doi: 10.1038/s41467-025-66867-6.
Structural basis of sodium ion-dependent carnitine transport by OCTN2
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- PMID: 41318751
- PMCID: PMC12780140
- DOI: 10.1038/s41467-025-66867-6
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Structural basis of sodium ion-dependent carnitine transport by OCTN2
Nat Commun.
.
Abstract
Carnitine is essential for the import of long-chain fatty acids into mitochondria, where they are used for energy production. The carnitine transporter OCTN2 (novel organic cation transporter 2, SLC22A5) mediates carnitine uptake across the plasma membrane and as such facilitates fatty acid metabolism in most tissues. OCTN2 dysfunction causes systemic primary carnitine deficiency (SPCD), a potentially lethal disorder. Despite its importance in metabolism, the mechanism of high-affinity, sodium ion-dependent transport by OCTN2 is unclear. Here we report cryo-EM structures of human OCTN2 in three conformations: inward-facing ligand-free, occluded carnitine- and Na+-bound, and inward-facing ipratropium-bound. These structures define key interactions responsible for carnitine transport and identify an allosterically coupled Na+ binding site housed within an aqueous cavity, separate from the carnitine-binding site. Combined with electrophysiology data, we provide a framework for understanding variants associated with SPCD and insight into how OCTN2 functions as the primary human carnitine transporter.
© 2025. The Author(s).
Conflict of interest statement
Competing interests: The authors declare no competing interests.
Figures
a OCTN2 facilitates the Na+-dependent transport of carnitine into the cell, which is then used for the transport of fatty acids (via the carnitine palmitoyltransferase I-containing complex; CPTI, and the carnitine-acylcarnitine translocase; CACT) to the mitochondrial matrix for β-oxidation. b Cryo-EM maps of OCTN2 in the inward-facing substrate-free (left), occluded (middle) and inward-facing ipratropium-bound (right) conformations, contoured at 7.5σ as calculated by ChimeraX and coloured according to domain: the intracellular helix; purple, the N-terminal transmembrane bundle; blue, the extracellular domain; green and the C-terminal transmembrane bundle orange. Inset Cryo-EM density of carnitine and ipratropium, from sharpened maps contoured at 8σ. c
Left: Cartoon representations of the occluded conformation of OCTN2, with helices coloured as per the schematic above showing the topology. d Surface cutaways of the inward-facing (left) and occluded (right) conformations. Na+ in each conformation is shown in purple, within an enclosed cavity, as indicated. In the occluded conformation, carnitine is also bound within an enclosed cavity, which is separate from the Na+-cavity.
a View of the Na+- and carnitine- binding sites. Left Na+ coordinated by S28, N32 and N210, as well as a water molecule below (W1) Middle Cutaway view of OCTN2 looking down from the ECD, showing the central carnitine-binding site and the Na+-binding site situated in the N-terminal bundle. Right View of the carnitine-binding site, showing the ionic interaction between the carboxylate of carnitine and R471, hydrogen bonding interactions (blue dashes) with Y447 and Q207 via a water molecule, and the conserved aromatic cage around the positively charged quaternary ammonium moiety (cation- interactions shown by yellow dashes). Helices are coloured according to Fig. 1c. b Representation of the Na+ and carnitine binding cavities, coloured by electrostatic surface potential as calculated by ChimeraX, with cryo-EM density corresponding to carnitine, Na+ and water molecules (W2, W3, W4) shown as mesh contoured at 7.5σ. Residues binding Na+ and putative linking residues (Q207 and Y211) are depicted as sticks. c 2d representation of the Na+ and carnitine binding sites. Red asterisks indicate positions within each binding site where variants are associated with SPCD per Koleske et al.. d Current voltage (IV) relationships measured in oocytes expressing hOCTN2 upon application of 100 µM L -carnitine in the presence of either 100 mM Na+ (grey) or Cs+(gold). e Carnitine concentration-response relationships were measured by applying l -carnitine (1−6000 µM) to oocytes expressing hOCTN2 in the presence of differing Na+ concentrations (10, 30, 40, 60 and 100 mM), with darker blue curves corresponding to higher Na+. Currents elicited at each carnitine concentration were measured at −60 mV and fitted to a Michaelis-Menten curve with GraphPad Prism. f Carnitine concentration-response curves and apparent affinities were determined for hOCTN2 mutant transporters. l -Carnitine concentrations ranging from 1 to 3000 µM were applied to oocytes expressing hOCTN2 mutants of interest. Current responses were measured at −60 mV and fitted to a Michaelis–Menten curve with GraphPad Prism. Replicates were measured in 5 oocytes (n = 5) across at least 2 batches of oocytes. Error bars represent SEM.
a Structural superposition of the inward-facing and occluded conformations. The TMs of the inward-facing (IFS) structure are coloured red and light blue, while the TMs of the occluded structure are coloured orange and light purple. The N-terminal bundle is coloured blue/purple and the C-terminal bundle is coloured orange/red. Dashed boxes correspond to views in panels (b–d). b Intracellular gate interactions. In the IFS (top), a charge network is observed in the N-terminal bundle, and in the occluded conformation (bottom), R459 (TM11) interacts with this network. c Gating interactions at the intracellular helix (ICH) domain where a salt bridge forms between R282 and D519 in the occluded conformation. Red asterisks indicate positions of variants associated with SPCD. d Extracellular gate interactions, where Y482 and D139 H-bond in the IFS (top) and move further apart in the occluded conformation (bottom). Cryo-EM density for residues detailed in (b–d) is shown in Supplementary Fig. 5. e Residues on TM1 (purple) and TM7 (brown) form a hydrophobic extracellular gate above the carnitine-binding site. As well as gating, residues on TM1 form the Na+-binding site. The PNGFxG motif observed in the Na+-dependent hOCTN2, hOCTN1 and mOCTN3 is indicated. Proline and glycine residues are coloured grey. f Flexibility of TM11 (orange) and formation of a hydrogen-bonding network with TM2 (blue) in the occluded carnitine-bound structure. Arrows and transparent overlay illustrates TM11 bending during the inward-occluded conformational change. The position of the conserved G473/P478 motif is indicated.
a Cutaway shows ipratropium bound within the solvent-accessible cavity in the inward-facing conformation. b Representation of ipratropium-OCTN2 interactions, showing cation- interactions between the aromatic cage residues and the quaternary ammonium group on the tropane ring, along with a weak hydrogen bond between Q207 and the ipratropium hydroxyl group. c Ipratropium inhibition response curves were measured by competing increasing concentrations of ipratropium (3–6000 μM) against carnitine at the approximate EC50 of 10 μM. The current measured at −60 mV was fitted to a three parameter inhibitor dose response with Graphpad Prism. Replicates were measured in five oocytes (n = 5) across at least two batches of oocytes. Error bars represent ±SEM.
We propose that OCTN2 follows a variation of the MFS-type transport cycle, in which Na+-binding within the N-terminal bundle (blue) allosterically modulates the transporter. We hypothesise that Na+ binds first (similar to other Na+-solute symporters,) and this primes the transporter for subsequent carnitine binding, which involves the critical R471 within the C-terminal bundle (orange). Na+-binding is likely to influence the conformational dynamics associated with extracellular gating, although kinetic evidence for the binding order remains to be established. Like other OCT and OAT transporters, electrostatic interactions (circles) govern the intracellular gate and aromatic gating residues (rectangles) that surround the binding pocket (effectively “thin” gates). The role of the extracellular domain (ECD; green) in the transport cycle is unclear, as the structural data here does not support significant interaction with the C-terminal bundle.
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