Fig. 2: The occluded conformation substrate- and Na⁺ ion-binding sites.

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.¹¹. 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.