The modulation of ion channel activity by lipids is increasingly recognized as a fundamental component of cellular signalling. The transient receptor potential mucolipin (TRPML) channel family belongs to the TRP superfamily1,2 and is composed of three members: TRPML1–TRPML3. TRPMLs are the major Ca2+-permeable channels on late endosomes and lysosomes (LEL). They regulate the release of Ca2+ from organelles, which is important for various physiological processes, including organelle trafficking and fusion3. Loss-of-function mutations in the MCOLN1 gene, which encodes TRPML1, cause the neurodegenerative lysosomal storage disorder mucolipidosis type IV, and a gain-of-function mutation (Ala419Pro) in TRPML3 gives rise to the varitint–waddler (Va) mouse phenotype4,5,6. Notably, TRPML channels are activated by the low-abundance and LEL-enriched signalling lipid phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2), whereas other phosphoinositides such as PtdIns(4,5)P2, which is enriched in plasma membranes, inhibit TRPMLs7,8. Conserved basic residues at the N terminus of the channel are important for activation by PtdIns(3,5)P2 and inhibition by PtdIns(4,5)P28. However, owing to a lack of structural information, the mechanism by which TRPML channels recognize PtdIns(3,5)P2 and increase their Ca2+ conductance remains unclear. Here we present the cryo-electron microscopy (cryo-EM) structure of a full-length TRPML3 channel from the common marmoset (Callithrix jacchus) at an overall resolution of 2.9 Å. Our structure reveals not only the molecular basis of ion conduction but also the unique architecture of TRPMLs, wherein the voltage sensor-like domain is linked to the pore via a cytosolic domain that we term the mucolipin domain. Combined with functional studies, these data suggest that the mucolipin domain is responsible for PtdIns(3,5)P2 binding and subsequent channel activation, and that it acts as a ‘gating pulley’ for lipid-dependent TRPML gating.
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EM data were collected at The Scripps Research Institute (TSRI) electron microscopy facility. We thank C.-G. Cheong and S. Thomas for initial TRPML3 biochemistry, J.-C. Ducom for computational support, B. Anderson for microscope support, S. Chowdhury for aiding in preliminary EM analyses, A. Kuk for help with the docking study, and R. Brennan and M. Schumacher for providing access to ITC equipment. This work was supported by the National Institutes of Health (NIH) (R35NS097241 to S.-Y.L., DP2EB020402 to G.C.L., R01NS055293 and R01NS074257 to D.R.). G.C.L is supported as a Searle Scholar and a Pew Scholar. M.A.H. was supported by a Helen Hay Whitney Foundation postdoctoral fellowship. Computational analyses of EM data were performed using shared instrumentation funded by NIH S10OD021634.
The authors declare no competing financial interests.
Reviewer Information Nature thanks 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
Secondary structure elements are indicated by rectangles (helices) and arrows (β-strands). Coloured as in Fig. 1. Residues with sequence conservation are highlighted in grey, the selectivity filter in green, the p-helix in yellow, the pH-sensing histidines in red, and putative PtdInsP2 binding residues in blue. Potential glycosylation sites are indicated by an orange branch.
Extended Data Figure 2 Functional characterization of the TRPML3 channel and representative ITC raw data and binding isotherms for diC8-PtdIns(3,5)P2 interacting with TRPML3 mutants.
