 1.2, 15.2 ± 1.0, 16.1 ± 0.9 and −2.0 ± 1.0, and Z = 0.78 ± 0.04, 0.82 ± 0.03, 0.89 ± 0.02 and 0.84 ± 0.05 for voltage activation in 0.05, 0.2, 2.0 and 10 μM cytosolic PtdIns(3,5)P2, respectively, in which V1/2 is the membrane potential for half maximum activation and Z is apparent valence. All data points are mean ± s.e.m. (n = 5 independent experiments). d, Luminal pH modulates the voltage activation of MmTPC1. Whole-cell currents of MmTPC1 recorded in the presence of 2 μM cytosolic PtdIns(3,5)P2 with a varying luminal (bath) pH of 7.4, 6.0 or 4.6. Sample traces were obtained from the same patch. The experiments were repeated five times independently with similar results. e, G/GmaxV curves of MmTPC1 at various luminal pH values. Boltzmann fit yields V1/2 = 16.2 ± 0.8 mV, Z = 0.91 ± 0.02 at pH 7.4, V1/2 = 38.2 ± 1.2 mV, Z = 0.95 ± 0.02 at pH 6.0. All data points were normalized against Gmax obtained at 100 mV activation voltage and pH 7.4. All data points are mean ± s.e.m. (n = 5 independent experiments). f, Sample traces of whole-cell currents with 150 mM Na+ in the pipette solution, and either 150 mM Na+ or 145 mM K+ and 5 mM Na+ in the bath solution, and the IV curves generated from the tail currents of the sample traces. g, Sample traces of whole-cell currents with 150 mM Na+ in the pipette solution and 150 mM Na+ or 100 mM Ca2+ in the bath solution, and the IV curves generated from the tail currents of the sample traces. Data in f and g were recorded with 10 μM PtdIns(3,5)P2 in the pipette at pH 7.4 and both experiments were repeated five times independently with similar results.

Source data

Extended Data Figure 3 Structure determination of PtdIns(3,5)P2-bound MmTPC1.

a, Representative electron micrograph of PtdIns(3,5)P2-bound MmTPC1; 2,348 micrographs were used for structure determination. b, 2D class averages. c, Euler angle distribution of particles used in the final 3D reconstruction, with the heights of the cylinders corresponding to the number of particles. d, Final density maps coloured by local resolution. e, Gold-standard FSC curves of the final 3D reconstructions. f, FSC curves for cross-validation between the models and the maps. Curves for model versus summed map in black (sum), for model versus half map in blue (work) and for model versus half map not used for refinement in red (free). g, Flowchart of electron microscopy data processing for PtdIns(3,5)P2-bound MmTPC1 particles.

Extended Data Figure 4 Structure determination of apo MmTPC1.

a, Representative electron micrograph of apo MmTPC1; 2,937 micrographs were used for structure determination. b, 2D class averages. c, Euler angle distribution of particles used in the final 3D reconstruction, with the heights of the cylinders corresponding to the number of particles. d, Final density maps coloured by local resolution. e, Gold-standard FSC curves of the final 3D reconstructions. f, FSC curves for cross-validation between the models and the maps. Curves for model versus summed map in black (sum), for model versus half map in blue (work) and for model versus half map not used for refinement in red (free). g, Flowchart of electron microscopy data processing for apo MmTPC1 particles.

Extended Data Figure 5 Sample electron microscopy density maps (blue mesh) for MmTPC1.

a-d, Sample electron microscopy density maps for various parts of PtdIns(3,5)P2-bound MmTPC1: IS1–IS6 and filter I (a), IIS1–IS6 and filter II (b), NAGs of Asn600 and Asn612 (c), and PtdIns(3,5)P2-binding site (d). The maps are low-pass filtered to 3.2 Å and sharpened with a temperature factor of −105 Å2. e, f, Sample electron microscopy density maps for the key parts of apo MmTPC1: ligand binding site (e) and S6 helices (f). The maps are low-pass filtered to 3.4 Å and sharpened with a temperature factor of −98.5 Å2. Residues discussed in main text are labelled in red.

Extended Data Figure 6 Structure comparison between MmTPC1 and AtTPC1.

a, Superposition of the overall structures of MmTPC1 (blue) and AtTPC1(salmon). b, Superposition of the pore regions. c, Superposition of VSD1 domains. The comparison of the VSD2 domains is shown in Fig. 3f. d, Superposition of cytosolic soluble domains.

Extended Data Figure 7 Sample traces of whole-cell currents for Asn648Ala and Asn649Ala filter mutants.

The pipette solution contained 150 mM Na+ and the bath solution contained 150 mM Na+, or 145 mM K+ and 5 mM Na+. The tail currents were used to generate the IV curves shown in Fig. 2g. The experiments were repeated five times independently with similar results.

Extended Data Figure 8 Voltage-sensing domains.

a, Superimposition of MmTPC1 VSD1 structures in the PtdIns(3,5)P2-bound (green) and apo (pink) states with S1 helices removed for clarity. The MmTPC1 VSD1 lacks some key features of canonical voltage sensors: the conserved aromatic residue on S2 and acidic residue on S3 that form the gating-charge transfer centre become Val152 and Lys177, respectively, in MmTPC1; the conserved basic residue at the R5 position becomes Phe209 in MmTPC1; no arginine from IS4 is positioned in the gating-charge transfer centre. b, Superimposition of MmTPC1 VSD2 structures in the PtdIns(3,5)P2-bound (orange) and apo (cyan) states. c, Sample traces of voltage activation of MmTPC1 and its IS4 arginine mutations, recorded in whole-cell configuration with 2 μM PtdIns(3,5)P2 in the pipette. Peak tail currents were used to generate the G/GmaxV curves shown in Fig. 3c. The experiments were repeated five times independently with similar results. d, Sample traces of voltage activation of Arg546Gln mutation, recorded in whole-cell configuration with 2 μM and 100 μM PtdIns(3,5)P2 in the pipette. The experiments were repeated five times independently with similar results.

Extended Data Figure 9 PtdIns(3,5)P2-binding in MmTPC1.

a, Model of bound PtdIns(3,5)P2 (left) and its electron microscopy density (right). Density of two other membrane lipid molecules (blue mesh in left panel) was also observed near PtdIns(3,5)P2 in the structure. b, Current density of mutations at the PtdIns(3,5)P2-binding site measured at −100 mV in whole-cell recordings. All mutants were generated on the background of Arg540Gln mutant, which was used as control. All data points are mean ± s.e.m. with the number of independent experiments for each mutant shown in parentheses. c, Sample IV curves of Arg540Gln mutant recorded in excised patches with varying concentrations of PtdIns(3,5)P2 in the bath (cytosolic). The experiments were repeated five times independently with similar results. Currents at −100 mV were used to generate the concentration-dependent PtdIns(3,5)P2 activation curve shown in Fig. 4c. Imax is the current recorded at −100 mV with 10 μM PtdIns(3,5)P2 in the bath. d, Structural comparison at the ligand-binding site between the PtdIns(3,5)P2-bound (green) and apo (salmon) states.

Extended Data Table 1 Cryo-EM data collection and model statistics

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She, J., Guo, J., Chen, Q. et al. Structural insights into the voltage and phospholipid activation of the mammalian TPC1 channel. Nature 556, 130–134 (2018). https://doi.org/10.1038/nature26139

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