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Functional characterization of genetic polymorphisms in the organic cation transporter OCT1 with a special focus on the substrate-specific effects of the M420del polymorphism

dc.contributor.advisorTzvetkov, Mladen Dr.
dc.contributor.authorSeitz, Tina
dc.titleFunctional characterization of genetic polymorphisms in the organic cation transporter OCT1 with a special focus on the substrate-specific effects of the M420del polymorphismde
dc.title.translatedFunctional characterization of genetic polymorphisms in the organic cation transporter OCT1 with a special focus on the substrate-specific effects of the M420del polymorphismde
dc.contributor.refereeBrockmöller, Jürgen Prof. Dr.
dc.description.abstractengThe human organic cation transporter 1 (OCT1) is strongly expressed in the sinusoidal membrane of hepatocytes. OCT1 mediates the uptake of drugs and exogenous substances with cationic or weak basic structures into the liver. The OCT1 gene has high genetic variability with five common amino acid substitutions R61C, C88R, G401S, G465R and the deletion of M420del that are known to cause loss of OCT1. The loss of OCT1 function decreases the hepatic uptake of drugs like morphine, tropisetron, and o-desmethyl-tramadol (the active metabolite of tramadol) and increases their plasma concentrations. This may result in increased efficacy of these drugs, but also increases the risk of adverse effects. In Caucasians, 9% of the individuals carry two inactive OCT1 alleles and further 42% carry only one inactive OCT1 allele. Recent next-generation resequencing analyses of 53 global populations as well as the existing data of 14 populations of the 1000 Genomes project led to the identification of ad¬di¬tional amino acid substitutions predicted to affect OCT1 function. In the first part of this thesis, 19 amino acid variants in OCT1 (including the 5 common, 5 novel, and 9 population-specific) were functionally characterized using a broad spectrum of structurally diverse OCT1 substrates. The aim was to generate a world map of genetically-determined loss of OCT1 function. This world map might shed light on the potential role of OCT1 polymorphisms in interethnic differences in drug therapy. Furthermore, the global distribution of loss of OCT1 function might point to a selection pressure for either retention or loss of OCT1 activity. Targeted genomic integration was used to generate HEK293 cell lines over¬ex¬pressing OCT1 allelic variants. The cells were used to measure the effect of the variants on the uptake of the model substrates MPP+, TEA+, ASP+ as well as of the drugs morphine, metformin, tropisetron, debrisoquine, and O-desmethyltramadol. The subcellular localization of OCT1 was analyzed by western blot analysis and immunofluorescence staining detected by confocal microscopy. Fifteen major OCT1 alleles causing a more than 50 % decrease or increase of the wild type transport activity were identified. An additional 6 sub-alleles were identified that did not substantially affect the transport properties of the major allele. Four major alleles OCT1*5 (G465R/M420del), OCT1*6 (C88R/M420del), OCT1*12 (S29L), and OCT1*15 (E284K) showed complete substrate-wide loss of activity caused by improper membrane localization of the protein. Three alleles OCT1*3 (R61C), OCT1*4 (G401S), and OCT1*14 (R206C/M420del) showed strong substrate-wide decrease in transport activity. Of these, OCT1*3 and OCT1*14 showed reduced plasma membrane localization. A substantial number of alleles (5 out of 19 tested) showed substrate-specific loss of activity: OCT1*2 (M420del), OCT1*7 (S14F), OCT1*10 (S189L), OCT1*11 (I449T), and OCT1*13 (T245M). Two alleles OCT1*8 (R488M) and OCT1*9 (P117L) showed more than 50% increase in activity for at least one substrate tested. A world map of genetically-determined loss of OCT1 function, which was generated based on these analyses, showed strong variability in the loss of OCT1 function among different world regions. Almost all individuals in East Asia and Oceania carry two active OCT1 alleles. In contrast, more than 80% of the Surui Indians, a Native American tribe in the Amazon, carried two loss-of-function alleles. These findings should be taken into consideration for recommendations of individualized adjustment of drug medication in specific populations. This work provides functional analysis of existing OCT1 allelic variants on a broad spectrum of structurally diverse substances revealing strong differences in the effect of these variants on transporter function. The high number of substrate-specific loss of function variants suggests that it is not sufficient to test single OCT1 substrates in order to predict the effect of OCT1 variants. Among the substrate-specific loss-of-function alleles OCT1*2 (M420del) was by far the most common one. It was ubiquitously observed across tested populations and was the only loss-of-function OCT1 variant observed in Surui Indians. In the second part of the thesis the structural mechanism underlying the highly substrate-specific effects of M420del was analyzed. First, it could be shown that the substrate-specific effects of M420del are not caused by an unspecific reduction of the protein chain but rather due to the deletion of the amino acid at codon 420. The transport activity of the mutants L427del and H428del, which were expected to show transport activity similar to M420del, did not differ from wild type. The mutant insertion of alanine after proline425 (A426ins) on M420del background, which was expected to restore wild type activity, showed the similar activity as M420del. Furthermore, the mutant M420A showed no differences in the uptake kinetics compared to M420del using structurally different OCT1 substrates. Furthermore, neither isoleucine, nor threonine, nor cysteine at position 420 could restore wild type transport activity. This data confirmed that the effects of M420del are caused by a specific loss of the methionine side chain. Finally, interactions of methionine420 with amino acids in the 7th transmembrane domain (L364 and H367) were analyzed. An interaction between M420 and L364 or H367 could not be confirmed, as a mutation of these amino acids resulted in a general reduction of OCT1 uptake. Analyses of interactions between methioine420 and amino acids known to be involved in substrate binding and translocation suggest complex interaction of M420del with D474, but not with W217 and F159. Taken together, it could be shown that the substrate-specific effects of M420del are caused by the specific loss of the methionine side chain and indirect interactions with essential transport domains were suggested. Further experiments applying detailed homology modeling are needed to reveal the exact structural mechanism how M420del affects OCT1
dc.contributor.coRefereeBurckhardt, Gerhard Prof. Dr.
dc.contributor.thirdRefereeNikolaev, Viacheslav Dr.
dc.contributor.thirdRefereeHahn, Heidi Prof. Dr.
dc.contributor.thirdRefereeJarry, Hubertus Prof. Dr.
dc.contributor.thirdRefereeDressel, Ralf Prof. Dr.
dc.subject.enggenetic variabilityde
dc.affiliation.instituteMedizinische Fakultät
dc.subject.gokfullMedizin (PPN619874732)de
dc.subject.gokfullMolekulare Medizinde

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