Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Molecular basis of neurosteroid and anticonvulsant regulation of TRPM3

Nature Structural & Molecular Biology volume 32, pages 828–840 (2025)Cite this article

Subjects

Abstract

Transient receptor potential channel subfamily M member 3 (TRPM3) is a Ca2+-permeable cation channel activated by the neurosteroid pregnenolone sulfate (PregS) or heat, serving as a nociceptor in the peripheral sensory system. Recent discoveries of autosomal dominant neurodevelopmental disorders caused by gain-of-function mutations in TRPM3 highlight its role in the central nervous system. Notably, the TRPM3 inhibitor primidone, an anticonvulsant, has proven effective in treating patients with TRPM3-linked neurological disorders and in mouse models of thermal nociception. However, our understanding of neurosteroids, inhibitors and disease mutations on TRPM3 is limited. Here we present cryogenic electron microscopy structures of the mouse TRPM3 in complex with cholesteryl hemisuccinate, primidone and PregS with the synthetic agonist CIM 0216. Our studies identify the binding sites for the neurosteroid, synthetic agonist and inhibitor and offer insights into their effects and disease mutations on TRPM3 gating, aiding future drug development.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Functional characterization of TRPM3 agonism and inhibition and cryo-EM structure determination.
Fig. 2: Structural basis of primidone-dependent TRPM3 inhibition.
Fig. 3: Structural basis of PregS and CHS recognition by TRPM3.
Fig. 4: CIM binding and rearrangement of the VSLD salt bridge interaction network.
Fig. 5: PregS and CIM dependent conformational changes, part I.
Fig. 6: PregS and CIM dependent conformational changes, part II.
Fig. 7: Structural basis of GOF disease mutations and mechanisms of ligand binding and gating.

Similar content being viewed by others

Data availability

For the CHS-bound TRPM3, the primidone-bound TRPM3 and the PregS/CIM-bound TRPM3 structures, the coordinates have been deposited in the Protein Data Bank (PDB) under IDs PDB 9B29, PDB 9B28 and PDB 9B2A; the cryo-EM density maps have been deposited in the Electron Microscopy Data Bank (EMDB) under IDs EMD-44101, EMD-44100 and EMD-44102. The molecular dynamics simulation data used in this study are available from the Zenodo OpenAIRE database (https://zenodo.org/doi/10.5281/zenodo.13369813) ref. 79. Source data are provided with this paper.

Code availability

For molecular dynamics simulations, the following software packages were used for performing simulations and analysis: CHARMM-GUI (free to academic users), OpenMM (v.7.7; open source), CHARMM (v.45b1; free to academic users) and VMD (v.1.9; open source).

References

  1. Su, S., Yudin, Y., Kim, N., Tao, Y. X. & Rohacs, T. TRPM3 channels play roles in heat hypersensitivity and spontaneous pain after nerve injury. J. Neurosci. 41, 2457–2474 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Aloi, V. D. et al. TRPM3 as a novel target to alleviate acute oxaliplatin-induced peripheral neuropathic pain. PAIN 164, 2060–2069 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Vriens, J., Nilius, B. & Voets, T. Peripheral thermosensation in mammals. Nat. Rev. Neurosci. 15, 573–589 (2014).

    Article  CAS  PubMed  Google Scholar 

  4. Vriens, J. et al. TRPM3 is a nociceptor channel involved in the detection of noxious heat. Neuron 70, 482–494 (2011).

    Article  CAS  PubMed  Google Scholar 

  5. Held, K. et al. Activation of TRPM3 by a potent synthetic ligand reveals a role in peptide release. Proc. Natl Acad. Sci. USA 112, E1363–E1372 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Vandewauw, I. et al. A TRP channel trio mediates acute noxious heat sensing. Nature 555, 662–666 (2018).

    Article  CAS  PubMed  Google Scholar 

  7. Wagner, T. F. J. et al. Transient receptor potential M3 channels are ionotropic steroid receptors in pancreatic β cells. Nat. Cell Biol. 10, 1421–1430 (2008).

    Article  CAS  PubMed  Google Scholar 

  8. Straub, I. et al. Flavanones that selectively inhibit TRPM3 attenuate thermal nociception in vivo. Mol. Pharmacol. 84, 736–750 (2013).

