Molecular and cellular physiology of organic cation transporter 2

Abstract

Organic cation transporters play a critical role in mediating the distribution of cationic pharmaceuticals. Indeed, organic cation transporter (OCT)2 is the initial step in the renal secretion of organic cations and consequently plays a defining role in establishing the pharmacokinetics of many cationic drugs. Although a hallmark of OCTs is their broad selectivity, this characteristic also makes them targets for unwanted, adverse drug-drug interactions (DDIs), making them a focus for efforts to develop models of ligand interaction that could predict and preempt these adverse interactions. This review discusses the molecular characteristics of these transporters as well as the evidence that established the OCTs as key players in the distribution of organic cations. However, the primary focus is the present understanding of the complexity of ligand interaction with OCTs, particularly OCT2, including evidence for the presence of multiple ligand-binding sites and the influence of substrate structure on the affinity of the transporter for inhibitory ligands. This leads to a discussion of the complexities associated with the development of protocols for assessing the inhibitory potential of new molecular entities to perpetrate unwanted DDIs, the criteria that should be considered in the interpretation of the results of such protocols, and the challenges associated with development of models capable of predicting unwanted DDIs.

Keywords: kidney; kinetics; organic cation; transport.

Fig. 1.

Influence of substrate identity on the degree of inhibition of organic cation transporter 2 (OCT2)-mediated transport. The 3 test substrates were metformin (12 µM), cimetidine (30 nM), and 1-methyl-4-phenylpyridinium (MPP; 12 nM). Each point represents the average of 30-s uptakes of substrate (expressed as %control uptake) from 2 experiments (each measured in triplicate), measured in the presence and absence (control) of a 20 µM concentration of 1 of 150 to 200 structurally distinct test inhibitors. The resulting inhibitory profiles are presented as 3 paired comparisons: inhibition of metformin transport versus inhibition of cimetidine transport (A), metformin transport vs. MPP transport (B), and cimetidine transport vs. MPP transport (C). The lines of unity are represented by dashed red lines (modified from Ref. 85).

Fig. 2.

Homology model of human organic cation transporter 2 (OCT2), based on the outwardly facing structure of unoccluded GLUT3 (18, 21), generated using I-Tasser (http://zhanglab.ccmb.med.umich.edu/I-TASSER/). A: face-on view from the extracellular side of the protein. B: side view, with the extracellular aspect toward the top. Italicized numbers indicate transmembrane helices 1 through 12. Amino acid residues experimentally determined to be involved in ligand interaction are shown in stick form (with solvent accessible surfaces) and color-coded to reflect structurally distinct binding interactions. The poorly resolved long extracellular loop between helices 1 and 2 is not displayed. Images shown were prepared using Pymol 2.3.

Fig. 3.

The kinetic basis for inhibitory interactions of metformin and 1-methyl-4-phenylpyridinium (MPP) with organic cation transporter 2 (OCT2). A: the kinetics of OCT2-mediated MPP transport determined in the absence (●) and presence (○) of 1 mM metformin. Each point represents the average (± SE) of initial rates of transport, determined from the time courses of net MPP accumulation, measured in 5 separate experiments. B: relationship between increasing metformin concentration and the rate of metformin transport, determined in the absence (0 µM; solid symbols) and presence of unlabeled MPP (1, 3, and 5 µM; ○, △, and □). Each point is the average (±SE) of rates (calculated from 60 s net uptakes) of transport measured in 3 separate experiments, each performed in triplicate. In both A and B, the red dashed lines show the predicted kinetic profiles derived from optimizing the model laid out in C. The configuration of binding sites on the transporter T₀ and their binding to substrate 1 (S₁; metformin) and substrate 2 (S₂; MPP) is shown in the diagram at right. “Inhib.” represents the inhibitory MPP binding site. The reversible transitions between the binding states are represented by double arrows. The associated constants (K₁, K₂, etc.) represent the ratios of the backward to the forward rate constants for each binding step. The available transport steps for substrate uptake are indicated by single arrows. The associated constants (k₁, k₂, etc.) represent the rate constants for the corresponding uptake. Because initial uptake rates are considered, cellular release of substrate is not considered (modified from Ref. 84).