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.

Organic cations comprise a significant part of medically relevant drugs and endogenous substances. Such substances need organic cation transporters for efficient transfer via cell membranes. However, the membrane transporters of most natural or synthetic organic cations are still unknown. To identify these transporters, genes of 10 known OCTs and 18 orphan solute carriers (SLC) were overexpressed in HEK293 cells and characterized concerning their transport activities with a broad spectrum of low molecular weight substances emphasizing organic cations. Several SLC35 transporters and SLC38A10 significantly enhanced the transport of numerous relatively hydrophobic organic cations. Significant organic cation transport activities have been found in gene families classified as transporters of other substance classes. For instance, SLC35G3 and SLC38A10 significantly accelerated the uptake of several cations, such as clonidine, 3,4-methylenedioxymethamphetamine, and nicotine, which are known as substrates of a thus far genetically unidentified proton/organic cation antiporter. The transporters SLC35G4 and SLC35F5 stood out by their significantly increased choline uptake, and several other SLC transported choline together with a broader spectrum of organic cations. Overall, there are many more polyspecific organic cation transporters than previously estimated. Several transporters had one predominant substrate but accepted some other cationic substrates, and others showed no particular preference for one substrate but transported several organic cations. The role of these transporters in biology and drug therapy remains to be elucidated.

The multidrug and toxin extrusion proteins MATE1 and MATE2K may determine the pharmacokinetics and drug-drug interactions of many drugs. However, their substrate spectrum and synergy with organic cation transporters OCT1 and OCT2 remain incompletely understood. Therefore, we screened 590 predominantly positively charged, low molecular weight compounds for transport via these four transporters in HEK293 cells using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). MATE1 and MATE2K transported 164 and 114 compounds, respectively, with significant overlap. High-affinity substrates included berberine, pentamidine, and amisulpride, while epinephrine and atenolol had the highest V_max. Despite less than 16% sequence homology, there was high overlap among MATE1/-2K and OCT1/-2 substrates. Neither isolated physicochemical properties nor their linear combinations predicted the substrates of these organic cation transporters. However, machine learning classifiers using 15 parameters allowed 69 to 87% correct prediction. The large number of substrates indicates a possibly broad role of multidrug and toxin extrusion (MATE) transporters in pharmacokinetics and drug interactions.

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.

Most central nervous system (CNS) active drugs are organic cations, which need carrier proteins for efficient transfer through the blood–brain barrier (BBB). A genetically still unidentified proton organic cation (H⁺/OC) antiporter is found in several tissues, including endothelial cells of the BBB. We characterized the substrate spectrum of the H⁺/OC antiporter and the overlap in substrate spectrum with OCTN1, OCTN2 or OCT3 by screening 87 potential substrates for transport activity. Based on high antiport rates, 45 of the tested substances were substrates of the H⁺/OC antiporter. They included antidepressants (like tranylcypromine or nortriptyline), antipsychotics (like levomepromazine) and local anaesthetics. Concentration‐dependent transport was confirmed for 38 of the substrates. Transport uptake depending on a pH gradient across the cell membrane confirmed that 43 drugs were indeed substrates of the H⁺/OC antiporter. However, the patterns of pH dependence differed between the substrates, possibly indicating different modes of transport or the existence of multiple antiporter proteins. The substrate overlap between the H⁺/OC antiporter and OCTN1, OCTN2 or OCT3 was minimal, indicating that the latter three are not the proteins underlying the H⁺/OC antiporter activity. Overall, about 50% of positively charged drugs may be substrates of the antiporter, which may be the most important membrane transport protein for many drugs.

Carrier-mediated uptake of choline is rate-limiting for acetylcholine biosynthesis and various other biological processes. To date, 16 solute carrier (SLC) proteins have been identified that may facilitate choline permeation across the outer cell membrane. However, their biochemical functions have not yet been experimentally compared. We overexpressed 16 SLC proteins with known choline-transporting capacity and compared their choline transport kinetics. Additionally, we evaluated their capacity to transport choline analogues as well as metabolites involved in its biosynthesis and degradation or modulators of cholinergic neurotransmission to gain insight into the biological functions of the SLCs. Furthermore, we investigated whether the transporters could be distinguished by their sensitivity to inhibition by hemicholinium-3 and other substances. Of the 16 SLCs tested, seven (SLC5A7, SLC35F2, SLC35F3, SLC35F4, SLC25F5, SLC35G4, and SLC44A5) exhibited K_M values for choline transport in the range of 12 to 50 µM, closely aligning with physiological plasma choline concentrations. Among them, SLC5A7 displayed over tenfold higher intrinsic clearance than any of the others. SLC22A1-3 were confirmed as choline transporters, albeit with low affinity. Hemicholinium-3 most strongly inhibited SLC5A7 and also significantly inhibited SLC35F2-5 and SLC35G4. Choline transport by these six transporters was inhibited by about 50 % at 100 µM decynium-22 and verapamil.. In humans, multiple SLCs may contribute to cellular choline uptake, depending on physiologic conditions and their yet incompletely characterized expression patterns. The present data may also enhance our understanding of inherited and environmental modulation of these transporters with possible consequence, for instance, on motor and cognitive functions.