    Article  CAS  PubMed  Google Scholar 

  9. Krügel, U., Straub, I., Beckmann, H. & Schaefer, M. Primidone inhibits TRPM3 and attenuates thermal nociception in vivo. PAIN 158, 856–867 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Gavva, N. R. et al. The vanilloid receptor TRPV1 is tonically activated in vivo and involved in body temperature regulation. J. Neurosci. 27, 3366–3374 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Gavva, N. R. et al. Pharmacological blockade of the vanilloid receptor TRPV1 elicits marked hyperthermia in humans. PAIN 136, 202–210 (2008).

    Article  CAS  PubMed  Google Scholar 

  12. Burglen, L. et al. Gain-of-function variants in the ion channel gene TRPM3 underlie a spectrum of neurodevelopmental disorders. Elife 12, e81032 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Van Hoeymissen, E. et al. Gain of channel function and modified gating properties in TRPM3 mutants causing intellectual disability and epilepsy. Elife 9, e57190 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Sundaramurthi, J. C. et al. De novo TRPM3 missense variant associated with neurodevelopmental delay and manifestations of cerebral palsy. Cold Spring Harb. Mol. Case Stud. 9, a006293 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Dyment, D., et al. TRPM3-related neurodevelopmental disorder. In GeneReviews® [Internet] (eds. Adam, M.P. et al.) (Univ. of Washington, 2023).

  16. Lines, M. A. et al. Phenotypic spectrum of the recurrent TRPM3 p.(Val837Met) substitution in seven individuals with global developmental delay and hypotonia. Am. J. Med. Genet. A 188, 1667–1675 (2022).

    Article  CAS  PubMed  Google Scholar 

  17. Kang, Q. et al. A Chinese patient with developmental and epileptic encephalopathies (DEE) carrying a TRPM3 gene mutation: a paediatric case report. BMC Pediatr. 21, 256 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Becker, L. L. et al. Primidone improves symptoms in TRPM3-linked developmental and epileptic encephalopathy with spike-and-wave activation in sleep. Epilepsia 64, e61–e68 (2023).

    Article  CAS  PubMed  Google Scholar 

  19. Harteneck, C. Pregnenolone sulfate: from steroid metabolite to TRP channel ligand. Molecules 18, 12012–12028 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Reddy, D. S. Neurosteroids: endogenous role in the human brain and therapeutic potentials. Prog. Brain Res. 186, 113–137 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Reddy, D. S. & Rogawski, M. A. Neurosteroids as endogenous regulators of seizure susceptibility and use in the treatment of epilepsy. Epilepsia 51, 84 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Belelli, D., Hogenkamp, D., Gee, K. W. & Lambert, J. J. Realising the therapeutic potential of neuroactive steroid modulators of the GABAA receptor. Neurobiol. Stress 12, 100207 (2020).

    Article  PubMed  Google Scholar 

  23. Wong, P., Sze, Y., Chang, C. C., Lee, J. & Zhang, X. Pregnenolone sulfate normalizes schizophrenia-like behaviors in dopamine transporter knockout mice through the AKT/GSK3β pathway. Transl. Psychiatry 5, e528 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Seljeset, S., Liebowitz, S., Bright, D. P. & Smart, T. G. Pre- and postsynaptic modulation of hippocampal inhibitory synaptic transmission by pregnenolone sulphate. Neuropharmacology 233, 109530 (2023).

    Article  CAS  PubMed  Google Scholar 

  25. Vriens, J. et al. Opening of an alternative ion permeation pathway in a nociceptor TRP channel. Nat. Chem. Biol. 10, 188–195 (2014).

    Article  CAS  PubMed  Google Scholar 

  26. Badheka, D. et al. Inhibition of transient receptor potential melastatin 3 ion channels by G-protein βγ subunits. Elife 6, e26147 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  27. Dembla, S. et al. Anti-nociceptive action of peripheral mu-opioid receptors by G-beta-gamma protein-mediated inhibition of TRPM3 channels. Elife 6, e26280 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Quallo, T., Alkhatib, O., Gentry, C., Andersson, D. A. & Bevan, S. G protein βγ subunits inhibit TRPM3 ion channels in sensory neurons. Elife 6, e26138 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Zhao, C. & MacKinnon, R. Structural and functional analyses of a GPCR-inhibited ion channel TRPM3. Neuron 111, 81–91.e7 (2023).

