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Persistent activation of TRPM4 triggers necrotic cell death characterized by sodium overload

Nature Chemical Biology volume 21, pages 1238–1249 (2025) Cite this article

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Abstract

Sodium influx and overload are frequently observed in human tissue injuries. Whether sodium overload imposes a causative effect on necrotic cell death and the mechanism involved are unclear. Here we identify necrocide 1 (NC1) as a compound that induces necrotic cell death through sodium overload, termed NECSO for necrosis by sodium overload. NC1 targets the transient receptor potential cation channel subfamily M member 4 (TRPM4), a nonselective monovalent cation channel, to promote Na+ influx and necrosis. TRPM4-deficient cells are resistant to NC1-induced NECSO. NC1 specifically activates human TRPM4, not mouse TRPM4, because of differences in a transmembrane region, as revealed by domain swapping and molecular docking. Gain-of-function mutations in human TRPM4 linked to cardiac arrhythmias show increased vulnerability to NECSO triggered by NC1 or 2-deoxy-d-glucose. Chemical screening identified NECSO inhibitors that block necrosis induced by NC1 or energy depletion. These findings provide insights into regulated Na+ influx-mediated necrosis and its implications for disease.

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Fig. 1: NC1 triggers a distinct cell death pathway dependent on TRPM4.
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Fig. 2: Sodium influx is an essential event for NC1-induced cell death.
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Fig. 3: NC1 specifically targets hTRPM4 through interaction with the TM region.
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Fig. 4: Docking and mutagenesis analysis reveal critical residues of TRPM4 for NC1 activation.
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Fig. 5: Energy-depletion-induced cell death shares features of NECSO.
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Fig. 6: DHPs and CLT function as small-molecule inhibitors of NECSO.
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Data availability

All original data that support the findings of this study are provided in the article and Supplementary Information. All specific and stable reagents generated in this study are available from the corresponding authors without restriction. Source data are provided with this paper.

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Acknowledgements

We thank the Core Facility of Basic Medical Science, Shanghai Jiaotong University School of Medicine and Instrumental Analysis Center of Shanghai Jiaotong University for technical support. The computations in this paper were run on the π2.0 cluster and supported by bioinformatics platform of the Center for High Performance Computing at Shanghai Jiao Tong University. This work was partially supported by SJTU Kunpeng and Ascend Center of Excellence. We thank Y. Jiang (University of Texas Southwestern Medical Center) for helpful discussion. We thank N. Huang (Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University) for the use of Schrödinger software. We express our gratitude to J. Guo (Zhejiang University), A. C. Y. Chang and B. Li (Shanghai Jiaotong University) for providing reagents. The work was supported in part by grants from the Ministry of Science and Technology of the People’s Republic of China (2023YFA0914900), National Natural Science Foundation of China (92254307, M-0140 and 91957204), the Fundamental Research Funds for the Central Universities, the Shanghai Sailing Program (20YF1422300) to W.F. and Zhiyuan Future Scholar Program (zirc2023-13) to Z. Zhao. The work was also supported by the innovative research team of high-level local universities in Shanghai (SHSMU-ZDCX20212000) and Shanghai Frontiers Science Center of Cellular Homeostasis and Human Diseases.

Author information

Author notes

  1. These authors contributed equally: Wan Fu, Jianghuang Wang, Tianyu Li, Yuhui Qiao.

Authors and Affiliations

  1. Institute for Translational Medicine on Cell Fate and Disease, Shanghai Ninth People’s Hospital, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Department of Pathophysiology, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Wan Fu, Jianghuang Wang, Yuhui Qiao, Zili Zhang, Xiaomin Zhang, Mingkai He, Yan Su, Ziye Zhao, Shen Zhang & Qing Zhong

  2. Shanghai Key Laboratory of Maternal Fetal Medicine, Shanghai Institute of Maternal-Fetal Medicine and Gynecologic Oncology, Department of Anesthesiology, Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University, Shanghai, China