a, b, Representative whole-cell current traces recorded with repeated voltage ramps (from −100 to +100 mV; 400 ms; 5-s intervals between ramps) from HEK293T cells transfected with TRPML3WT (a) or TRPML3NQ (b) at basal currents in 140 mM Na (orange), 0 mM Na (black), and during application of either 20 μM (green) or 80 μM (blue) of the TRPML3 agonist SN-2. c, Averaged inward current sizes at −100 mV normalized to cell capacitance (pA/pF) (TRPML3WT: n = 6 biologically independent experiments; TRPML3NQ: n = 6; open circles represent individual experimental data points). No significant differences (NS) in the current density at 20 μM (P = 0.89) and 80 μM (P = 0.75) SN-2 was determined between TRPML3WT and TRPML3NQ (two-tailed Student’s t-test, P > 0.05). Confidence intervals (95%): −2.43 (low)/1.88(high) for 0 Na+, −6.18/4.31 for 140 Na+, −62.69/56.84 for 20 μM SN-2 and −150.61/121.17 for 80 μM SN-2. Bar graph and error bars denote means ± s.e.m. d, e, Whole lysosomal current–voltage traces from TRPML3WT (d) and TRPML3NQ R58A (e), in the presence and absence of PtdIns(3,5)P2. Average inward currents are shown in Fig. 3f. f, For each TRPML3 mutant, raw ITC data and fitted binding isotherms are shown. Putative binding site mutants (Arg58Ala, Lys62Ala, Tyr342Ala, Arg305Ala and Lys52Ala/Arg58Ala/Lys62Ala) show a substantially reduced binding affinity while two negative control mutations (Lys59Ala and Lys326Ala) do not affect binding appreciably. NQ refers to N138Q mutant background. All titrations were performed in triplicate (technical replicates). Representative data are shown. Mean thermodynamic parameters for triplicate titrations are shown in Extended Data Table 2. Mean Kd values for each triplicate are as follows: NQ 2.5 μM, NQ R58A 11.8 μM, NQ K59A 4.4 μM, NQ K62A 11.3 μM, NQ R305A 11.8 μM, NQ K326A 4.0 μM, NQ Y342A 9.4 μM, NQ K52A/R58A/K62A not determined. Owing to the low heat associated with binding in many TRPML3 mutants, only the Kd values for NQ, NQ K59A, and NQ K326A were reliably measured.
a, b, Representative micrograph of TRPML3 in vitreous ice (a). We collected 2,259 movies of TRPML3. Only images exhibiting Thon rings beyond 4 Å were used for image processing, as assessed by a 1D plot (b). c, Per-frame radiation damage weighting was applied by estimating the average per-frame B factor for all movies in the TRPML3 data set (above). The frequency-dependent weights used to generate the final stack of summed particle images are shown below. d, Representative 2D class averages. e, Initial classification of particles into three 3D classes (II), one of which (class 1) was used for subsequent 3D refinement. After performing the particle polishing step in RELION (III), per-particle CTF was estimated, particles were re-extracted with a box size of 512 × 512, and then refined in RELION to produce the final reconstruction (IV). f, Fourier shell correlation plot calculated from independently refined half-maps. g, FSC curves calculated between the atomic model and the final map (black line), and between the model and each half-map (orange and blue lines). h, Local resolution estimates of the final reconstructions calculated using BSOFT45. i, Worm representation with the ASU coloured according to the per-residue Cα root mean square deviation (r.m.s.d.) value (Å), the rest of the molecule is coloured wheat. j, Histogram of the per-residue Cα r.m.s.d. values calculated from the top 10 refined atomic models with the mean per-residue Cα r.m.s.d. value shown as a black vertical bar.
The structural elements are shown in cartoon representation with side chains as sticks, coloured as in Fig. 1. The cryo-EM density is shown as blue mesh.
a, Four lipid densities per protomer were resolved in the cryo-EM density and corresponding lipid molecules were built. An additional lipid density was observed near S4 of the VSLD and S5 of the pore domain, but the lipid molecule was not built owing to ambiguity of the density. b, Two cholesteryl hemisuccinate (CHS) molecules were built in the crevice formed by S5 and S6, and the third CHS molecule was fit alongside the N-terminal end of S1. In between the CHS molecules an elongated density was observed, into which a long lipid tail was built, probably from either a phospholipid or a fatty acid molecule. c, d, Lipid densities in analogous locations were reported for TRPV1 (c, PDB ID: 5IRZ14) and PKD2 (d, PDB ID: 5MKE12). e, f, PtdIns(3,5)P2 was docked onto the TRPML3 structure, showing that the phosphoinositol headgroup docks to the basic pocket between the VSLD and MLD interacting primarily with the side-chains of Lys52, Arg58, Lys62, and Tyr342, while the acyl chains penetrate through a tunnel in the cytosolic domain to a cavity formed by S3, S4 and S6. Residues interacting with the docked PtdIns(3,5)P2 are shown in green (f). g, PtdIns(3,5)P2 (blue sticks) docked to a location in the TRPML3 structure (blue cartoon representation) distinct from the phosphoinositide (green sticks) found in the TRPV1 structure (PDB ID: 5IRZ14, green cartoon representation) in the pocket formed by S3–S5.