    Article  CAS  PubMed  Google Scholar 

  30. Behrendt, M. et al. The structural basis for an on–off switch controlling Gβγ-mediated inhibition of TRPM3 channels. Proc. Natl Acad. Sci. USA 117, 29090–29100 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Oberwinkler, J., Lis, A., Giehl, K. M., Flockerzi, V. & Philipp, S. E. Alternative splicing switches the divalent cation selectivity of TRPM3 channels. J. Biol. Chem. 280, 22540–22548 (2005).

    Article  CAS  PubMed  Google Scholar 

  32. Held, K. et al. Pharmacological properties of TRPM3 isoforms are determined by the length of the pore loop. Br. J. Pharmacol. 179, 3560–3575 (2022).

    Article  CAS  PubMed  Google Scholar 

  33. Yin, Y. et al. Structure of the cold- and menthol-sensing ion channel TRPM8. Science 359, 237–241 (2018).

    Article  CAS  PubMed  Google Scholar 

  34. Winkler, P. A., Huang, Y., Sun, W., Du, J. & Lu, W. Electron cryo-microscopy structure of a human TRPM4 channel. Nature 552, 200–204 (2017).

    Article  CAS  PubMed  Google Scholar 

  35. Diver, M. M., Cheng, Y. & Julius, D. Structural insights into TRPM8 inhibition and desensitization. Science 365, 1434–1440 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Naylor, J. et al. Pregnenolone sulphate- and cholesterol-regulated TRPM3 channels coupled to vascular smooth muscle secretion and contraction. Circ. Res. 106, 1507–1515 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Hirschi, M. et al. Cryo-electron microscopy structure of the lysosomal calcium-permeable channel TRPML3. Nature 550, 411–414 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Autzen, H. E. et al. Structure of the human TRPM4 ion channel in a lipid nanodisc. Science 359, 228–232 (2018).

    Article  CAS  PubMed  Google Scholar 

  39. Kwon, D. H. et al. Heat-dependent opening of TRPV1 in the presence of capsaicin. Nat. Struct. Mol. Biol. 28, 554–563 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Majeed, Y. et al. Cis-isomerism and other chemical requirements of steroidal agonists and partial agonists acting at TRPM3 channels. Br. J. Pharmacol. 161, 430–441 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Yin, Y. et al. Visualizing structural transitions of ligand-dependent gating of the TRPM2 channel. Nat. Commun. 10, 3740 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Zubcevic, L., Le, S., Yang, H. & Lee, S. Y. Conformational plasticity in the selectivity filter of the TRPV2 ion channel. Nat. Struct. Mol. Biol. 25, 405–415 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kwon, D. H., Zhang, F., Fedor, J. G., Suo, Y. & Lee, S. Y. Vanilloid-dependent TRPV1 opening trajectory from cryoEM ensemble analysis. Nat. Commun. 13, 2874 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Pumroy, R. A. et al. Molecular mechanism of TRPV2 channel modulation by cannabidiol. Elife 8, e48792 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Zubcevic, L., Borschel, W. F., Hsu, A. L., Borgnia, M. J. & Lee, S. Y. Regulatory switch at the cytoplasmic interface controls TRPV channel gating. Elife 8, e47746 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Zhao, S., Yudin, Y. & Rohacs, T. Disease-associated mutations in the human TRPM3 render the channel overactive via two distinct mechanisms. Elife 9, e47746 (2020).

    Article  Google Scholar 

  47. Zhao, Y., McVeigh, B. M. & Moiseenkova-Bell, V. Y. Structural pharmacology of TRP channels. J. Mol. Biol. 433, 166914 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Song, K. et al. Structural basis for human TRPC5 channel inhibition by two distinct inhibitors. Elife 10, e63429 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Yin, Y. et al. Activation mechanism of the mouse cold-sensing TRPM8 channel by cooling agonist and PIP2. Science 378, eadd1268 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Smart, O. S., Neduvelil, J. G., Wang, X., Wallace, B. A. & Sansom, M. S. HOLE: a program for the analysis of the pore dimensions of ion channel structural models. J. Mol. Graph. 14, 354–360 (1996).

