Several orphan solute carriers functionally identified as organic cation transporters: substrates specificity compared with known cation transporters.

Organic cations comprise a significant part of medically relevant drugs and endogenous substances. Such substances need organic cation transporters (OCT) for efficient transfer via cell membranes. However, the membrane transporters of most natural or synthetic organic cations the membrane transporters 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.

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.