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Cited by 14Heavy Metal Exposure and ToxicityTrace Elements in HealthIron Metabolism and Disorders

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Molecular mechanisms of SLC30A10-mediated manganese transport

Open access
Shen, Xurui, Zhang, Jinlun Kylian, Sun, Peixin, Zhong, Huiwen, He, Rui, Wang, Shiliang, Guo, Xiaojun, Yang, Hanting·2025·Nature Communications
DOI 10.1038/s41467-025-63616-7PMC PMC12480533

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Abstract

Abstract Manganese (Mn) is an essential trace element required for various physiological processes, yet excessive levels induce oxidative stress and lead to severe health issues. The transporter SLC30A10 is crucial for maintaining Mn homeostasis by exporting Mn from cells to prevent toxic accumulation. Mutations in the SLC30A10 gene disrupt this function, resulting in manganese accumulation, which leads to disorders such as hypermanganesemia with dystonia 1 (HMNDYT1). Here, we show that SLC30A10 mediates Mn efflux under elevated Mn conditions, alleviating oxidative stress and preserving mitochondrial integrity. High-resolution cryo-EM structures reveal a unique Mn-binding site in SLC30A10, setting it apart from other SLC30 family transporters. We further demonstrate that the HMNDYT1-linked D40A mutation disrupts the binding and transport of Mn, identifying D40 as a potential therapeutic target. These findings provide structural insights into Mn transport and potential avenues for targeting Mn toxicity.

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Figures

Fig. 1 Functional characterisation and overall architecture of hSLC30A10. a Normalized Mn levels in SH-SY5Y cells (n = 5). b, c Flow cytometry detection (b) and statistical analysis (c; n = 3) of cell survival by Annexin V / PI. d Cell survival by Annexin V / PI in the absence or the presence of 100 μM MnCl2 for 24 h in Dox-inducible SLC30A10 overexpressed SH-SY5Y cells (n = 4). -Dox, without Dox treatment. +Dox, treated with 100 ng/ml Dox. e Normalized Mn levels in SLC30A10 transfected HEK293T cells by ICP-MS. Cells transfected with vector served as control. Data were normalized to control (n = 4). f In vitro Mn2+ transport assay of SLC30A10. Purified human SLC30A10 protein and calcein-salt were reconstituted into liposome. The fluorescence of calcein was quenched by Mn2+ that is transported into the liposome (n = 3). Transport was initiated by the addition of 100 μM MnCl₂. The fluorescence of MnCl2-free proteoliposome was measured to determine the background signal (F0). The fluorescence of 0 s was measured as maximum (max) fluorescence. The relative fluorescence was calculated using the equation (F/Fmax)-(1-F0/F0max). g, h Cryo-EM map of human SLC30A10 in the Mn2+-bound state in the side view. The two monomers are coloured in red and blue. i, j Cylinder representation of SLC30A10 in the Mn2+-bound state in the side view. Mn2+ is in purple spheres. The flexible region in the loops between TM4 and TM5 not observed in the structure are shown as dash line. k Cylinder representation of SLC30A10 in the Mn2+-bound state in the extracellular view. l Topology of SLC30A10 monomer. All bar graphs above represent mean ± SEM. Statistical analysis was performed using two-tailed unpaired t-test (a, c, e), one-way ANOVA with Dunnett’s multiple comparisons test (d), or two-way ANOVA with Sidak’s multiple comparisons test (f) (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns not significant). The experiments were independently repeated 3 times.
Fig. 1 Functional characterisation and overall architecture of hSLC30A10. a Normalized Mn levels in SH-SY5Y cells (n = 5). b, c Flow cytometry detection (b) and statistical analysis (c; n = 3) of cell survival by Annexin V / PI. d Cell survival by Annexin V / PI in the absence or the presence of 100 μM MnCl2 for 24 h in Dox-inducible SLC30A10 overexpressed SH-SY5Y cells (n = 4). -Dox, without Dox treatment. +Dox, treated with 100 ng/ml Dox. e Normalized Mn levels in SLC30A10 transfected HEK293T cells by ICP-MS. Cells transfected with vector served as control. Data were normalized to control (n = 4). f In vitro Mn2+ transport assay of SLC30A10. Purified human SLC30A10 protein and calcein-salt were reconstituted into liposome. The fluorescence of calcein was quenched by Mn2+ that is transported into the liposome (n = 3). Transport was initiated by the addition of 100 μM MnCl₂. The fluorescence of MnCl2-free proteoliposome was measured to determine the background signal (F0). The fluorescence of 0 s was measured as maximum (max) fluorescence. The relative fluorescence was calculated using the equation (F/Fmax)-(1-F0/F0max). g, h Cryo-EM map of human SLC30A10 in the Mn2+-bound state in the side view. The two monomers are coloured in red and blue. i, j Cylinder representation of SLC30A10 in the Mn2+-bound state in the side view. Mn2+ is in purple spheres. The flexible region in the loops between TM4 and TM5 not observed in the structure are shown as dash line. k Cylinder representation of SLC30A10 in the Mn2+-bound state in the extracellular view. l Topology of SLC30A10 monomer. All bar graphs above represent mean ± SEM. Statistical analysis was performed using two-tailed unpaired t-test (a, c, e), one-way ANOVA with Dunnett’s multiple comparisons test (d), or two-way ANOVA with Sidak’s multiple comparisons test (f) (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns not significant). The experiments were independently repeated 3 times.CC BY-NC-ND · PubMed Central
Fig. 2 Mn²⁺ binding site and coordination in SLC30A10. a Electrostatic surface potential of the SLC30A10 inward-facing (IF). Mn2+ is in purple spheres. Negatively to positively charged regions are coloured from red to blue. The black box indicates the focused area shown in (b, c). b Zoomed-in view of the Mn2+-binding site in the side view. The density is contoured at 1.7σ. c The density map of the Mn2+-binding site. The density is contoured at 2σ. Blue, Mn2+ bound; orange, Mn2+ free. d Time-dependent variation of the distance between Mn2+ and the coordinating residue atoms in MD simulation. e Transport ability of SLC30A10 WT and mutations in HEK293T cells (n = 3). Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. f Structural comparison of Mn2+-bound SLC30A10 (IF) and Zn2+-bound ZnT1 (IF, PDB code:8XMF). The SLC30A10 is shown in blue, and ZnT1 in grey. The boxes indicate the focused areas shown in (g, h). g Zoomed-in view of the Mn2+-binding site from the extracellular view. Blue, Mn2+-bound SLC30A10 (IF); grey, Zn2+-bound ZnT1 (IF). h Comparison of the ZnT1 ion-binding site with the corresponding residues in SLC30A10. Blue, Mn2+-bound SLC30A10 (IF); grey, Zn2+-bound ZnT1 (IF); Mn2+ is in purple sphere; Zn2+ is in pink sphere. i Sequence alignment of human SLC30A10, ZnT1, ZnT3, ZnT4, ZnT7 and ZnT8 across a portion of the TM2 and TM5 region. j Transport ability of SLC30A10 WT and mutations in HEK293T cells (n = 3). Statistical analysis was performed using one-tailed one-sample t-test. All σ values are derived from locally extracted and resampled maps surrounding the Mn²⁺-binding site. All bar graphs above represent mean ± SEM from three independent experiments (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns not significant).
Fig. 2 Mn²⁺ binding site and coordination in SLC30A10. a Electrostatic surface potential of the SLC30A10 inward-facing (IF). Mn2+ is in purple spheres. Negatively to positively charged regions are coloured from red to blue. The black box indicates the focused area shown in (b, c). b Zoomed-in view of the Mn2+-binding site in the side view. The density is contoured at 1.7σ. c The density map of the Mn2+-binding site. The density is contoured at 2σ. Blue, Mn2+ bound; orange, Mn2+ free. d Time-dependent variation of the distance between Mn2+ and the coordinating residue atoms in MD simulation. e Transport ability of SLC30A10 WT and mutations in HEK293T cells (n = 3). Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. f Structural comparison of Mn2+-bound SLC30A10 (IF) and Zn2+-bound ZnT1 (IF, PDB code:8XMF). The SLC30A10 is shown in blue, and ZnT1 in grey. The boxes indicate the focused areas shown in (g, h). g Zoomed-in view of the Mn2+-binding site from the extracellular view. Blue, Mn2+-bound SLC30A10 (IF); grey, Zn2+-bound ZnT1 (IF). h Comparison of the ZnT1 ion-binding site with the corresponding residues in SLC30A10. Blue, Mn2+-bound SLC30A10 (IF); grey, Zn2+-bound ZnT1 (IF); Mn2+ is in purple sphere; Zn2+ is in pink sphere. i Sequence alignment of human SLC30A10, ZnT1, ZnT3, ZnT4, ZnT7 and ZnT8 across a portion of the TM2 and TM5 region. j Transport ability of SLC30A10 WT and mutations in HEK293T cells (n = 3). Statistical analysis was performed using one-tailed one-sample t-test. All σ values are derived from locally extracted and resampled maps surrounding the Mn²⁺-binding site. All bar graphs above represent mean ± SEM from three independent experiments (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns not significant).CC BY-NC-ND · PubMed Central
Fig. 3 Conformational transition of SLC30A10. a Structural overlay of SLC30A10 inward-facing (IF) and outward-facing (OF). The IF conformation is coloured in blue, and OF conformation in green, respectively. The box indicates the focused area shown in panels (b−d). b−d Zoomed-in view of the overlay of IF and OF in sideview (b), intracellular view (c) and extracellular view (d). e Electrostatic surface potential of the transmembrane domain of SLC30A10 (OF). Negatively to positively charged regions are coloured from red to blue. f, g The OF conformation of SLC30A10 structure aligned with the ZnT1 (PDB code:8ZSZ), ZnT7 (PDB code:8J7W) and ZnT8 (PDB code:6XPE). SLC30A10, ZnT1, ZnT7 and ZnT8 are coloured in green, grey, yellow and purple, respectively. Zn2+ is in pink spheres.
Fig. 3 Conformational transition of SLC30A10. a Structural overlay of SLC30A10 inward-facing (IF) and outward-facing (OF). The IF conformation is coloured in blue, and OF conformation in green, respectively. The box indicates the focused area shown in panels (b−d). b−d Zoomed-in view of the overlay of IF and OF in sideview (b), intracellular view (c) and extracellular view (d). e Electrostatic surface potential of the transmembrane domain of SLC30A10 (OF). Negatively to positively charged regions are coloured from red to blue. f, g The OF conformation of SLC30A10 structure aligned with the ZnT1 (PDB code:8ZSZ), ZnT7 (PDB code:8J7W) and ZnT8 (PDB code:6XPE). SLC30A10, ZnT1, ZnT7 and ZnT8 are coloured in green, grey, yellow and purple, respectively. Zn2+ is in pink spheres.CC BY-NC-ND · PubMed Central
Fig. 4 Mn2+ binding site is crucial for the protective function of SLC30A10. a Proposed model for the Mn²⁺ transport mechanism of SLC30A10: The orientation and relative positions of helices and key residues are shown based on the structural data. Purple spheres represent Mn²⁺ ions. For clarity, only the transmembrane domain (TMD) of one protomer is shown. Mn²⁺ is attracted to a negatively charged cytoplasmic cavity (Red) and coordinated by D248, D40, N127, and S252. The ions are ultimately exported through an extracellular cavity, exiting the cell. b–e Statistical analysis of normalized Mn levels (b; n = 5), ROS levels (c; n = 3), mitochondrial membrane potential (d; n = 3) and ATP levels (e; n = 3) in the presence of 100 μM MnCl2 for 24 h in SH-SY5Y cells. f, g Flow cytometry detection (f) and statistical analysis (g; n = 3) of cell survival by Annexin V / PI staining in SH-SY5Y cells. All bar graphs above represent mean ± SEM from three independent experiments. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns not significant).
Fig. 4 Mn2+ binding site is crucial for the protective function of SLC30A10. a Proposed model for the Mn²⁺ transport mechanism of SLC30A10: The orientation and relative positions of helices and key residues are shown based on the structural data. Purple spheres represent Mn²⁺ ions. For clarity, only the transmembrane domain (TMD) of one protomer is shown. Mn²⁺ is attracted to a negatively charged cytoplasmic cavity (Red) and coordinated by D248, D40, N127, and S252. The ions are ultimately exported through an extracellular cavity, exiting the cell. b–e Statistical analysis of normalized Mn levels (b; n = 5), ROS levels (c; n = 3), mitochondrial membrane potential (d; n = 3) and ATP levels (e; n = 3) in the presence of 100 μM MnCl2 for 24 h in SH-SY5Y cells. f, g Flow cytometry detection (f) and statistical analysis (g; n = 3) of cell survival by Annexin V / PI staining in SH-SY5Y cells. All bar graphs above represent mean ± SEM from three independent experiments. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns not significant).CC BY-NC-ND · PubMed Central

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Citations

Cites

Research works printed but not linked: 2

  1. Kwakye, G. F., Li, D., Kabobel, O. A. & Bowman, A. B. Cellular fura-2 manganese extraction assay (CFMEA). Curr. Protoc. Toxicol. Chapter 12, Unit12–Unit18 (2011).
  2. Hess, B., Bekker, H., Berendsen, H. J. C. & Fraaije, J. G. E. M. LINCS: linear Constraint Solver Mol. Simul. 18, 1463–1472 (1997).

Metadata

Authors
Shen, X., Zhang, J. K., Sun, P., Zhong, H., He, R., Wang, S., Guo, X., & Yang, H.
Affiliations
Fudan University (CN)
Corresponding author
Yang, Hanting
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2025-09-29
Volume
16
Issue
1
Pages
8581-8581
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-025-63616-7
PMC
PMC12480533
OpenAlex
W4414601776
Cited by
14
References
59
Citations by year
2026: 13 · 2025: 1
FWCI
5.96
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by-nc-nd)
  • Research Square (acceptedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Ministry of Science and Technology of the People's Republic of China (STI2030-Major Projects 2022ZD0212600)
  • National Natural Science Foundation of China (32171216)
  • China Postdoctoral Science Foundation (2023M730689)
  • Fudan University (23216)
Topics
  • Physical Sciences › Environmental Science › Health, Toxicology and Mutagenesis › Heavy Metal Exposure and Toxicity
  • Health Sciences › Nursing › Nutrition and Dietetics › Trace Elements in Health
  • Health Sciences › Medicine › Hematology › Iron Metabolism and Disorders
Keywords
SLC30A10, manganese transport, manganese homeostasis, manganese efflux, manganese binding site, manganese accumulation, mitochondrial dysfunction, therapeutic target
MeSH terms
Zinc Transporter 8, Binding Sites, Biological Transport, Humans, Manganese, Models, Molecular, Mutation, Protein Binding, Ion Transport, Cryoelectron Microscopy, Cation Transport Proteins, HEK293 Cells

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