    Article  CAS  PubMed  Google Scholar 

  51. Goehring, A. et al. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat. Protoc. 9, 2574–2585 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Zivanov, J., Nakane, T. & Scheres, S. H. W. Estimation of high-order aberrations and anisotropic magnification from cryo-EM data sets in RELION-3.1. IUCrJ 7, 253–267 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  54. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Zivanov, J., Nakane, T. & Scheres, S. H. W. A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis. IUCrJ 6, 5–17 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Punjani, A., Zhang, H. & Fleet, D. J. Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction. Nat. Methods 17, 1214–1221 (2020).

    Article  CAS  PubMed  Google Scholar 

  58. Scheres, S. H. & Chen, S. Prevention of overfitting in cryo-EM structure determination. Nat. Methods 9, 853–854 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  60. Moriarty, N. W., Grosse-Kunstleve, R. W. & Adams, P. D. Electronic ligand builder and optimization workbench (eLBOW): a tool for ligand coordinate and restraint generation. Acta Crystallogr. D Biol. Crystallogr. 65, 1074–1080 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  PubMed  Google Scholar 

  63. Delano, W.L. The PyMol Molecular Graphics System (DeLano Scientific, 2002).

  64. Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).

    Article  CAS  PubMed  Google Scholar 

  65. Ren, Z. et al. Structural basis for inhibition and regulation of a chitin synthase from Candida albicans. Nat. Struct. Mol. Biol. 29, 653–664 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Joyce, L. R. et al. Identification of a novel cationic glycolipid in Streptococcus agalactiae that contributes to brain entry and meningitis. PLoS Biol. 20, e3001555 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Best, R. B. et al. Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone φ, ψ and side-chain χ1 and χ2 dihedral angles. J. Chem. Theory Comput. 8, 3257–3273 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Huang, J. & MacKerell, A. D. Jr. CHARMM36 all-atom additive protein force field: validation based on comparison to NMR data. J. Comput. Chem. 34, 2135–2145 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Klauda, J. B. et al. Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types. J. Phys. Chem. B 114, 7830–7843 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Huang, J. et al. CHARMM36m: an improved force field for folded and intrinsically disordered proteins. Nat. Methods 14, 71–73 (2017).

    Article  CAS  PubMed  Google Scholar 

  71. Durell, S. R., Brooks, B. R. & Ben-Naim, A. Solvent-induced forces between two hydrophilic groups. J. Phys. Chem. 98, 2198–2202 (1994).

    Article  CAS  Google Scholar 

  72. Vanommeslaeghe, K. et al. CHARMM general force field: a force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields. J. Comput. Chem. 31, 671–690 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Wu, E. L. et al. CHARMM-GUI membrane builder toward realistic biological membrane simulations. J. Comput. Chem. 35, 1997–2004 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Lee, J. et al. CHARMM-GUI input generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM/OpenMM simulations using the CHARMM36 additive force field. J. Chem. Theory Comput. 12, 405–413 (2016).

    Article  CAS  PubMed  Google Scholar 

  75. Jo, S., Kim, T., Iyer, V. G. & Im, W. CHARMM-GUI: a web-based graphical user interface for CHARMM. J. Comput. Chem. 29, 1859–1865 (2008).

    Article  CAS  PubMed  Google Scholar 

  76. Jo, S., Lim, J. B., Klauda, J. B. & Im, W. CHARMM-GUI membrane builder for mixed bilayers and its application to yeast membranes. Biophys. J. 97, 50–58 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Gao, Y. et al. CHARMM-GUI supports hydrogen mass repartitioning and different protonation states of phosphates in lipopolysaccharides. J. Chem. Inf. Model. 61, 831–839 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Eastman, P. et al. OpenMM 7: rapid development of high performance algorithms for molecular dynamics. PLoS Comput. Biol. 13, e1005659 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  79. Yin, Y. et al. MD simulations for “molecular basis of neurosteroid and anticonvulsant regulation of TRPM3”. Zenodo https://doi.org/10.5281/zenodo.13369813 (2024).

