The full spectrum of SLC22 OCT1 mutations illuminates the bridge between drug transporter biophysics and pharmacogenomics.

Mutations in transporters can impact an individual's response to drugs and cause many diseases. Few variants in transporters have been evaluated for their functional impact. Here, we combine saturation mutagenesis and multi-phenotypic screening to dissect the impact of 11,213 missense single-amino-acid deletions, and synonymous variants across the 554 residues of OCT1, a key liver xenobiotic transporter. By quantifying in parallel expression and substrate uptake, we find that most variants exert their primary effect on protein abundance, a phenotype not commonly measured alongside function. Using our mutagenesis results combined with structure prediction and molecular dynamic simulations, we develop accurate structure-function models of the entire transport cycle, providing biophysical characterization of all known and possible human OCT1 polymorphisms. This work provides a complete functional map of OCT1 variants along with a framework for integrating functional genomics, biophysical modeling, and human genetics to predict variant effects on disease and drug efficacy.

Targeted mutagenesis of negatively charged amino acids outlining the substrate translocation path within the human organic cation transporter 3.

Recently published cryo-EM structures of human organic cation transporters of the SLC22 family revealed seven, sequentially arranged glutamic and aspartic acid residues, which may be relevant for interactions with positively charged substrates. We analyzed the functional consequences of removing those negative charges by creating D155N, E232Q, D382N, E390Q, E451Q, E459Q, and D478N mutants of OCT3. E232Q, E459Q, and D478N resulted in a lack of localization in the outer cell membrane and no relevant uptake activity. However, D155N and E451Q showed a substrate-specific loss of transport activity, whereas E390Q had no remaining activity despite correct membrane localization. In contrast, D382N showed almost wild-type-like uptake. D155 is located at the entrance to the substrate binding pocket and could, therefore be involved in guiding cationic substrates towards the inside of the binding pocket. For E390, we confirm its critical function for transporter function as it was recently shown for the corresponding position in OCT1. Interestingly, E451 seems to be located at the bottom of the binding pocket in the outward-open confirmation of the transporter. Substrate-specific loss of transport activity of the E451Q variant suggests an essential role in the transport cycle of specific substances as part of an opportunistic binding site. In general, our study highlights the impact of the cryo-EM structures in guiding mutagenesis studies to understand the molecular level of transporter-ligand interactions, and it also confirms the importance of testing multiple substrates in mutagenesis studies of polyspecific OCTs.

Identification and characterization of an endogenous biomarker of the renal vectorial transport (OCT2-MATE1).

The renal tubular organic cation transporter 2 (OCT2) and multidrug and toxin extrusion protein 1 (MATE1) mediate the vectorial elimination of many drugs and toxins from the kidney, and endogenous biomarkers for vectorial transport (OCT2-MATE1) would allow more accurate drug dosing and help to characterize drug-drug interactions and toxicity. Human serum uptake in OCT2-overexpressing cells and metabolomics analysis were carried out. Potential biomarkers were verified in vitro and in vivo. The specificity of biomarkers was validated in renal transporter overexpressing cells and the sensitivity was investigated by Km . The results showed that the uptake of thiamine, histamine, and 5-hydroxytryptamine was significantly increased in OCT2-overexpressing cells. In vitro assays confirmed that thiamine, histamine, and 5-hydroxytryptamine were substrates of both OCT2 and MATE1. In vivo measurements indicated that the serum thiamine level was increased significantly in the presence of the rOCT2 inhibitor cimetidine, and the level in renal tissue was increased significantly by the rMATE1 inhibitor pyrimethamine. There were no significant changes in the uptake or efflux of thiamine in cell lines overexpressed OAT1, OAT2, OAT3, MRP4, organic anion transporting polypeptide 4C1, P-gp, peptide transporter 2, urate transporter 1, and OAT4. The Km for thiamine with OCT2 and MATE1 were 71.2 and 10.8 µM, respectively. In addition, the cumulative excretion of thiamine at 2 and 4 h was strongly correlated with metformin excretion (R2  > 0.6). Thus, thiamine is preferentially secreted by the OCT2 and MATE1 in renal tubules and can provide a reference value for evaluating the function of the renal tubular OCT2-MATE1.

Stereoselectivity in Cell Uptake by SLC22 Organic Cation Transporters 1, 2, and 3.