    Tianyu Li & Zhiqiang Liu

  3. Anesthesia and Brain Function Research Institute, Tongji University School of Medicine, Shanghai, China

    Tianyu Li & Zhiqiang Liu

  4. State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    Tianyu Li & Yang Li

  5. University of Chinese Academy of Sciences, Beijing, China

    Tianyu Li & Yang Li

  6. Zhiyuan College, Shanghai Jiao Tong University, Shanghai, China

    Ziye Zhao

  7. Network and Information Center, Shanghai Jiao Tong University, Shanghai, China

    Chen Li & James Lin

  8. School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, China

    Ronghua Xiao, Yujun Han & Yang Li

  9. Hainan Academy of Medical Sciences, School of Basic Medicine and Life Science, Hainan Medical University, Haikou, China

    Guoqiang Chen

  10. National Clinical Research Center for Aging and Medicine, Huashan Hospital, Fudan University, Shanghai, China

    Yang Li

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  1. Wan Fu
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Contributions

W.F. and J.W. performed most of the biological and biochemical experiments. T.L. performed electrophysiological experiments with the help of R.X., Y.H. and Z.L. J.W. performed the molecular docking, MD simulations and mutagenesis analysis. Y.Q. and Z. Zhang performed the chemical inhibitor screening and helped with some of the cell experiments. X.Z., M.H., Y.S. and Z. Zhao helped with the cell biological experiments. C.L. performed the CRISPR screening data analysis with guidance of J.L. S.Z. and G.C. helped in data analysis. W.F. and Q.Z. conceptualized the project and designed the experiments. W.F., J.W. and Q.Z. analyzed the data and wrote the paper with the help of all authors. Y.L. and Q.Z. supervised the project. All authors discussed the results and commented on the paper.

Corresponding authors

Correspondence to Yang Li or Qing Zhong.

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Nature Chemical Biology thanks János Almássy, Kengo Watanabe and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Different death-inducing effect of the stereoisomers NC1 and NC1i.

a, Chemical structure of compound NC1 and NC1i. b, IC50 of NC1 and NC1i in various cell lines treated for 48 h. c, Live-cell images of MCF7 cells treated with 50 nM NC1 in medium containing SytoxGreen. Scale bars, 25 μm. d, Heatmap of viability of cells pretreated for 1 h with 20 μM Z-VAD-FMK (Z-VAD), 10 μM Necrostatin-1 (Nec-1), 50 μM Chloroquine (CQ), 50 μM 3-Methyladenine (3-MA), 1 μM Ferrostatin-1 (Fer-1), 1 μM Liproxstatin-1 (Liprox-1), or 100 μM Deferoxamine (DFO) and then treated with either 100 nM NC1, RSL3, 50 ng/ml TNF-α + 1 μM Smac Mimetic (TS), or TS + 20 μM Z-VAD (TSZ) for 24 h. Viability data represent mean of n = 3 replicates from one representative of three independent experiments. Micrograph data show one representative out of three replicates from three independent experiments.

Source data

Extended Data Fig. 2 NC1, rather than NC1i, triggers TRPM4 activation.

a, Typical whole-cell current traces recorded from HEK293T cells stably expressing hTRPM4 channel and stimulating voltage steps used in the experiment is on the top (left panel). Subsequently, whole-cell current traces recorded after 1 μM NC1 application (middle panel), followed by inhibition with 50 μM 9-ph inhibitor (right panel). Three figures were recorded from one same patch. b, The current density-voltage relation of hTRPM4 steady-state current with vehicle or NC1i application. c, Statistics of hTRPM4 steady-state whole-cell current density with vehicle, NC1i or NC1 application at +100 mV in b. d-g, Typical current traces of hTRPM4 channel treated vehicle or 1 μM NC1 by using outside-out (d) or inside-out (f) patch. Statistics of hTRPM4 outside-out (e) or inside-out (g) current. h, The time course of TRPM4 current-densities in HEK293T-TRPM4 cells activated by 1 μM NC1 was followed by NC1 washout using bath solution after 420 seconds. The whole-cell currents were recorded at −100 and +100 mV. Electrophysiological data represent mean ± sem of (n = 5 for b, 11 for NC1 and 5 for NC1i in c, 8 for e, 4 for g, 8 for h) independent cells. Two-way ANOVA with post Sidak test (GraphPad Prism 9.0.0).

