@phdthesis{Meyer2020,
  author    = {Marleen J. Meyer},
  title     = {Effects of ligand structure, amino acid substitutions, and species differences on the function of organic cation transporter OCT1: utilizing polyspecificity for understanding structure-function relationships},
  journal    = {Effekte von Ligandenstruktur, Aminos{\"a}ureaustauschen und Speziesunterschieden auf die Funktion des organischen Kationentransporters OCT1 – Nutzen von Polyspezifit{\"a}t zum Verst{\"a}ndnis von Struktur-zu-Funktionsbeziehungen},
  url       = {https://nbn-resolving.org/urn:nbn:de:gbv:9-opus-41825},
  pages     = {227},
  year      = {2020},
  abstract  = {Organic cation transporter OCT1 is strongly expressed in the sinusoidal membrane of hepatocytes. OCT1 mediates the uptake of weakly basic and cationic compounds from the blood into the liver and may thereby facilitate the first step in hepatic metabolism or excretion of many cationic drugs. OCT1 is a polyspecific transporter and has a very broad spectrum of structurally highly diverse ligands (substrates and inhibitors). The exact transport mechanism and the amino acids involved in polyspecific ligand binding of OCT1 are poorly understood. The aim of this work was to utilize the polyspecificity to better understand the structure-function relationships of OCT1 and to gain first insights into potential mechanisms conferring the polyspecificity. We followed two strategies, analyzing the effects of variability in both ligand and transporter structure on OCT1 function. The effects of ligand structure were analyzed by comparing uptake and inhibitory potencies of structurally similar drugs of the group of opioids. The effects of transporter structure were analyzed by comparing the effects of variability caused by naturally occurring genetic variants or artificial mutations on OCT1 uptake and inhibition of several substrates. Most importantly, the effects of interspecies variability in transporter structure were analyzed by comparing uptake kinetics between human and mouse OCT1 orthologs. To this end, we used stably or transiently transfected HEK293 cells overexpressing OCT1 and different chimeric and mutant variants thereof. Focusing on OCT1 ligands, we compared the uptake and inhibitory potencies of structurally similar opioids. Only minor changes of the ligand structure strongly affected the interaction with OCT1. The presence of the ether linkage between C4 and C5 of the morphinan ring was associated with reduced OCT1 inhibitory potencies, while passive membrane permeability was the major negative determinant of OCT1-mediated uptake among structurally highly similar morphinan opioids. Only minor structural changes strongly increased the inhibitory potency by 28-fold from the lowest IC50 of 2004 µM for oxycodone to 72 µM for morphine. Additional removal of the ether linkage between C4-C5 increased the inhibitory potency by a total of 313-fold to the lowest IC50 of 6 µM for dextrorphan. Consequently, our data demonstrates that despite its polyspecificity, OCT1-mediated uptake and inhibition of this uptake is still somewhat very specific. Focusing on OCT1 protein structure, we first analyzed the effects of variability caused by naturally occurring genetic variants on OCT1 uptake and inhibition. OCT1 transport was strongly affected by OCT1 genetic variants and these effects were often substrate-specific. Correlation of these effects revealed several substrates that were similarly affected by the variants and may therefore be suggested to share similar or overlapping binding sites in OCT1. In addition, the effects of the genetic variants OCT1*2 and OCT1*3 on different substrates correlated well which may suggest that the structural variability caused by these two variants similarly affects substrate uptake. OCT1 genetic variants also affected the inhibition of OCT1, with both substrate and genotype-specific differences. Ranitidine inhibited the uptake of several substrates, among them the clinically relevant drugs metformin and morphine. Moreover, the inhibition was more potent (about 2-fold) on the uptake mediated by the common genetic variant OCT1*2 than on the uptake mediated by the reference OCT1*1. Second, we analyzed the effects of artificial mutations of key amino acids. Tyr222 and Asp475 in rat OCT1 had strongly substrate-specific and also species-specific effects on both OCT1-mediated uptake and inhibition. Mutation of these amino acids strongly decreased OCT1-mediated uptake, which further underscored an important role especially of Asp475. Interestingly, despite a proposed essential role of this amino acid, we observed Asp475-independent transport. This transport was observed in mouse, but not in human OCT1 and was substrate-specific. TMH10 was identified to be involved in determining the Asp475-independent uptake of mouse OCT1. Finally and most importantly, we analyzed the effects of sequence differences between human and mouse OCT1 on the transport kinetics of several OCT1 substrates. The transport kinetics differed strongly between human and mouse OCT1 orthologs. These differences were substrate-specific and affected both the affinity (KM) and capacity (vmax) of transport. Human OCT1 had an 8-fold higher capacity of trospium transport, while mouse OCT1 had an 8-fold higher capacity of fenoterol transport. Furthermore, mouse OCT1 had a 5-fold higher affinity for metformin transport compared to human OCT1. The difference between Phe32 in human and Leu32 in mouse OCT1 in TMH1 was identified to confer a higher capacity of transport by human compared to mouse OCT1, while the difference between Cys36 in human and Tyr36 in mouse OCT1 in TMH1 was identified to confer a higher capacity of transport by mouse compared to human OCT1. Furthermore, Leu155 in human OCT1, corresponding to Val156 in mouse OCT1 in TMH2, in concert with TMH3 were identified to confer the differences in affinity for metformin transport between the species. It may be speculated that ligand binding in OCT1 involves a core binding region that includes Asp474/475 and that polyspecific ligand binding is enabled by providing further binding partners (different amino acids) in more peripheral regions that different ligands can selectively interact with. This mechanism may also be a first step in explaining the substrate-specific effects of genetic variants with clinical relevance. Based on our findings, these “polyspecificity regions” may include TMH1, TMH2, and TMH3. Further analyses are warranted to characterize and narrow down these regions to unravel the structure-function relationships and with that the polyspecificity of OCT1. To summarize, variability in both ligand and transporter structure strongly affected OCT1 function and we were able to identify ligand structures that affect inhibitory potency and protein structures that confer species-specific differences in OCT1 transport. This work emphasizes again the complexity of OCT1 transport and structure-function relationships. We also showed that, in spite of the difficulties for experimental analysis and data interpretation that arise from the polyspecific nature of OCT1, polyspecificity can also be used as a tool to better understand the structure-function relationships of this transporter.},
  language  = {en}
}