Transient receptor potential melastatin 2 (TRPM2) is a calcium-permeable, non-selective cation channel that has an essential role in diverse physiological processes such as core body temperature regulation, immune response and apoptosis1,2,3,4. TRPM2 is polymodal and can be activated by a wide range of stimuli1,2,3,4,5,6,7, including temperature, oxidative stress and NAD+-related metabolites such as ADP-ribose (ADPR). Its activation results in both Ca2+ entry across the plasma membrane and Ca2+ release from lysosomes8, and has been linked to diseases such as ischaemia-reperfusion injury, bipolar disorder and Alzheimer’s disease9,10,11. Here we report the cryo-electron microscopy structures of the zebrafish TRPM2 in the apo resting (closed) state and in the ADPR/Ca2+-bound active (open) state, in which the characteristic NUDT9-H domains hang underneath the MHR1/2 domain. We identify an ADPR-binding site located in the bi-lobed structure of the MHR1/2 domain. Our results provide an insight into the mechanism of activation of the TRPM channel family and define a framework for the development of therapeutic agents to treat neurodegenerative diseases and temperature-related pathological conditions.
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The cryo-electron microscopy density map and coordinates of EDTA–TRPM2 and ADPR/Ca2+–TRPM2 have been deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-8901 and EMD-7999 and in the Research Collaboratory for Structural Bioinformatics Protein Data Bank under accession codes 6DRK and 6DRJ. All other data relating to this study are available from the corresponding author upon reasonable request.
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We thank G. Zhao and X. Meng for support with data collection at the David Van Andel Advanced Cryo-Electron Microscopy Suite, the HPC team in the Van Andel Research Institute (VARI) for computational support, C. Xu for help with SerialEM, and D. Nadziejka for technical editing. This work was supported by internal VARI funding.
Nature thanks A. H. Guse, A. Ward and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
Extended Data Fig. 1 The cryo-electron microscopy data processing workflow, using the data of EDTA–TRPM2 as an example.
Particles were auto-picked using Gautomatch and visually checked in RELION. After removing false positives, particles were subjected to two rounds of 2D classification in RELION. The contrast transfer function values of individual particles from selected 2D class averages were estimated using Gctf. For 3D classification in RELION, a reference model was generated using CryoSparc. Initial 3D refinement was carried out using RELION. Particles were further refined using cisTEM54.
Extended Data Fig. 2 Cryo-electron microscopy analysis of full-length DrTRPM2 in the presence of EDTA and ADPR/Ca2+.
a, e, Representative electron micrograph of EDTA–TRPM2 (a) and ADPR/Ca2+–TRPM2 (e). b, f, Selected 2D class averages of the electron micrographs of EDTA–TRPM2 (b) and ADPR/Ca2+–TRPM2 (f). c, g, The gold-standard Fourier shell correlation curves for the electron microscopy maps of EDTA–TRPM2 (c) and ADPR/Ca2+–TRPM2 (g) are shown in black, and the Fourier shell correlation curves between the atomic model and the final electron microscopy maps are shown in blue. d, h, Angular distribution of particles used for the refinement of EDTA–TRPM2 (d) and ADPR/Ca2+–TRPM2 (h).
Extended Data Fig. 3 Representative densities of the reconstruction of EDTA–TRPM2 and ADPR/Ca2+–TRPM2.
a, b, Local resolution estimation of the structure of EDTA–TRPM2. The map is coloured according to local resolution estimation. The unsharpened reconstructions are shown as transparent envelopes. c, Representative densities of EDTA–TRPM2. d, e, Local resolution estimation of the structure of ADPR/Ca2+–TRPM2. f, Representative densities of ADPR/Ca2+–TRPM2.
a, Domain organization of TRPM2. Dashed lines and cylinders denote regions that have not been modelled. b, c, Cartoon representation of one subunit of the EDTA–TRPM2 structure. d, e, Cartoon representation of one subunit of the ADPR/Ca2+–TRPM2 structure. ADPR and Ca2+ are shown as spheres.
a, Sequence alignment of zebrafish (Dr), starlet sea anemone (Nv) and human (Hs) TRPM2 NUDT9-H domains with human NUDT9 using Clustal Omega55. b, c, Superimposition of the zebrafish ADPR/Ca2+–TRPM2 NUDT9-H domain (red) with human NUDT9 (green, PDB ID: 1Q33). Cap and core regions are indicated. d, e, Superimposition of the NUDT9-H domains of the zebrafish ADPR/Ca2+–TRPM2 structure (red) and the EDTA–TRPM2 structure (blue).
