Structural basis of organic cation transporter-3 inhibition

Abstract

Organic cation transporters (OCTs) facilitate the translocation of catecholamines, drugs and xenobiotics across the plasma membrane in various tissues throughout the human body. OCT3 plays a key role in low-affinity, high-capacity uptake of monoamines in most tissues including heart, brain and liver. Its deregulation plays a role in diseases. Despite its importance, the structural basis of OCT3 function and its inhibition has remained enigmatic. Here we describe the cryo-EM structure of human OCT3 at 3.2 Å resolution. Structures of OCT3 bound to two inhibitors, corticosterone and decynium-22, define the ligand binding pocket and reveal common features of major facilitator transporter inhibitors. In addition, we relate the functional characteristics of an extensive collection of previously uncharacterized human genetic variants to structural features, thereby providing a basis for understanding the impact of OCT3 polymorphisms.

Fig. 1. Structure and function of OCT3.

a Schematic representation of OCT3; key features of the transporter are illustrated in the panel. b OCT3 transport inhibition by decynium-22 (D22) and corticosterone (CORT). The values correspond to mean ± SD; n = 3 represents three biologically independent experiments that are conducted with three technical repeats. c A scheme depicting the topology and the secondary structure elements of OCT3. d, e The cryo-EM map (d) and model of OCT3 in nanodiscs at 3.2 Å resolution. The colors of the protein correspond to those in c; annular lipids are colored gray. f, g Same as d, e, for OCT3-D22 complex at 3.6 Å resolution (D22 colored violet). h, i Same as f, g, for OCT3-CORT complex at 3.7 Å resolution (CORT colored green). Source Data are available as a Source Data file.

Fig. 2. Comparison of the apo-state and the ligand-bound states of OCT3.

a Structural alignment of the three indicated states of OCT3 shows an overall high degree of similarity. b Same as in a, view from the extracellular space. The positions of each transmembrane (TM) helix are indicated. The residues flanking the extended extracellular loop 1 (ECL1) are indicated with boxes (L42 and V139). The translocation pathway / ligand binding site is indicated by an asterisk. c A sliced view of D22, showing the inhibitor buried deep in the substrate translocation pathway. d The expanded views of the D22 binding site in different orientations (left the same orientation as the one in c), indicating the residues within 4 Å distance of the inhibitor. e, f Same as c, d, for OCT3-CORT. The TM domains containing the residues in close proximity to the ligand are indicated in the right-most panel (d and f). g, h 2D interaction plot showing the residues interacting with D22 and CORT.

Fig. 3. Molecular basis of OCT3 ligand specificity.

a–d Comparison between OCT3-D22 and four different MFS transporter structures in outward-facing, ligand bound states, including FucP (PDB ID: 3o7q), ENT1 (PDB ID: 6ob7), LmrP (PDB ID: 6t1z) and GLUT3 (PDB ID: 4zwc). The dotted lines/boxes show the zoomed in views of the isolated ligands (left, same orientation as in the main panel; right – rotated ~90°). e Inhibition of OCT1, 2 and 3 transport by D22 and CORT (mean ± SD; n = 3). n = 3 represent three biologically independent experiments for each cell line. f Sequence alignment of OCT1-3, OCTN1 and OCTN2, indicating the positions of the key OCT3 residues involved in ligand binding (and varied among the homologs): F36, F250 and F450. g Comparison of the CORT- and D22-bound states in the experimentally determined OCT3 structures (white) with the OCT1 and OCT2 homology models (black). Source Data are available as a Source Data file.

Fig. 4. Functional analysis of OCT3 mutants.

a Distribution of the OCT3 genetic variants, mapped on the structure of OCT3. b A heat map illustrating the effects of mutations. The scale bar indicates the increase (green) or decrease (magenta) of V_max, K_m, IC₅₀, membrane expression and NFRET compared to wild-type, as detailed in Materials and Methods. Arrows indicate the variants selected for detailed characterization. c View of OCT3 from the extracellular space, with the side-chains of the selected residues shown as sticks. d Uptake of MPP⁺ by the wild-type OCT3 (WT) and by the selected variants expressed in HEK293 cells (Supplementary Table 5). e, f Inhibition of MPP⁺ transport by CORT (e) and D22 (f). The WT is indicated with a dotted line. The values in d–f correspond to mean ± SD, n = 3–4 represents biologically independent experiments performed with three technical repeats.

Fig. 5. The possible underlying causes of function disruption in genetic variants of OCT3.

a Distance between the side chains of W223 and Y227 or Q247, indicating the orientational dynamics of the sidechain of W223. b Local environment of W223, showing average water molecules occupancy in its proximity. c Donor-acceptor hydrogen bond distance between Y461 and T351, confirming the long-range effect of the bound inhibitor on the local structural stability of the Y461 site. d Zoom onto Y461, showing the hydrogen bond to T351 and averaged water occupancy in its proximity. e Distance of the side chain of R212 to residue T157 and Q215, highlighting the oscillation of the interaction pattern in the apo state compared to the stable conformation in the inhibitor bound states. f Close-up of R212, emphasizing on the hydrogen bond network formed with residues Q215, T157 and water molecules. Averaged spatial occupancy of water is represented as a density colored according to the legend.