Stereoselectivity in the Membrane Transport of Phenylethylamine Derivatives by Human Monoamine Transporters and Organic Cation Transporters 1, 2, and 3

Abstract

Stereoselectivity is well known and very pronounced in drug metabolism and receptor binding. However, much less is known about stereoselectivity in drug membrane transport. Here, we characterized the stereoselective cell uptake of chiral phenylethylamine derivatives by human monoamine transporters (NET, DAT, and SERT) and organic cation transporters (OCT1, OCT2, and OCT3). Stereoselectivity differed extensively between closely related transporters. High-affinity monoamine transporters (MATs) showed up to 2.4-fold stereoselective uptake of norepinephrine and epinephrine as well as of numerous analogs. While NET and DAT preferentially transported (S)-norepinephrine, SERT preferred the (R)-enantiomer. In contrast, NET and DAT showed higher transport for (R)-epinephrine and SERT for (S)-epinephrine. Generally, MAT stereoselectivity was lower than expected from their high affinity to several catecholamines and from the high stereoselectivity of some inhibitors used as antidepressants. Additionally, the OCTs differed strongly in their stereoselectivity. While OCT1 showed almost no stereoselective uptake, OCT2 was characterized by a roughly 2-fold preference for most (R)-enantiomers of the phenylethylamines. In contrast, OCT3 transported norphenylephrine and phenylephrine with 3.9-fold and 3.3-fold preference for their (R)-enantiomers, respectively, while the para-hydroxylated octopamine and synephrine showed no stereoselective OCT3 transport. Altogether, our data demonstrate that stereoselectivity is highly transporter-to-substrate specific and highly diverse even between homologous transporters.

Keywords: chiral HPLC; monoamine transporters; neurotransmitter transport; organic cation transporters; phenylethylamines; stereoselective drug transport.

Figure 1

Chemical relationship of investigated substances. Apart from salsolinol, all substances were beta-hydroxylated phenylethylamines. All substances differed only by single structural alterations, including hydroxylation of the phenolic ring, methylation of the primary amino group and methylation of the para-hydroxy group. Salsolinol is characterized by a second ring which may be formed by condensation of dopamine with acetaldehyde. Chiral centers are marked with an asterix (*).

Figure 2

Stereoselective uptake of norepinephrine (NE) and epinephrine (E) by MATs and OCTs. Transporter-overexpressing HEK293 cells were incubated for two minutes with increasing concentrations of racemic norepinephrine and epinephrine. Data are provided as mean ± SEM of three independent experiments. Note that different scaling had to be applied in the graphs, particularly due to the substantially higher affinity of NET in comparison to the other transporters.

Figure 3

Maximum transport capacities, v_max, of the investigated structural analogues of norepinephrine and epinephrine by MATs and OCTs. Data is presented as mean ± SEM of at least three independent experiments. Missing bars indicate no saturable net uptake by the respective transporters.

Figure 4

Chemical structures of single ring-hydroxylated phenylethylamine analogues (A). Stereoselective uptake analyses by OCT3 (B). Data is provided as mean ± SEM of three independent experiments.

Figure 5

Analysis of stereoselectivity in the kinetic parameters K_m, v_max and Cl_int. Kinetic constants of the (R)-enantiomer uptake are shown on the ordinate whereas kinetic constants of the (S)-enantiomer are shown on the abscissa. The bisections are highlighted by the dashed lines, and stereoselectivity is indicated by how strongly each point deviates from these bisections.

Figure 6

Influence of the A270S polymorphism on the stereoselectivity of monoamine transport by OCT2 (A). HEK293 cells stably overexpressing wild-type or mutant OCT2 were incubated with 100 μM racemic substance for two minutes. Data is shown as mean ± SEM of four independent experiments. Stereoselectivity and selectivity between both transporter variants are summarized in Table S4. Comparison of the net uptake by both transporter variants (B). The bisection indicates equal uptake, and the thin grey lines illustrate increased or decreased uptake, respectively. Correlation of stereoselectivity expressed as ratio of the uptake of the (R)- to the (S)-enantiomers (C). The bisection (dashed line) indicated equal stereoselectivity whereas the solid line illustrated the regression function.