Source data

Extended Data Fig. 3 NECSO is accompanied with Membrane depolarization and cell edema.

a, Dose-dependent cytotoxicity of NC1 in MCF7 cells pretreated with 20μM NKCC inhibitors (Furosemide, Bumetanid and Azosemide) or KATP inhibitors (PNU 37883A and Glipizide). b, A typical membrane potential of MCF7 cells treated with 1 μM NC1. c, Statistics of membrane potential values of MCF7 cells recorded 10 minutes after exposed to vehicle or NC1 in b. d, Representative confocal images of MCF7 cells loaded with 5 μM DiBAC4(3) followed by treatment with 50 nM NC1 for 1.5 h. e, Statistics of fold change for DiBAC4(3) fluorescence intensity, mean ± s.d. Each dot represents an individual image in d. f, Representative confocal images of MCF7 cells treated with DMSO, 50 or 100 nM NC1 for 3 h and then loaded with chloride indicator MQAE. Scale bars, 10 μm. g, Statistics of fold change MQAE fluorescence intensity, mean ± s.d. Each dot represents an individual image in f. h, Dose-dependent cytotoxicity of NC1 in MCF7 cells pretreated with multiple inhibitors of chloride anion channels. i, Transmission electron microscopy (TEM) of MCF7 cells treated with 100 nM NC1 at different time points as indicated. Scale bars, 500 nm. Red arrowheads in images of 0.5, 1.5 and 2.5 h represented swollen mitochondria, endoplasmic reticulum and Golgi apparatus respectively, and highlighted swollen nuclear membrane (above) and broken cell membrane (below) in images of 4 h. j, Cell death assay measured by LDH release of MCF7 cells treated with NC1 for 24 h in medium containing different doses of osmo-protectant D-mannitol. Viability and cell death data represent mean ± s.d. of n = 3 replicates from one representative of three independent experiments. Electrophysiological data represent mean ± sem of n = 10 independent cells. Micrograph data show one representative out of three replicates from three independent experiments. One-way ANOVA for statistics in c, e, g (GraphPad Prism 9.0.0).

Source data

Extended Data Fig. 4 Different responses to NC1 of TRPM4 from multiple species.

a, IC50 of NC1 in various cell lines of different species treated for 48 h. b, Representative confocal images of HeLa cells with expression of various species TRPM4, treated with 1 μM NC1 for 24 h. PI staining indicates the dead cell. c, Statistics of the percentage of NC1-responsive cells in TRPM4-EGFP transfected cells in b, mean ± s.d. from 50 cells from three independent experiments. d, Summary results of sensitivity to NC1-induced current density of different species TRPM4 at +100 mV in whole-cell patch clamp experiments. e, Representative confocal images of HeLa cells transfected with different TRPM family protein, subsequently treated with 1 μM NC1 for 24 h. PI staining indicates the dead cell. f, Histogram summarizes the percentage of NC1-responsive cells in TRPM4-EGFP transfected cells in e, mean ± s.d. from 50 cells from three independent experiments. Micrograph data show one representative out of three replicates from three independent experiments.

Source data

Extended Data Fig. 5 Distinct responses to NC1 of TRPM4 human-mouse chimeras.

a, Schematic representation of the three major domains and their corresponding amino acid sequence numbers in hTPPM4 (upper panel). Each structural component is labeled and color-coded in detail (lower panel). b, Representative confocal images of HeLa cells transfected with different TRPM4 chimera, treated with 1 μM NC1 for 24 h. c, Histogram summarizes the percentage of NC1-responsive cells with TRPM4-EGFP expression in b, mean ± s.d. from 50 cells from three independent experiments. Micrograph data show one representative out of three replicates from three independent experiments.

Source data

Extended Data Fig. 6 Model representations of ligand binding sites in vanilloid binding pocket (VBP).

The overall architecture of the indicated TRP channels, and one subunit is highlighted. Ligands are presented with spheres (upper panel). Ligands are shown as sticks. Oxygen is shown in red, nitrogen in blue, sulfur in yellow, fluorine in light blue, bromine in dark red, and chloride in green (lower panel).