Download references

Acknowledgements

Cryo-EM data were collected at the Shared Materials Instrumentation Facility (SMIF) at Duke University and the National Cancer Institute (NCI)’s National Cryo-EM facility (NCEF) at the Frederick National Laboratory for Cancer Research. We thank A. Wier at NCEF and N. Bhattacharya at SMIF for assistance with microscope operation. We thank J. Fedor for critical paper reading and S. Yang for help with data collection. This work was supported by the National Institutes of Health (R35NS097241 to S.-Y.L. and R01GM138472 to W.I.), the National Institute of Health Intramural Research Program; National Institute of Environmental Health Sciences (ZIC ES103326 to M.J.B) and by the NCI’s NCEF under contract 75N91019D00024.

Author information

Authors and Affiliations

  1. Department of Biochemistry, Duke University School of Medicine, Durham, NC, USA

    Ying Yin, Cheon-Gyu Park, Ziqiang Guan, Hyuk-Joon Lee, Feng Zhang, Mario J. Borgnia & Seok-Yong Lee

  2. Department of Biological Sciences, Lehigh University, Bethlehem, PA, USA

    Shasha Feng & Wonpil Im

  3. Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC, USA

    Kedar Sharma & Mario J. Borgnia

Authors
  1. Ying Yin
    View author publications

    Search author on:PubMed Google Scholar

  2. Cheon-Gyu Park
    View author publications

    Search author on:PubMed Google Scholar

  3. Shasha Feng
    View author publications

    Search author on:PubMed Google Scholar

  4. Ziqiang Guan
    View author publications

    Search author on:PubMed Google Scholar

  5. Hyuk-Joon Lee
    View author publications

    Search author on:PubMed Google Scholar

  6. Feng Zhang
    View author publications

    Search author on:PubMed Google Scholar

  7. Kedar Sharma
    View author publications

    Search author on:PubMed Google Scholar

  8. Mario J. Borgnia
    View author publications

    Search author on:PubMed Google Scholar

  9. Wonpil Im
    View author publications

    Search author on:PubMed Google Scholar

  10. Seok-Yong Lee
    View author publications

    Search author on:PubMed Google Scholar

Contributions

Y.Y. conducted biochemical preparation, cryo-EM experiments, single-particle 3D reconstruction and model building under the guidance of S.-Y.L. C.-G.P. and F.Z. carried out all the electrophysiological recordings under the guidance of S.-Y.L. S.F. carried out all the molecular dynamics simulations under the guidance of W.I. Z.G., H.-J.L. and Y.Y. conducted mass spectrometry experiments. K.S. helped with part of the grid screening under the guidance of M.J.B. S.-Y.L. wrote the paper with input from the other authors.

Corresponding author

Correspondence to Seok-Yong Lee.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Functional characterization of TRPM3 channel activation by various agonists.

a, Representative currents at –60 mV (lower traces) and +60 mV (upper traces) from HEK293T cells expressing the mTRPM3α2 WT channels (left) and from GFP-only transfected cells (middle), in response to 200 μM PregS (blue), 200 μM Nif (orange) and 1 μM CIM (green) as indicated. Summary (right) of current amplitude in response to 200 μM PregS, 200 μM Nif and 1 μM CIM, respectively, from HEK293T cells expressing mTRPM3α2 WT (filled circles, n = 4 biological replicates; mean ± s.e.m.) and from cells transfected only with GFP (open circles, n = 3 biological replicates; mean ± s.e.m.) at +60 mV. P values from two-way ANOVA followed by Sidak post-hoc test are shown. b, Left: representative currents at –60 mV (lower traces) and +60 mV (upper traces) from HEK293T cells transfected only with GFP, in response to 200 μM PregS (blue) and 1 μM CIM (green) as indicated. Right: Summary of current amplitude in response to 200 μM PregS and 1 μM CIM from HEK293T cells transfected only with GFP at +60 mV (n = 3 biological replicates; mean ± s.e.m.). c, Left: representative traces at –60 mV (lower traces) and +60 mV (upper traces) from HEK293T cells transfected only with GFP, in response to 200 μM PregS (blue) and 200 μM Nif (orange) as indicated. Summary of current amplitude in response to 200 μM PregS and 200 μM Nif from HEK293T cells transfected only with GFP at +60 mV (n = 3 biological replicates; mean ± s.e.m.).