a, The ECD from TRPML3 (red ribbon) superimposes well on the crystal structure of the ECD from TRPML1 (brown ribbon, PDB ID: 5TJA15), with a Cα r.m.s.d. of 1.8 Å. Views are shown from the membrane plane (left) and the extracytosolic side of the membrane (right). b, The polycystin domain from PKD2 (blue ribbon, PDB ID: 5T4D13) adopts a similar fold to the ECD from TRPML3 (red ribbon). The structural elements of PKD2 are labelled. c, The TRPML3 ECD lies ~9 Å away from the channel domain, which leads to limited interactions between the ECD and VSLD. d, The PKD2 TOP/polycystin domain lies directly on top of the channel domain (PDB ID: 5T4D13) and forms extensive interactions with the pore. e, More extensive interactions are formed between S1 and the remainder of the channel in TRPML3 than in other TRP channels (buried surface areas ~3,000 Å2 and 1,500–2,000 Å2, respectively). The interaction interface is shown in green. f, In many TRP channels a short 310-helix is present in S4. By contrast, S4 in TRPML3 is completely α-helical, suggesting a more static nature.
a, Four cryo-EM density peaks observed near Asp458 and Asp459 are attributed to water molecules, based on coordination distances (~3.2 Å). TRPML3 is shown in cartoon representation with the selectivity filter residues shown as sticks. Cryo-EM density is shown in orange mesh for water molecules (red spheres) and red mesh for sodium ions (purple spheres). b, Interatomic distances (Å) between ions and coordinating side-chain and backbone atoms. c–e, Selectivity filter comparison of TRPML3 (c) with TRPV1 (PDB ID: 3J5P22) (d) and TRPV6 (PDB ID: 5IWP21) (e), with respective ions shown as spheres (purple for Na+ and green for Ca2+).
Extended Data Figure 8 The locations of the varitint-waddler and ML-IV-causing mutations in the TRPML3 structure.
a, Overview of the TRPML3 structure with the location of the varitint-waddler mutation Ala419Pro indicated by a green sphere and the p-helix coloured magenta. TRPML3 is shown as cartoon representation, coloured as in Fig. 1. b, Detailed view of the location of Ala419. Ala419 is positioned in the middle of S5 near the p-helix on S6, such that mutation to a proline is likely to disrupt normal S6 bending, locking it in an ‘open gate’ conformation. c, d, Overview of the TRPML3 structure with the location of ML-IV-causing mutations indicated by red spheres, shown from the membrane plane (c) and the extracytosolic side of the membrane (d). Unless otherwise indicated, residues are labelled using TRPML1 numbering. The locations of varitint-waddler mutations are represented by cyan spheres. While the mutations on the ECD are associated with mild phenotypes and affect channel assembly, there are many missense mutations that cause more severe phenotypes and are localized to the channel region. These mutations can be categorized into three groups according to location: mutations around pore helix 1 (group 1), mutations around S5 near A419 (group 2), and mutations within the VSLD around the PtdInsP2-binding site (group 3). On the basis of our structural and functional studies, we can infer that group 1 mutations potentially disrupt ion conduction and selectivity, group 2 mutations disturb gating, and group 3 mutations affect either PtdInsP2 binding (R403C in TRPML1) or S2 motion associated with gating (T308P and D362Y in TRPML1). Future structural and functional studies of these mutations will shed light on the molecular basis of pathogenic mutations that lead to ML-IV.
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Hirschi, M., Herzik Jr, M., Wie, J. et al. Cryo-electron microscopy structure of the lysosomal calcium-permeable channel TRPML3. Nature 550, 411–414 (2017). https://doi.org/10.1038/nature24055
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