Stereoselectivity can be most relevant in drug metabolism and receptor binding. Although drug membrane transport might be equally important for small-molecule pharmacokinetics, the extent of stereoselectivity in membrane transport is largely unknown. Here, we characterized the stereoselective transport of 18 substrates of SLC22 organic cation transporters (OCTs) 1, 2, and 3. OCT2 and OCT3 showed highly stereoselective cell uptake with several substrates and, interestingly, often with opposite stereoselectivity. In contrast, transport by OCT1 was less stereoselective, although (R)-tamsulosin was transported by OCT1 with higher apparent affinity than the (S)-enantiomer. Using OCT1 and CYP2D6 co-overexpressing cells, an additive effect of the stereoselectivities was demonstrated. This indicates that pharmacokinetic stereoselectivity may be the result of combined effects in transport and metabolism. This study highlights that the pronounced polyspecificity of OCTs not contradicts stereoselectivity in the transport. Nevertheless, stereoselectivity is highly substrate-specific and for most substrates and OCTs, there was no major selectivity.

Stereoselective Inhibition of High- and Low-Affinity Organic Cation Transporters.

Many drugs have chiral centers and are therapeutically applied as racemates. Thus, the stereoselectivity in their interactions with membrane transporters needs to be addressed. Here, we studied stereoselectivity in inhibiting organic cation transporters (OCTs) 1, 2, and 3 and the high-affinity monoamine transporters (MATs) NET and SERT. Selectivity by the inhibition of 35 pairs of enantiomers significantly varied among the three closely related OCTs. OCT1 inhibition was nonselective in almost all cases, whereas OCT2 was stereoselectively inhibited by 45% of the analyzed drugs. However, the stereoselectivity of the OCT2 was only moderate with the highest selectivity observed for pramipexole. The (R)-enantiomer inhibited OCT2 4-fold more than the (S)-enantiomer. OCT3 showed the greatest stereoselectivity in its inhibition. (R)-Tolterodine and (S)-zolmitriptan inhibited OCT3 11-fold and 25-fold more than their respective counterparts. Interestingly, in most cases, the pharmacodynamically active enantiomer was also the stronger OCT inhibitor. In addition, stereoselectivity in the OCT inhibition appeared not to depend on the transported substrate. For high-affinity MATs, our data confirmed the stereoselective inhibition of NET and SERT by several antidepressants. However, the stereoselectivity measured here was generally lower than that reported in the literature. Unexpectedly, the high-affinity MATs were not significantly more stereoselectively inhibited than the polyspecific OCTs. Combining our in vitro OCT inhibition data with available stereoselective pharmacokinetic analyses revealed different risks of drug-drug interactions, especially at OCT2. For the tricyclic antidepressant doxepine, only the (E)-isomer showed an increased risk of drug-drug interactions according to guidelines from regulatory authorities for renal transporters. However, most chiral drugs show only minor stereoselectivity in the inhibition of OCTs in vitro, which is unlikely to translate into clinical consequences.

Structural basis of promiscuous substrate transport by Organic Cation Transporter 1.

Organic Cation Transporter 1 (OCT1) plays a crucial role in hepatic metabolism by mediating the uptake of a range of metabolites and drugs. Genetic variations can alter the efficacy and safety of compounds transported by OCT1, such as those used for cardiovascular, oncological, and psychological indications. Despite its importance in drug pharmacokinetics, the substrate selectivity and underlying structural mechanisms of OCT1 remain poorly understood. Here, we present cryo-EM structures of full-length human OCT1 in the inward-open conformation, both ligand-free and drug-bound, indicating the basis for its broad substrate recognition. Comparison of our structures with those of outward-open OCTs provides molecular insight into the alternating access mechanism of OCTs. We observe that hydrophobic gates stabilize the inward-facing conformation, whereas charge neutralization in the binding pocket facilitates the release of cationic substrates. These findings provide a framework for understanding the structural basis of the promiscuity of drug binding and substrate translocation in OCT1.

Stereoselective study of fluoxetine and norfluoxetine across the blood-brain barrier mediated by organic cation transporter 1/3 in rats using an enantioselective UPLC-MS/MS method.

Fluoxetine (FLT) is a widely used antidepressant in clinical practice, which can be metabolized into active norfluoxetine (NFLT) in vivo. The stereoselectivity of FLT and NFLT enantiomers across the blood-brain barrier (BBB) is still to be clarified. In this study, accurate and reliable UPLC-MS/MS enantioselective analysis was established in rat plasma and brain. The characteristics of FLT and NFLT enantiomers across the BBB were studied by chemical knockout of rat transporters. We found that the dominant enantiomers of FLT and NFLT were S-FLT and R-NFLT, respectively, both in plasma and in brain. The FLT and NFLT enantiomers showed significant stereoselectivity across the BBB, and S-FLT and S-NFLT were the dominant configurations across the BBB. Chemical knockout of organic cation transporter 1 (OCT1) and OCT3 can affect the ratio of plasma FLT and NFLT enantiomers into the brain, suggesting that OCT1/3 is stereoselective for FLT and NFLT transport across the BBB.