Source data

Extended Data Fig. 2 Cryo-EM data processing.

a–c, Cryo-EM data processing workflows, including representative 2D classification images, for the primidone-bound TRPM3 (a), the CHS-bound TRPM3 (b) and the PregS/CIM-bound TRPM3 (c) structures.

Extended Data Fig. 3 Cryo-EM data validation.

a, Representative micrographs with the physical pixel size indicated. b, Fourier shell correlation (FSC) curves of the final 3D reconstruction with different masking calculated in CryoSPARC. c, Local resolution estimation of the final 3D reconstruction. d, Orientation distribution diagram of the final 3D reconstruction. e, FSC curves between model and full- or half-maps calculated in PHENIX. Results are shown for the primidone-bound TRPM3 (left), the CHS-bound TRPM3 (middle) and the PregS/CIM-bound TRPM3 (right) structures.

Extended Data Fig. 4 Quality of cryo-EM maps.

a–c, Representative cryo-EM densities (meshes) for various structural elements in the primidone-bound TRPM3 (a, green), the CHS-bound TRPM3 (b, light blue) and the PregS/CIM-bound TRPM3 (c, magenta) structures. Residue ranges are specified in parentheses. d, EM densities (meshes) for primidone, CHS1 and CHS2 in the primidone-bound TRPM3 reconstruction (green), CHS1 and CHS2 in the CHS-bound TRPM3 reconstruction (light blue), CIM, PregS1 and PregS2 in the PregS/CIM-bound TRPM3 reconstruction (magenta).

Extended Data Fig. 5 Architecture of the TRPM3 channel and comparison with the published TRPM3 structure.

a, Structure model of the CHS-bound TRPM3 with a three-layer architecture. Each layer is labeled as transmembrane domain (TMD), top and bottom layers of the cytosolic domain (CD top and CD bottom). Protomers are colored blue, green, gold and red. b,c, Topology (b) and structure (c) of a single TRPM3 protomer. d, Comparisons of the TMDs between the published TRPM3 structure (PDB 8ED7) and the CHS-bound TRPM3 structure (left) and between the published TRPM3 structure and the PregS/CIM-bound TRPM3 structure (right). e, Side-by-side comparison of the published TRPM3 (PDB 8ED7), the CHS-bound TRPM3 and the PregS/CIM-bound TRPM3 structures.

Extended Data Fig. 6 Functional characterization of TRPM3.

a, Representative currents of the mutant mTRPM3α2 channels in HEK293T cells at –60 mV (lower traces) and +60 mV (upper traces) in response to 100 μM PregS (blue), 100 μM primidone (gold) and 50 μM ruthenium red (RR) (red) as indicated. b, Representative TEVC recording on the WT and mutant mTRPM3α2 channels in oocytes at –60 mV (lower traces) and +60 mV (upper traces) in response to 200 μM PregS (blue), 200 μM primidone (gold) and 50 μM RR (red) as indicated. c, Summary of current inhibition by 200 μM primidone in WT and mutant mTRPM3α2 channels at +60 mV (n = 8,7,6,8,8 and 8 biological replicates, respectively; mean ± s.e.m.). P values from one-way ANOVA followed by Dunnett’s post-hoc test for differences between WT and each mutant are shown. d, Representative currents of the mutant mTRPM3α2 channels in HEK293T cells at –60 mV (lower traces) and +60 mV (upper traces) in response to 200 μM Nif (orange) and 200 μM PregS (blue) as indicated. e, Representative currents of the mutant mTRPM3α2 channels in HEK293T cells at –60 mV (lower traces) and +60 mV (upper traces) in response to 200 μM PregS (blue), 200 μM Nif (orange) and 1 μM CIM (green) as indicated. Dotted lines in a,b,d,e indicate the zero-current level.

Source data

Extended Data Fig. 7 Identification of lipids and small molecules bound to TRPM3 by liquid chromatography–high-resolution mass spectrometry analysis.

a, Chemical structures and expected exact masses of pregnenolone sulfate (PregS), cholesteryl hemisuccinate (CHS) and CIM 0216 (CIM). b,c, PregS is detected in the +PregS sample (b) but not in the -PregS sample (c). d,e, PregS is detected in the +PregS sample (d) and CHS is detected in the -PregS sample (e). f,g, CHS is not detected in the +PregS sample (f) but is detected in the -PregS sample (g). h,i, CIM is not detected in the control sample (h) but is detected in the +CIM sample (i). Inset: mass spectrum showing the [M + H]+ and [M+Na]+ ions of CIM at m/z 348.176 and m/z 370.155, respectively. Normal LC–MS analysis of PregS was performed in the negative ion mode. Reverse LC–MS analysis of CIM was performed in the positive ion mode. EIC, extracted ion chromatogram.

