The human organic cation transporter 1 (OCT1) is expressed in the liver and mediates hepatocellular uptake of organic cations. However, some studies have indicated that OCT1 could transport neutral or even anionic substrates. This capability is interesting concerning protein-substrate interactions and the clinical relevance of OCT1. To better understand the transport of neutral, anionic, or zwitterionic substrates, we used HEK293 cells overexpressing wild-type OCT1 and a variant in which we changed the putative substrate binding site (aspartate474) to a neutral amino acid. The uncharged drugs trimethoprim, lamivudine, and emtricitabine were good substrates of hOCT1. However, the uncharged drugs zalcitabine and lamotrigine, and the anionic levofloxacin, and prostaglandins E2 and F2α, were transported with lower activity. Finally, we could detect only extremely weak transport rates of acyclovir, ganciclovir, and stachydrine. Deleting aspartate474 had a similar transport-lowering effect on anionic substrates as on cationic substrates, indicating that aspartate474 might be relevant for intra-protein, rather than substrate-protein, interactions. Cellular uptake of the atypical substrates by the naturally occurring frequent variants OCT1*2 (methionine420del) and OCT1*3 (arginine61cysteine) was similarly reduced, as it is known for typical organic cations. Thus, to comprehensively understand the substrate spectrum and transport mechanisms of OCT1, one should also look at organic anions.

An over‐the‐counter product berberine (a major alkaloid in goldenseal) is a substrate of the uptake transporter OCT1 and the metabolizing enzyme CYP2D6. The two genes exhibit common functional polymorphisms. Approximately 9% of Europeans and white Americans are either poor CYP2D6 metabolizers or poor OCT1 transporters. In this study, we investigated the effects of OCT1 and CYP2D6 polymorphisms on berberine pharmacokinetics in humans. We confirmed in vitro that berberine is an OCT1 substrate (K_M of 7.0 μM, CL_int of 306 ± 29 μL/min/mg). Common OCT1 alleles *3 to *6 showed uptake reduced by at least 65% and Oct1/2 knockout mice showed 3.2‐fold higher AUCs in liver perfusion experiments. However, in humans, poor OCT1 transporters did not show any differences in berberine pharmacokinetics compared with reference participants. In contrast, CYP2D6 polymorphisms significantly affected berberine metabolism, but exclusively in females. Females who were poor CYP2D6 metabolizers had an 80% lower M1‐to‐berberine ratio. General linear model analyses suggest strong synergistic, rather than additive, effects between female sex and CYP2D6 genotype. Overall, berberine displayed low oral bioavailability, yet females had a 2.8‐fold higher AUC and a 3.6‐fold higher C_max than males (P < 0.001). These effects were only partially attributable to the sex‐CYP2D6 genotype interaction. In conclusion, despite berberine being an OCT1 substrate, OCT1 deficiency did not affect berberine pharmacokinetics in humans. In contrast, CYP2D6 emerges as a critical enzyme for berberine metabolism in females, but not in males, highlighting sex‐specific differences. We suggest that factors beyond CYP2D6 metabolism are determining berberine's systemic exposure, especially in males (NCT05463003).

OCT1 Polyspecificity—Friend or Foe?

Transporters of the SLC22 family, such as organic cation transporter 1 (OCT1), possess very broad substrate specificity. It is unclear to what extent the inhibitory potencies of OCT1 depend on the substrate used. Here, we describe a multisubstrate drug cocktail that allows for the simultaneous testing of drug-drug interactions using 8 different victim drugs: fenoterol, salbutamol, sumatriptan, zolmitriptan, ipratropium, trospium, methylnaltrexone, and metformin. There were no significant differences in Michaelis constant (K_M) and v_max of the OCT1-mediated uptake of the substrates alone or in the cocktail. Depending on the victim drug analyzed, we observed 6.7-fold differences in the inhibitory potency of fenoterol (IC₅₀ of 0.75 μM for metformin and 5.1 μM for sumatriptan). Similarly, the inhibitory potency of verapamil varied 6.7-fold (IC₅₀ of 1.3 μM for zolmitriptan and 8.7 μM for ipratropium). Two groups of inhibitors showed strong correlations in their victim-dependent inhibitory potencies. Group 1 comprised verapamil, quinidine, fenoterol, and ipratropium, and group 2 comprised metformin, sumatriptan, and trimethoprim. By comparing OCT1 paralogs and orthologs, the broadest substrate spectra were observed for OCT1 and multidrug and toxin extrusion 1, followed by OCT2, multidrug and toxin extrusion 2-K, and OCT3. In contrast, organic cation transporters novel 1 and organic cation transporters novel 2 exhibited very narrow substrate specificity, transporting only L-carnitine and L-ergothioneine, respectively. In conclusion, OCT1 demonstrates substantial differences in inhibitory potencies, depending on the victim drug used. We developed a cocktail approach that enables rapid screening for such differences, facilitating the identification of drug-drug interactions at the early stages of drug development. This approach can be extended to other transporters with broad substrate specificity. SIGNIFICANCE STATEMENT: Polyspecific transporters have a broad substrate-binding cavity with no defined single binding position. Consequently, inhibitors may exhibit different inhibitory potencies depending on the victim drug used for testing. Here, we demonstrate this for organic cation transporter 1 (OCT1, SLC22A1) and presents a drug cocktail designed to identify varying inhibitory potencies in vitro and prevent false-negative drug-drug interaction results during early drug development. This approach can be extended to other polyspecific drug transporters.

OCT1 and OCT2 are polyspecific membrane transporters that are involved in hepatic and renal drug clearance in humans and mice. In this study, we cloned dog OCT1 and OCT2 and compared their function to the human and mouse orthologs. We used liver and kidney RNA to clone dog OCT1 and OCT2. The cloned and the publicly available RNA-Seq sequences differed from the annotated exon-intron structure of OCT1 in the dog genome CanFam3.1. An additional exon between exons 2 and 3 was identified and confirmed by sequencing in six additional dog breeds. Next, dog OCT1 and OCT2 were stably overexpressed in HEK293 cells and the transport kinetics of five drugs were analyzed. We observed strong differences in the transport kinetics between dog and human orthologs. Dog OCT1 transported fenoterol with 12.9-fold higher capacity but 14.3-fold lower affinity (higher K_M) than human OCT1. Human OCT1 transported ipratropium with 5.2-fold higher capacity but 8.4-fold lower affinity than dog OCT1. Compared to human OCT2, dog OCT2 showed 10-fold lower transport of fenoterol and butylscopolamine. In conclusion, the functional characterization of dog OCT1 and OCT2 reported here may have implications when using dogs as pre-clinical models as well as for drug therapy in dogs.

Hepatic organic cation transporter 1 (OCT1, SLC22A1) has a broad spectrum of structurally diverse substrates, including drugs like morphine, metformin, sumatriptan, and fenoterol. Here, we leveraged existing cryo-EM data to identify amino acids that constrain the binding pocket toward the intracellular lumen and performed functional analyses to determine their roles in OCT1 transport, in general, and in substrate specificity, in particular. We mutated W217, W354, and I446 and analyzed their effects on the transport of 27 structurally highly diverse substrates in HEK293 cells. The W354A mutant resulted in a complete loss of function for all tested substrates, whereas only the W354Y mutant partially retained OCT1 activity. Molecular dynamics simulations and functional analyses suggest that W354 affects OCT1 transport by forming a hydrogen bond with N453 in transmembrane helix 10, thereby stabilizing OCT1 in its outward open conformation. W217A showed strongly substrate-dependent effects, with the aromatic ring of tryptophan being the key property affecting transport. Smaller, less lipophilic substrates were more strongly affected. The substrate-specific effects of the W217A mutant correlated significantly with those of the F244A mutant. The double mutant W217A/F244A strongly reduced uptake of all tested substrates, indicating a compensatory mechanism between these two amino acids. I446 showed only marginal effects on OCT1 transport. Our data suggest that W354 is crucial for OCT1 function by mediating the switch from an inward-occluded to inward-open conformation but is not directly involved in substrate interaction. In contrast, W217, along with the previously known F244, contributes to the substrate specificity of OCT1.