Molecular basis of polyspecific drug and xenobiotic recognition by OCT1 and OCT2.

A wide range of endogenous and xenobiotic organic ions require facilitated transport systems to cross the plasma membrane for their disposition. In mammals, organic cation transporter (OCT) subtypes 1 and 2 (OCT1 and OCT2, also known as SLC22A1 and SLC22A2, respectively) are polyspecific transporters responsible for the uptake and clearance of structurally diverse cationic compounds in the liver and kidneys, respectively. Notably, it is well established that human OCT1 and OCT2 play central roles in the pharmacokinetics and drug-drug interactions of many prescription medications, including metformin. Despite their importance, the basis of polyspecific cationic drug recognition and the alternating access mechanism for OCTs have remained a mystery. Here we present four cryo-electron microscopy structures of apo, substrate-bound and drug-bound OCT1 and OCT2 consensus variants, in outward-facing and outward-occluded states. Together with functional experiments, in silico docking and molecular dynamics simulations, these structures uncover general principles of organic cation recognition by OCTs and provide insights into extracellular gate occlusion. Our findings set the stage for a comprehensive structure-based understanding of OCT-mediated drug-drug interactions, which will prove critical in the preclinical evaluation of emerging therapeutics.

Combined and independent effects of OCT1 and CYP2D6 on the cellular disposition of drugs.

The organic cation transporter 1 (OCT1) mediates the cell uptake and cytochrome P450 2D6 (CYP2D6) the metabolism of many cationic substrates. Activities of OCT1 and CYP2D6 are affected by enormous genetic variation and frequent drug-drug interactions. Single or combined deficiency of OCT1 and CYP2D6 might result in dramatic differences in systemic exposure, adverse drug reactions, and efficacy. Thus, one should know what drugs are affected to what extent by OCT1, CYP2D6 or both. Here, we compiled all data on CYP2D6 and OCT1 drug substrates. Among 246 CYP2D6 substrates and 132 OCT1 substrates, we identified 31 shared substrates. In OCT1 and CYP2D6 single and double-transfected cells, we studied which, OCT1 or CYP2D6, is more critical for a given drug and whether there are additive, antagonistic or synergistic effects. In general, OCT1 substrates were more hydrophilic than CYP2D6 substrates and smaller in size. Inhibition studies showed unexpectedly pronounced inhibition of substrate depletion by shared OCT1/CYP2D6 inhibitors. In conclusion, there is a distinct overlap in the OCT1/CYP2D6 substrate and inhibitor spectra, so in vivo pharmacokinetics and -dynamics of shared substrates may be significantly affected by frequent OCT1- and CYP2D6-polymorphisms and by comedication with shared inhibitors.

Interaction of buspirone and its major metabolites with human organic cation transporters.

Buspirone, a cationic drug, is an anxiolytic and antidepressant drug. However, whether buspirone and its metabolites are interacted with organic cationic transporter remains uncertain. In this study, we examined the interaction of buspirone and its major metabolites 1-(2-pyrimidinyl)piperazine (1-PP) and 6-hydroxybuspirone (6'-OH-Bu) with hOCTs using human hepatocellular carcinoma (HepG2), human colorectal adenocarcinoma (Caco-2) cells, and S2 cells expressing OCT1 (S2hOCT1), 2 (S2hOCT2), or 3 (S2hOCT3). Coadministration of buspirone and fluorescent 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+ ) was examined using HepG2 cells, and [3 H]-1-methyl-4-phenylpyridinium (MPP+ ) transport was assessed in S2 cell overexpressing hOCTs. The results showed that ASP+ transport was suppressed by buspirone with an IC50 of 26.3 ± 2.9 µM without any cytotoxic effects in HepG2 expressing hOCTs cells. Consistently, buspirone strongly inhibited [3 H]-MPP+ uptake by S2hOCT1, S2hOCT2, and S2hOCT3 cells with an IC50s of 89.0 ± 1.3 µM, 43.7 ± 7.5 µM, and 20.4 ± 1.0 µM, respectively. Nonetheless, 6'-OH-Bu and 1-PP caused weak or no inhibition on ASP+ and [3 H]-MPP+ transport. These findings suggest the potential interaction of buspirone with organic cation drugs that are handled by hOCT3. However, further clinical relevance is needed to support these findings for preventing drug-drug interaction in patients who take prescribed drugs together with buspirone.