Extended Data Fig. 6 Comparison of NvTRPM2 with DrTRPM2 (EDTA–TRPM2 and ADPR/Ca2+–TRPM2) and HsTRPM4, and comparison of the gate and selectivity filter of EDTA–TRPM2 with those of HsTRPM4 (PDB: 5WP6).
a–c, Superimposition of EDTA–TRPM2 (a, blue, r.m.s.d = 50 Å, overall, main chain atoms only), ADPR/Ca2+–TRPM2 (b, red, r.m.s.d = 50 Å, overall, main chain atoms only) and HsTRPM4 (c, green, r.m.s.d = 47.5 Å, overall, main chain atoms only) with NvTRPM2 (yellow). The NUDT9-H domain is completely invisible in NvTRPM2. d–f, Superimposition of the MHR1/2 domains of EDTA–TRPM2 (d, blue), ADPR/Ca2+–TRPM2 (e, red) and HsTRPM4 (f, green) with NvTRPM2 (yellow). g–i, Superimposition of the transmembrane domains of EDTA–TRPM2 (g, blue), ADPR/Ca2+–TRPM2 (h, red) and HsTRPM4 (i, green) with NvTRPM2 (yellow). The transmembrane domain of NvTRPM2 is distinct from EDTA–TRPM2 and ADPR/Ca2+–TRPM2 but very similar to that of HsTRPM4. j, k, Comparison of the gates of TRPM2 (blue) and TRPM4 (green) viewed from the intracellular side of the membrane (j), or viewed parallel to the membrane (k). Only two subunits are shown in (k) for clarity. l, m, Comparison of the selectivity filters of DrTRPM2 and HsTRPM4 viewed from the extracellular side of the membrane (l), or viewed parallel to the membrane (m). Only two subunits are shown in m for clarity. The superimposition was performed by aligning the P loop and S6 (residues 958–1050 in HsTRPM4 and residues 978–1069 in DrTRPM2). The Cα atoms of the residues in the selectivity filter and gate are shown as spheres.
Extended Data Fig. 7 Secondary structure arrangement of DrTRPM2 and sequence alignment of TRPM family channels.
The secondary structure prediction of DrTRPM2 was performed using the JPred online server56. The sequences (TRPM2 from zebrafish; TRPM2, TRPM4, TRPM5 and TRPM8 from human; TRPM2 from starlet sea anemone) were aligned using Clustal Omega55. Residues that are involved in ADPR binding and calcium binding are marked with red and black triangles, respectively. The cap and core regions of the NUDT9-H domain are indicated.
a, b, Alanine mutations were introduced within the ADPR-binding pocket and showed expression levels comparable to that of wild-type TRPM2, observed from both transfected cells (a) and fluorescence-detection size-exclusion chromatography (b). c, d, Electrophysiological experiments were carried out to measure the amplitude of agonist-induced current in inside-out patches pulled from HEK293 cells, with c showing the representative current and d showing the statistics of current amplitude and cell numbers. At +60 mV, robust current (4.87 ± 0.55 nA, n = 9 cells) could be detected when applying 0.1 mM ADPR and 1 mM Ca2+ onto inside-out patches expressing wild-type TRPM2. Single mutations (K154A, Y271A, E274A, R278A and R334A) each show robust channel activation (n = 5 cells, 8 cells, 8 cells, 7 cells and 8 cells for the corresponding mutants) and—other than K154A receptors, which did not show a markedly reduced current amplitude—mean current amplitudes for the mutated receptors were around two- to threefold smaller compared to the wild type. Introducing double mutations R278A/R334A (n = 7 cells) to the receptor nearly abolished ADPR/Ca2+-induced current. Deletion of the NUDT9-H domain (∆NUDT9-H) (n = 6 cells) also nearly abolished the ADPR/Ca2+-induced current. Data are shown as mean ± s.e.m.
a, Densities of the putative Ca2+-binding site and adjacent residues. b, Comparison of the Ca2+-binding site in DrTRPM2 (red) and HsTRPM4 (white). Residues coordinating the Ca2+ ion are indicated, with residues of HsTRPM4 shown in parentheses. c, Sequence alignment of the putative Ca2+-binding site within the TRPM family. Residues coordinating the Ca2+ ion are marked with a black triangle.