Functional characterization of solute carriers with emphasis on proton-coupled organic cation antiporters
by Kyra-Elisa Maria Redeker
Date of Examination:2025-06-25
Date of issue:2025-07-04
Advisor:Prof. Dr. Jürgen Brockmöller
Referee:Prof. Dr. Jürgen Brockmöller
Referee:Prof. Dr. Bert De Groot
Referee:Prof. Dr. Dr. Ingolf Cascorbi
Sponsor:Deutsche Forschungsgemeinschaft (DFG - project number 461080000)
Persistent Address:
http://dx.doi.org/10.53846/goediss-11368
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Abstract
English
Solute carriers (SLCs) play an important role in the uptake of natural compounds and medically relevant drugs into various tissues, including the blood-brain barrier (BBB). Many cationic drugs rely on carrier-mediated uptake across the BBB due to their charge and lipophilicity, but the specific membrane transporters responsible for their uptake remain largely unknown. Instead, based on several physiological criteria, many of these substances were identified as substrates of the proton-coupled organic cation (H+/OC) antiporter. However, the H+/OC antiporter is biochemically and genetically still not well characterized. The aim of this project was to characterize several known and novel solute carriers with potential transport activity for organic cations. A comprehensive analysis of these transporters is expected to enhance our understanding of their biological functions and may provide valuable insight into organic cation transport. Such knowledge may also elucidate the mechanisms underlying the translocation of clinically important drugs across the BBB, with implications for both research and clinics. For instance, identifying the specific transporters involved might allow a better understanding of human genomic variation and drug-drug interactions. The substrate spectrum of the genetically not yet identified H+/OC antiporter was further expanded in hCMEC cells, a model of the BBB endothelium. New substrates were identified based on antiport transport activity, pH dependence, and concentration-dependent uptake. Most of the newly identified substrates were psychoactive drugs from various therapeutic classes, including antidepressants and antipsychotics. Although these compounds were potential substrates for known organic cation transporters expressed at the BBB, such as OCT3, OCTN1, and OCTN2, they were not transported by any of these three transporters. Similarly, the multidrug and toxin extrusion (MATE) proteins 1 and 2, which are proton-coupled antiporters, showed minimal overlap with the antiporter activity observed in the hCMEC cell line. Nonetheless, over 100 new substrates of these MATE transporters were identified. A comparison of their substrate spectrum with the well- characterized organic cation transporters (OCT) 1 and 2 revealed a high overlap, despite a small sequence homology. While none of the typical physicochemical characteristics of these molecules alone could reliably explain their substrate status, machine learning classifiers provided promising results: it allowed to predict between 68% and 88% of the substrates, depending on the transporter. Surprisingly, the transporter OCT1 was also capable of efficiently transporting several uncharged or even negatively charged compounds, such as lamivudine and emtricitabine, and to a lesser extent, compounds like prostaglandins E2 and F2α. To gain insight into the substrate polyspecificty of OCTs, cryogenic electron microscopy recently became a most important tool. In case of OCT3, seven sequentially arranged negatively charged amino acids had been proposed to be relevant in the substrate translocation path. Our findings confirmed the critical role of E390 as an orthosteric binding site. Additionally, we identified E451 as potential opportunistic binding site, as the removal of the negative charge resulted in substrate-specific loss of transport activity. Further, several previously orphan or poorly characterized SLCs were identified and characterized as organic cation transporters. Notably, SLC35G3 and SLC38A10 demonstrated moderate uptake of several known H+/OC antiporter substrates, such as clonidine and nicotine. A prominent finding was the uptake of choline by multiple SLCs. SLC35F2, -3, -4, -5 and SLC35G4 exhibited KM values for choline uptake between 12 to 50 μM, which is in the range of physiological plasma concentrations of choline in humans. In summary, I characterized several known and novel organic cation transporters regarding their substrate spectrum and specificity. The application of machine learning models to analyze the biochemical properties of substrates is promising for identifying distinct molecular features associated with transporter recognition. Collectively, these findings contribute to a more comprehensive understanding of organic cation transporters and the functionally important but yet genetically unidentified H+/OC antiporter.
Keywords: Solute carrier; Proton-organic cation antiporter; Organic cation transport; Substrate polyspecificity; Choline transport; Pharmacology