Extended Data Fig. 8 Comparison of ligand binding at the outer pore in TRP channels.

a, Cholesteryl hemisuccinate (CHS) (yellow, left) and pregnenolone sulfate (PregS) (purple, right) binding at site 1 and site 2 in TRPM3 channel identified in the current study. PregS at site 2 is shown as semi-transparent. b, CHS and phospholipid binding in the TRPML3 channel. c, Binding of the detergent glyco-diosgenin (GDN) in TRPM3 (left, yellow), CHS in TRPM4 (middle, yellow) and CHS in TRPM8 (right, yellow). d, Binding of the inhibitor HC-070 (left, green) and the putative diacylglycerol (DAG) (middle, yellow) in the TRPC5 channel. Binding of the agonist AM-0883 (right, green) in the TRPC6 channel. PDB IDs for the published structures in b–d are indicated. Ligands are shown as spheres, with agonists/inhibitors colored green, CHS/phospholipids/GDN/DAG colored yellow and PregS colored purple.

Extended Data Fig. 9 All-atom molecular dynamics simulation studies of TRPM3.

a, Conformational ensemble of PregS, CHS and cholesterol binding at site 1 and site 2, with twelve replicas shown. b,d, The starting configurations of CIM ligand in pose 1 (b) and pose 2 (d), with a schematic showing the orientation of the three ring systems. c,e, Conformational ensemble overlay of the CIM ligand in pose 1 (c) and pose 2 (e) after 1200-ns simulation. Twelve replicas (3 replicas x 4 subunits) are overlaid with two views shown. Pose 1 leads to more consistent ligand-binding configurations, as indicated by the well aligned oxazole and amide. f, Whole-molecule ligand r.m.s.d. of CIM in pose 1 and pose 2. g, Oxazole ring-based ligand r.m.s.d. of CIM in pose 1 and pose 2. The difference is salient between the two poses, as the ligand r.m.s.d. is more stable in pose 1, consistent with the structural overlay in (c).

Extended Data Fig. 10 Potential ion permeation pathways within VSLDs and disease mutation mapping on the TRPM3 structures.

a, Dot surface representations of the potential ion permeation pathways within the VSLDs in both the CHS-bound and the PregS/CIM-bound states. Regions inaccessible to water (pore radius <1.15 Å) are colored red. b–e, In silico mutagenesis (cyan) on both the CHS-bound and the PregS/CIM-bound TRPM3 structures. (b,c) Both L759 (L769 in human, pre-S1) and V992 (V1002 in human, S4b) interact with the TRP domain. However, the changes are subtle, making the increased activity of these mutations not entirely clear. (d) P1092 (P1102 in human) is located in S6 at the outer pore interface, comprising part of the CHS (or cholesterol) site 2 in the CHS-bound or the primidone-bound TRPM3 structures. In the PregS/CIM-bound TRPM3 structure, S6 rotates to position P1092 away from the PregS site 2 to form an interaction with E1057 on PHb. Therefore, P1092Q likely destabilizes the apo (closed) state by reducing cholesterol binding and stabilizes the PregS/CIM-bound conformation through interaction with PHb. CHS at site 2 of the CHS-bound structure is shown as yellow sticks (left panels). (e) N1116 (N1126 in human) is located toward the C-terminus of S6, adjacent to the neighboring S6. N1116D can interact electrostatically with K1122′ of the neighboring S6 only in the PregS/CIM-bound conformation. The salt bridge interaction between N1116D and K1122′ in the PregS/CIM-bound state is shown in the PregS/CIM N1116D panel.

Supplementary information

Source data

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yin, Y., Park, CG., Feng, S. et al. Molecular basis of neurosteroid and anticonvulsant regulation of TRPM3. Nat Struct Mol Biol 32, 828–840 (2025). https://doi.org/10.1038/s41594-024-01463-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Version of record:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41594-024-01463-8

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing