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Structure and function of the human mitochondrial MRS2 channel

Nature Structural & Molecular Biology volume 32, pages 459–468 (2025)Cite this article

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Abstract

The human mitochondrial RNA splicing 2 protein (MRS2) has been implicated in Mg2+ transport across mitochondrial inner membranes, thus having an important role in Mg2+ homeostasis critical for mitochondrial integrity and function. However, the molecular mechanisms underlying its fundamental channel properties such as ion selectivity and regulation remain unclear. Here we present a structural and functional investigation of MRS2. Cryo-electron microscopy structures in various ionic conditions reveal a pentameric channel architecture and the molecular basis of ion permeation and potential regulation mechanisms. Electrophysiological analyses demonstrate that MRS2 is a Ca2+-regulated, nonselective channel permeable to Mg2+, Ca2+, Na+ and K+, which contrasts with its prokaryotic ortholog, CorA, operating as a Mg2+-gated Mg2+ channel. Moreover, a conserved arginine ring within the pore of MRS2 functions to restrict cation movements, thus preventing the channel from collapsing the proton motive force that drives mitochondrial adenosine triphosphate synthesis. Together, our results provide a molecular framework for further understanding MRS2 in mitochondrial function and disease.

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Fig. 1: Structure of human MRS2.
Fig. 2: Mg2+ and Ca2+ recognition.
Fig. 3: The role of the R332 ring on MRS2 function.
Fig. 4: Ion permeation properties.
Fig. 5: Divalent cation-binding sites.

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Data availability

The raw images were deposited to the EM Public Image Archive with accession codes EMPIAR-12018, EMPIAR-12019 and EMPIAR-12035. The cryo-EM maps were deposited to EM Data Bank with accession codes EMD-41587, EMD-41588 and EMD-41589. Atomic coordinates were deposited to the PDB with accession codes 8TS1, 8TS2 and 8TS3. A previously published structure used for comparison was obtained from the PDB with accession code 4I0U. Previously published cryo-EM maps referred to are available from the EM Data Bank with accession codes EMD-41624, EMD-41628, EMD-35630 and EMD-35631. Source data are provided with this paper.

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Acknowledgements

This work was partly supported by National Institutes of Health grants R01NS109307 and R01GM143440 (to P.Y.) and R01GM144485 (to M.T.). We thank T.-Y. Liu and Y.-L. Huang for technical assistance. We thank the staff scientists at Washington University Center for Cellular Imaging for data acquisition. Some of this work was performed at the Simons EM Center at the New York Structural Biology Center, with major support from the Simons Foundation (SF349247).

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  1. These authors contributed equally: Zhihui He, Yung-Chi Tu.

Authors and Affiliations

  1. Department of Cell Biology and Physiology, Washington University School of Medicine, Saint Louis, MO, USA

    Zhihui He, Jonathan Mount, Jingying Zhang & Peng Yuan

  2. Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    Yung-Chi Tu, Chen-Wei Tsai & Ming-Feng Tsai

  3. Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, USA

    Yung-Chi Tu, Chen-Wei Tsai & Ming-Feng Tsai

  4. Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Jonathan Mount, Jingying Zhang & Peng Yuan

  5. Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Peng Yuan

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Contributions

Z.H. performed protein biochemistry. Z.H., J.M. and J.Z. collected cryo-EM images. J.M., J.Z. and P.Y. conducted cryo-EM structure determination and analysis. Y.T. and C.T. performed electrophysiology experiments. M.T. and P.Y. supervised the project and wrote the paper. All authors edited the paper.

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Correspondence to Ming-Feng Tsai or Peng Yuan.

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

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Extended data

Extended Data Fig. 1 Cryo-EM data processing and validation.

a-c, Image processing and map validation of MRS2Mg (a), MRS2EDTA (b), and MRS2Ca (c). Representative micrographs and 2D classes are shown. The final 3D reconstructions are colored by local resolution. Also shown are Fourier shell correlations (FSC) and orientation distribution of particles used in the final reconstruction.

Extended Data Fig. 2 Cryo-EM density.

a, Ribbon representation of a single subunit of MRS2Mg and cryo-EM density. b, Segments of the final refined model of MRS2Mg and the corresponding cryo-EM densities.

Extended Data Fig. 3 Structural comparison of human MRS2 and TmCorA.

a-b, A single subunit of human MRS2 (a) and TmCorA (b, PDB ID: 4I0U). The N-terminal α/β domain is also highlighted for comparison. c, Divalent ion binding sites in human MRS2 (left panels) and TmCorA (right panels). Overlays of sites 1 and 2 are also shown (MRS2 in green and red; CorA in gray).