Selective Inhibition of Organic Cation Transporter 1 by Benzoylpaeoniflorin Attenuates Hepatic Lipid Accumulation through AMPK Activation.

Organic cation transporter 1 (OCT1) is a liver-specific transporter and plays an essential role in drug disposition and hepatic lipid metabolism. Therefore, inhibition of OCT1 may not only lead to drug-drug interactions but also represent a potential therapy for fatty liver diseases. In this study, we systematically investigated the inhibitory effect of 200 natural products on OCT1-mediated uptake of 4,4-dimethylaminostyryl-N-methylpyridinium (ASP+) and identified 10 potent OCT1 inhibitors. The selectivity of these inhibitors over OCT2 was evaluated using both in vitro uptake assays and in silico molecular docking analyses. Importantly, benzoylpaeoniflorin was identified as the most potent OCT1 inhibitor with the highest selectivity over OCT2. Additionally, benzoylpaeoniflorin prevented lipid accumulation in hepatocytes, with concomitant activation of AMPK and down-regulation of lipogenic genes, such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). To conclude, our findings are of significant value in understanding OCT1-based natural product-drug interactions and provide a natural source of OCT1 inhibitors which may hold promise for treating fatty liver diseases.

Structural basis of organic cation transporter-3 inhibition.

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.

Atypical Substrates of the Organic Cation Transporter 1.

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.

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

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.

Substrates and Inhibitors of the Organic Cation Transporter 3 and Comparison with OCT1 and OCT2.

Organic cation transporters (OCTs) 1, 2, and 3 facilitate cellular uptake of structurally diverse endogenous and exogenous substances. However, their substrate and inhibitor specificity are not fully understood. We performed a broad in vitro screening for OCT3 substrates and inhibitors, allowing us to compare the substrate spectra and to study the relationship between transport and inhibition of transport. Generally, substrates were smaller and more hydrophilic than OCT3 inhibitors. The best model-based predictor of transport was the positive charge, while the best predictor of inhibition was the aromatic ring count. OCT3 inhibition was well correlated between different model substrates. Substrates of OCT3 were mainly weak inhibitors, and the best inhibitors were not substrates. As tested with 264 substances, OCT3 transport had significantly more overlap with OCT2 than OCT1. Our data further substantiate that specificity of OCT transport varies with minor substitutions rather than with the general scaffolds of substrates.

Effects of single-nucleotide polymorphism on the pharmacokinetics and pharmacodynamics of metformin.

INTRODUCTION: Metformin has been recognized as the first-choice drug for type 2 diabetes mellitus (T2DM). The potency of metformin in the treatment of type 2 diabetes has always been in the spotlight and shown significant individual differences. Based on previous studies, the efficacy of metformin is related to the single-nucleotide polymorphisms of transporter genes carried by patients, amongst which a variety of gene polymorphisms of transporter and target protein genes affect the effectiveness and adverse repercussion of metformin. AREAS COVERED: Here, we reviewed the current knowledge about gene polymorphisms impacting metformin efficacy based on transporter and drug target proteins. EXPERT OPINION: The reason for the difference in clinical drug potency of metformin can be attributed to the gene polymorphism of drug transporters and drug target proteins in the human body. Substantial evidence shows that genetic polymorphisms in transporters such as organic cation transporter 1 (OCT1) and organic cation transporter 2 (OCT2) affect the glucose-lowering effectiveness of metformin. However, optimization of individualized dosing regimens of metformin is necessary to clarify the role of several polymorphisms.

Substrates of the Human Brain Proton-Organic Cation Antiporter and Comparison with Organic Cation Transporter 1 Activities.

Many organic cations (OCs) may be transported through membranes by a genetically still uncharacterized proton-organic cation (H + OC) antiporter. Here, we characterized an extended substrate spectrum of this antiporter. We studied the uptake of 72 drugs in hCMEC/D3 cells as a model of the human blood-brain barrier. All 72 drugs were tested with exchange transport assays and the transport of 26 of the drugs was studied in more detail concerning concentration-dependent uptake and susceptibility to specific inhibitors. According to exchange transport assays, 37 (51%) drugs were good substrates of the H + OC antiporter. From 26 drugs characterized in more detail, 23 were consistently identified as substrates of the H + OC antiporter in six different assays and transport kinetic constants could be identified with intrinsic clearances between 0.2 (ephedrine) and 201 (imipramine) mL × minute-1 × g protein-1. Excellent substrates of the H + OC antiporter were no substrates of organic cation transporter OCT1 and vice versa. Good substrates of the H + OC antiporter were more hydrophobic and had a lower topological polar surface area than non-substrates or OCT1 substrates. These data and further research on the H + OC antiporter may result in a better understanding of pharmacokinetics, drug-drug interactions and variations in pharmacokinetics.