Extended Data Fig. 7 Different cross-sections of Ligand NC1 binding pocket in hTRPM4 (PDB: 6BQV).

a, b, Overall surface presentation of TRPM4. Ligand NC1 is shown in green spheres presentation. Subunits are individually colored with gray black or gray white. The ligand binding pocket of NC1 is composed of residues from two adjacent subunits. The view is from the front (a) or the top (b).

Extended Data Fig. 8 Molecular dynamics (MD) simulation analyses of the binding of NC1 to TRPM4.

a, The simulated state diagram of the TRPM4-NC1 complex embedded in the cell membrane phospholipid bilayer. b, Summary results of structural stability analyses (root mean square deviation, RMSD) of channel-ligand complex following NC1 binding to TRPM4 channel in 100 ns bouts. c, Interaction energy calculations for NC1 binding to TRPM4 channel on trajectories with convergent conformations. d, Information on the binding pockets of NC1 with the TRPM4 channel was calculated after MD simulations. e, Energy contribution of residues within a 5Å distance of NC1. The mean values of energy are represented by blue bars, while the black lines depict the standard error.

Source data

Extended Data Fig. 9 NC1 activation of TRPM4 per se is not affected by calcium.

a, b, c, The conductance-voltage relation of whole-cell hTRPM4 current in HEK293T-TRPM4-EGFP cells treated vehicle or 1 μM NC1 with different concentrations of EGTA (0, 1, 10 mM) added in the patch pipette solutions. d, Statistics of whole-cell hTRPM4 current density at +100 mV in a, b, c. e, The current density-voltage relation of whole-cell hTRPM4 current in HEK293T-TRPM4 cells treated vehicle or 1 μM NC1 in the presence or absence of 1 μM intracellular Ca2+. NC1 further increase the current amplitude of TRPM4 with the presence of intracellular calcium. f, g, Dose-dependent cytotoxicity of NC1 in MCF7 cells pretreated with calcium ion chelators BAPTA-AM (f) or EGTA-AM (g). h, i, The whole-cell current-voltage relation of hTRPM4 E828K (h) or E1068Q (i) mutant expressed in HeLa cells (right panel). Summary data showing the maximum amplitude of currents activated by NC1 at +100 mV (left panel). j, Dose-dependent cytotoxicity of NC1 in TRPM4-KO MCF7 cells with re-expression of TRPM4 WT or mutants above. Viability data represent mean ± s.d. of n = 3 replicates from one representative of three independent experiments. Electrophysiological data represent mean ± sem of (n = 7 for a, e and i, 5 for b and h, 6 for c) independent cells. Immunoblot data show one representative out of three replicates from three independent experiments. Two-way ANOVA (GraphPad Prism 9.0.0) for statistics in d, h, i.

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Extended Data Fig. 10 NECSO inhibitors protect cells from energy depletion.

Heatmap depicting the cell death (measured by LDH release) of Cos7 cells stably expressing hTRPM4 which were pre-treated with different inhibitors followed by 2DG + NaN3 or NC1 exposure respectively for 16 h (left). Representative bright field images of the cellular morphology corresponding to each treatment condition as described above (right). Scale bars, 20 μm. Cell death and micrograph data show one representative out of three replicates from three independent experiments.

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Supplementary information

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Supplementary Figs. 1–8 and Tables 1 and 2.

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Supplementary Video 1 (download AVI )

Upon NC1 treating, MCF7 cells exhibited typical morphological characteristics of necrosis.

Supplementary Video 2 (download GIF )

The X and Y roll presentation of NC1 binding to the VBP pocket in hTRPM4 (PDB 6BQV).

Supplementary Data 1

The docking model of NC1/CLT binding to hTRPM4.

Supplementary Data 2

The docking model of NC1/CLT binding to hTRPM4.

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Uncropped scans of blots and gels in figures, supplementary figures and extended data.

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Fu, W., Wang, J., Li, T. et al. Persistent activation of TRPM4 triggers necrotic cell death characterized by sodium overload. Nat Chem Biol 21, 1238–1249 (2025). https://doi.org/10.1038/s41589-025-01841-3

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