Extended Data Fig. 4 Sequence alignment of MRS2 channels.

Multiple protein sequences, including Homo sapiens MRS2 (hMRS2, NCBI sequence: NP_065713.1), Mus musculus MRS2 (mMRS2, NCBI sequence: NP_001013407.2), Rattus norvegicus MRS2 (rMRS2, NCBI sequence: NP_076491.1), Danio rerio MRS2 (zMRS2, NCBI sequence: XP_693621.5), Saccharomyces cerevisiae MRS2 (ScMRS2, NCBI sequence: NP_014979.1), Schizosaccharomyces pombe MRS2 (SpMRS2, NCBI sequence: NP_596358.1), Arabidopsis thaliana MRS2 (AtMRS2, NCBI sequence: AAM62917.1),and Thermotoga maritima CorA (TmCorA, NCBI sequence: WP_004081315.1). Secondary structure elements on the basis of hMRS2 are indicated above the sequences. Critical amino acids are highlighted.

Extended Data Fig. 5 Ion densities.

a-b, Cryo-EM densities in the three ion binding sites for Mg2+ (a) and Ca2+ (b). Mg2+ and Ca2+ are shown as magenta and yellow spheres, respectively. c, Densities near the assigned Cl− binding site from Li et al. (EMD-35630 and EMD-35631). d, Densities near the two additional Mg2+ binding sites in the pore from Lai et al. (EMD-41624).

Extended Data Fig. 6 Cryptic densities near site 3 in MRS2EDTA.

Cryo-EM densities near site 3 in the MRS2EDTA reconstruction. Also shown are cryo-EM density maps of human MRS2 from previous studies (EMD-41628 and EMD-35631).

Extended Data Fig. 7 Control experiments for MRS2 function.

a, MRS2-specific Mg2+ currents. The traces show that Xenopus oocytes without MRS2RS expression (uninjected) or expressing the transmembrane subunits of the mitochondrial calcium uniporter (hMEWT) exhibit no Mg2+ currents. b, The effect of BAPTA injection on MRS2RS Ca2+ currents. 5 nmol of BAPTA was injected into oocytes through a third electrode as indicated by arrows. The maximal Ca2+ current amplitudes before and 40 s after BAPTA injection were compared, showing smaller currents after BAPTA injection, as summarized in the paired dot plot. This mimics the effect of mutating site 3 to abolish divalent cation binding in MRS2’s matrix domain. Similar results were obtained when Ca2+ was applied 2.5 min after BAPTA injection. Statistical analyses were performed using paired two-tailed t-test. c, Isolation of Ca2+-activated Cl− currents (CACC). The I-V relationship of Mg2+-conducting TmCorA was obtained before and after adding 1 mM niflumic acid (NA) in the same oocyte. Recordings from 6 oocytes were summed to create the ensemble I-V curve. Subtracting the I-V curve with NA from the I-V curve without NA reveals the outwardly-rectifying CACC that reverses at -20 mV. d-e, The effect of NA on the Ca2+ currents from TmCorA (d) or MRS2RS (e). f, Recovery of MRS2 from inactivation. Switching the solution directly from Ca2+ to Na+ leads to slow Na+-current increase (first red bar). However, Na+ currents rise more rapidly following a 1-min washout (second red bar). In this trace, and in a subset ( ~ 20%) of our MRS2RS recordings, we observed that Ca2+ currents would inactivate following a double-exponential time course with some residual currents. The residual currents might reflect Ca2+ currents from native Ca2+ channels in Xenopus oocytes. The double-exponential Ca2+ current decay suggests that there might be multiple intermediate states in the inactivation process, but the molecular nature of these states remain unclear currently.

Extended Data Fig. 8 MRS2 function without R332S.

a-b, MRS2WT shows Ca2+ inactivation (a) and conducts Na+ currents that are not inhibited by Mg2+ (b). c-d, The effects of introducing quad mutations into MRS2WT, as shown in representative traces (c) and a bar chart (d). Statistical analyses were performed using two-tailed t-test. n.s.: no significance. The exact number of independent repeats was provided above each bar. Data were presented as mean ± S.E.M.

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He, Z., Tu, YC., Tsai, CW. et al. Structure and function of the human mitochondrial MRS2 channel. Nat Struct Mol Biol 32, 459–468 (2025). https://doi.org/10.1038/s41594-024-01420-5

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