Structure and function of the human mitochondrial MRS2 channel

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.

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

a-c, Image processing and map validation of MRS2_Mg (a), MRS2_EDTA (b), and MRS2_ca (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 MRS2_Mg and cryo-EM density. b, Segments of the final refined model of MRS2_Mg 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 MRS2_EDTA.

Cryo-EM densities near site 3 in the MRS2_EDTA 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 MRS2_RS expression (uninjected) or expressing the transmembrane subunits of the mitochondrial calcium uniporter (hMEwT) exhibit no Mg²⁺ currents. b, The effect of BAPTA injection on MRS2_RS 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 MRS2_RS (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 MRS2_RS 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 ∣. Function of the wild-type MRS2 (without R332S mutation).

a-b, The wild-type MRS2 (MRS2_WT) shows Ca²⁺ inactivation (a) and conducts Na⁺ currents that are not inhibited by Mg²⁺ (b). c-d, The effects of introducing quad mutations into MRS2_WT, as shown in representative traces (c) and a bar chart (d). Statistical analyses were performed using two-tailed t-test.

Fig. 1 ∣. Structure of human MRS2.

a, Cryo-EM reconstruction of human MRS2. Each subunit is uniquely colored and the contour indicates membrane boundaries. b, The overall structure of human MRS2. c, The central ion pathway, estimated using the HOLE program and represented as colored dots (pore radius: 1.15 Å < green < 2.3 Å < blue). Two opposite subunits are shown for clarity. d, Cutaway view of the channel surface, colored by surface electrostatic potential (red, −5 kT/e; white, neutral; blue, +5 kT/e). e, Details of the pore-lining residues and the dimension of the central pore.

Fig. 2 ∣. Mg²⁺ and Ca²⁺ recognition.

a, Mg²⁺ ions (purple spheres) identified in the structure of MRS2 in the presence of Mg²⁺. Also shown are the details of the three Mg²⁺ ions (site 1 and 2) coordinated within the channel. Only two adjacent subunits are shown for clarity. b, Orthogonal view as in (a). c, Ca²⁺ ions (yellow spheres) in the MRS2_Ca structure.

Fig. 3 ∣. The role of the R332 ring on MRS2 function.

a, Mg²⁺ transport by TmCorA or MRS2. The western-blot image compares Xenopus-oocyte expression of TmCorA, MRS2_FL, and MRS_EM, all tagged with a C-terminal 1D4 sequence and detected with an anti-1D4 antibody (Histone: loading control). b, MRS2 Mg²⁺ currents induced by the R332S mutation. MRS2_RS exhibits ~60% expression level of MRS2_WT in oocytes. c, Inhibition of MRS2 activity by cobalt hexammine. Na⁺ currents were presented here, but similar inhibition was obtained when recording Mg²⁺ currents. d, Dose response of cobalt hexammine inhibition of MRS2 Na⁺ currents. Data points were fit with a saturation binding equation (black curve). Maximal inhibition achieved is ~50%, comparable with that obtained with TmCorA. Each data point represents 3 independent repeats. e, The effect of MRS2 on IMM potentials. WT or R332S MRS2_FL were expressed in HEK293 cells. The ratios of TMRM signals before (F) and after (F₀) adding FCCP were presented. The Western blot data compare WT and R332S expression in HEK cells, showing lower R332S expression similar to what we observed in oocytes (Tim23: loading control). f, Death of HEK cells induced by R332S MRS2_FL. Data were presented as mean ± S.E.M. (d) or individual data points (e-f). Statistical analyses in e-f were performed with paired, two-tailed t-test. The unit of Western blot molecular weight markers is kDa.

Fig. 4 ∣. Ion permeation properties.

a, Monovalent and divalent cation currents through MRS2_WT or MRS2_RS. Peak-level amplitudes of Ca²⁺ currents were presented in the bar chart. Data were presented as mean ± S.E.M. Statistical analyses were performed with unpaired, two-tailed t-test. The number of independent repeats was labeled above each bar. b, Ca²⁺ currents conducted by TmCorA or MRS2_RS. c, Recovery of MRS2_RS from inactivation. Na⁺ currents reached steady-state levels much more slowly when MRS2_RS was pre-exposed to Ca²⁺, but not Mg²⁺. d, The effect of Mg²⁺ on Na⁺ currents. e, Lack of Ca²⁺ inhibition of MRS2 Na⁺ currents. 500 μM of EGTA was added later in the recording to test if the trace amount of Ca²⁺ in the NaCl buffer can inhibit Na⁺ currents. f, A cartoon summarizing key functional properties of MRS2: (1) R332 impedes ion permeation, and (2) MRS2 acts as a Ca²⁺-regulated non-selective cation channel.

Fig. 5 ∣. Divalent cation-binding sites.

a-b, Functional impacts of site 1 or site 2 mutations on MRS2_RS. The Western-blot image shows that these mutations do not affect MRS2_RS expression. RS: MRS2_RS. NA: N362A; DS: D329S; DR: D329R. c, Mg²⁺ inactivation of TmCorA. d, The effect of site 3 mutations on MRS2_RS function (quad: E138A-E243A-D247A-E312A mutation). Western blot shows that these mutations do not reduce surface expression. Data in b and d were presented as mean ± S.E.M. Statistical analyses were performed with unpaired, two-tailed t-test. The number of independent repeats was labeled inside the parentheses above each bar. The unit of Western blot molecular weight markers is kDa.