A Highly Sensitive UPLC-MS/MS Method for the Quantification of the Organic Cation Transporters' Mediated Metformin Uptake and Its Inhibition in Cells.

Metformin is the gold standard substrate for evaluating potential inhibitors of the organic cation transporters (OCTs). Here, we established a UPLC-MS/MS assay to quantify metformin in cell pellets with a range of 0.05-50 ng/mL using 6-deuterated metformin as an internal standard.

Comprehensive characterization of the OCT1 phenylalanine-244-alanine substitution reveals highly substrate-dependent effects on transporter function.

Organic cation transporters (OCTs) can transport structurally highly diverse substrates. The molecular basis of this extensive polyspecificity has been further elucidated by cryo-EM. Apparently, in addition to negatively charged amino acids, aromatic residues may contribute to substrate binding and substrate selectivity.

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.

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.

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.

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.

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.

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).

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 screening for OCT3 substrates and inhibitors, allowing us to compare the substrate spectra and to study the relationship between transport and inhibition of transport.

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.

Relationships between Inhibition, Transport and Enhanced Transport via the Organic Cation Transporter 1.

The organic cation transporter 1 (OCT1, ) transports a large number of structurally diverse endogenous and exogenous substrates. There are numerous known competitive and non-competitive inhibitors of OCT1, but there are no studies systematically analyzing the relationship between transport, stimulation, and inhibition. Here, we tested in vitro OCT1 inhibition by OCT1 substrates and transport of OCT1 inhibitors under uniform analytical conditions.

Molecular basis for stereoselective transport of fenoterol by the organic cation transporters 1 and 2.

Stereoselectivity is important in many pharmacological processes but its impact on drug membrane transport is scarcely understood. Recent studies showed strong stereoselective effects in the cellular uptake of fenoterol by the organic cation transporters OCT1 and OCT2. To provide possible molecular explanations, homology models were developed and the putative interactions between fenoterol enantiomers and key residues explored in silico through computational docking, molecular dynamics simulations, and binding free energy calculations as well as in vitro by site-directed mutagenesis and cellular uptake assays.

Overlap and Specificity in the Substrate Spectra of Human Monoamine Transporters and Organic Cation Transporters 1, 2, and 3.

Human monoamine transporters (MATs) are cation transporters critically involved in neuronal signal transmission. While inhibitors of MATs have been intensively studied, their substrate spectra have received far less attention. Polyspecific organic cation transporters (OCTs), predominantly known for their role in hepatic and renal drug elimination, are also expressed in the central nervous system and might modulate monoaminergic signaling.

Cellular Uptake of Psychostimulants - Are High- and Low-Affinity Organic Cation Transporters Drug Traffickers?

Psychostimulants are used therapeutically and for illegal recreational purposes. Many of these are inhibitors of the presynaptic noradrenaline, dopamine, and serotonin transporters (NET, DAT, and SERT). According to their physicochemical properties, some might also be substrates of polyspecific organic cation transporters (OCTs) that mediate uptake in liver and kidneys for metabolism and excretion.

A double-Flp-in method for stable overexpression of two genes.

Overexpression of single genes in mammalian cells is widely used to investigate protein function in basic and applied biosciences and in drug research. A better understanding of interactions of two proteins is an important next step in the advancement of our understanding of complex biological systems. However, simultaneous and robust overexpression of two or more genes is challenging.