Molecular mechanisms of mitochondrial Ca2+ exchanger NCLX

Figures

• 

  Fig. 1. Overall structures of trimeric NCLX. a 3D reconstruction of the NCLX trimer shown in side and top views, with each subunit individually colored. b Cylinder representation of an NCLX subunit in side and top views. Transmembrane helices (TMs) 2-5 and 7-10 constitute the core of the exchanger, whereas TMs 1 and 6 together with the auxiliary helices (NH1-3 and MH1-4) form the auxiliary module that mediates alternating-access transitions. c Topology diagram of an NCLX subunit. d Top and side views of the trimerization assembly formed by TMs 9 and 10. e Zoomed-in view of the inter-subunit trimerization contacts within the TM10 helix bundle. f Zoomed-in view of the histidine cluster from the cytosolic side with density (grey surface) countered at 6σ. g Zoomed-in view of the inter-subunit contacts between TM10 and TM9 from a neighboring subunit.

• 

  Fig. 2. Ion-binding pocket of NCLX. a The two α-repeats (highlighted in blue) form the central ion-binding pocket of NCLX. b Zoomed-in view of the central pocket in trimeric NCLX, showing the density (grey mesh contoured at 6σ) at the Sca site modeled as a Ca²⁺ ion. c Comparison of the central ion-binding sites between NCLX (left) and NCX_Mj (right; PDB 3V5U). Key residues that differ between NCLX and NCX_Mj at the Sint, Sext and Smid, sites are labeled in red. d Partial sequence alignment of rat NCLX (UniProt ID: Q6AXS0), human NCLX (UniProt ID: Q6J4K2), human NCX1 (UniProt ID: P32418), NCX_Mj (UniProt ID: Q57556), and human NCKX (UniProt ID: O60721) at the α-repeats. Residues forming the ion-binding sites in NCX are boxed in red. Asterisks indicate key residues that differ between NCX and NCLX within the ion-binding pocket.

• 

  Fig. 3. Transition of cytosolic-facing NCLX from the occluded to the open state. a Structural comparison between trimeric (blue) and monomeric (pink) NCLX. The red arrow highlights the conformational change of TM2ab. For clarity, TM5 is omitted in this view. Inset: the backbone carbonyl of N149 shifts away from the SCa site. b Zoomed-in view of the central pocket of monomeric NCLX, showing the density (grey mesh contoured at 6σ) at the SCa site modeled as a Na⁺ ion. c Cross-section of the surface-rendered trimeric NCLX structure showing a deep cavity (red arrow) extending toward the SCa site, while the central ion-binding pocket remains occluded. d Cross-section of the surface-rendered monomeric NCLX structure showing that both the SCa site and the central ion-binding pocket are exposed to the cytosol through two independent solvent-accessible pathways (red arrows).

• 

  Fig. 4. Alternative access mechanism of NCLX. a Structural comparison between the cytosolic-facing NCLX from the trimer (blue) and the proposed matrix-facing NCLX (light gold). The core regions (light blue) remain unchanged between the two conformations. TMs 1 and 6, together with their associated auxiliary components, undergo a rigid-body sliding motion, as indicated by the red arrow. b Cartoon representation of the proposed alternating-access cycle of cation/Ca²⁺ exchange in NCLX. Although multiple monovalent cations may be required to exchange a single Ca²⁺ ion, the exact stoichiometry between monovalent cations and Ca²⁺ remains unresolved.

• 

  Fig. 5. Ca²⁺ uptake assays in exchanger-expressing HEK cells. a Sample traces of Ca²⁺ uptake in NCX1-expressing HEK cells using 150 mM NMDG⁺ as the monovalent salt and varying external [Ca²⁺] (left panel). Cytosolic [Ca²⁺] changes were monitored by Fura-2 fluorescence ratios at 510 nm (excitation 340/380 nm) following the addition of external Ca²⁺ (indicated by the arrow) and were quantified at the time point marked by the grey line (right panel). All data points shown here and the subsequent experiments represent mean ± SEM of n = 4 independent replicates. p values: 1.97 × 10−3 for 0.05 mM, 1.19 × 10−7 for 0.25 mM, 7.74 × 10−5 for 1 mM, and 1.50 × 10−6 for 5 mM. b Same experiment as in (a) performed with NCLX-expressing HEK cells. p values: 2.05 × 10−3 for 0.05 mM, 6.91 × 10−8 for 0.25 mM, 1.75 × 10−6 for 1 mM, and 5.90 × 10−6 for 5 mM. All traces in (a) and (b) are shown after background subtraction (Supplementary Fig. 8). c Ca²⁺ uptake in NCX1-expressing HEK cells with 0.05 mM external [Ca²⁺] and 150 mM of various monovalent salts. p values: 1.18 × 10−4 for K⁺, 2.26 × 10−4 for Li⁺, and 5.12 × 10−7 for Na⁺. d Ca²⁺ uptake in NCLX-expressing HEK cells with 0.25 mM external [Ca²⁺] and 150…