Free Cholesterol Affects the Function and Localization of Human Na+/Taurocholate Cotransporting Polypeptide (NTCP) and Organic Cation Transporter 1 (OCT1).

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are associated with obesity. They are accompanied by increased levels of free cholesterol in the liver. Most free cholesterol resides within the plasma membrane. We assessed the impact of adding or removing free cholesterol on the function and localization of two hepatocellular uptake transporters: the Na+/taurocholate cotransporting polypeptide (NTCP) and the organic cation transporter 1 (OCT1). We used a cholesterol-MCD complex (cholesterol) to add cholesterol and methyl-ß-cyclodextrin (MCD) to remove cholesterol. Our results demonstrate that adding cholesterol decreases NTCP capacity from 132 ± 20 to 69 ± 37 µL/mg/min and OCT1 capacity from 209 ± 66 to 125 ± 26 µL/mg/min. Removing cholesterol increased NTCP and OCT1 capacity to 224 ± 65 and 279 ± 20 µL/mg/min, respectively. In addition, adding cholesterol increased the localization of NTCP within lipid rafts, while adding or removing cholesterol increased OCT1 localization in lipid rafts. These results demonstrate that increased cholesterol levels can impair NTCP and OCT1 function, suggesting that the free cholesterol content of the liver can alter bile acid and drug uptake into the liver. This could explain the increased plasma bile acid levels in NAFLD and NASH patients and potentially lead to altered drug disposition.

Role of human organic cation transporter-1 (OCT-1/SLC22A1) in modulating the response to metformin in patients with type 2 diabetes.

BACKGROUND: Organic cation transporter 1 primarily governs the action of metformin in the liver. There are considerable inter-individual variations in metformin response. In light of this, it is crucial to obtain a greater understanding of the influence of OCT1 expression or polymorphism in the context of variable responses elicited by metformin treatment. RESULTS: We observed that the variable response to metformin in the responders and non-responders is independent of isoform variation and mRNA expression of OCT-1. We also observed an insignificant difference in the serum metformin levels of the patient groups. Further, molecular docking provided us with an insight into the hotspot regions of OCT-1 for metformin binding. Genotyping of these regions revealed SNPs 156T>C and 1222A>G in both the groups, while as 181C>T and 1201G>A were found only in non-responders. The 181T>C and 1222A>G changes were further found to alter OCT-1 structure in silico and affect metformin transport in vitro which was illustrated by their effect on the activation of AMPK, the marker for metformin activity. CONCLUSION: Taken together, our results corroborate the role of OCT-1 in the transport of metformin and also point at OCT1 genetic variations possibly affecting the transport of metformin into the cells and hence its subsequent action in responders and non-responders.

Amino acids in transmembrane helix 1 confer major functional differences between human and mouse orthologs of the polyspecific membrane transporter OCT1.

Organic cation transporter 1 (OCT1) is a membrane transporter that affects hepatic uptake of cationic and weakly basic drugs. OCT1 transports structurally highly diverse substrates. The mechanisms conferring this polyspecificity are unknown. Here, we analyzed differences in transport kinetics between human and mouse OCT1 orthologs to identify amino acids that contribute to the polyspecificity of OCT1. Following stable transfection of HEK293 cells, we observed more than twofold differences in the transport kinetics of 22 out of 28 tested substrates. We found that the ß2-adrenergic drug fenoterol was transported with eightfold higher affinity but at ninefold lower capacity by human OCT1. In contrast, the anticholinergic drug trospium was transported with 11-fold higher affinity but at ninefold lower capacity by mouse Oct1. Using human-mouse chimeric constructs and site-directed mutagenesis, we identified nonconserved amino acids Cys36 and Phe32 as responsible for the species-specific differences in fenoterol and trospium uptake. Substitution of Cys36 (human) to Tyr36 (mouse) caused a reversal of the affinity and capacity of fenoterol but not trospium uptake. Substitution of Phe32 to Leu32 caused reversal of trospium but not fenoterol uptake kinetics. Comparison of the uptake of structurally similar ß2-adrenergics and molecular docking analyses indicated the second phenol ring, 3.3 to 4.8 Å from the protonated amino group, as essential for the affinity for fenoterol conferred by Cys36. This is the first study to report single amino acids as determinants of OCT1 polyspecificity. Our findings suggest that structure-function data of OCT1 is not directly transferrable between substrates or species.