%0 Journal Article %1 gorboulev2018assay %A Gorboulev, V. %A Rehman, S. %A Albert, C. M. %A Roth, U. %A Meyer, M. J. %A Tzvetkov, M. V. %A Mueller, T. D. %A Koepsell, H. %D 2018 %J Mol Pharmacol %K 1-Methyl-4-phenylpyridinium/metabolism/pharmacology myOwn uni_network %N 4 %P 402-415 %R 10.1124/mol.117.110767 %T Assay Conditions Influence Affinities of Rat Organic Cation Transporter 1: Analysis of Mutagenesis in the Modeled Outward-Facing Cleft by Measuring Effects of Substrates and Inhibitors on Initial Uptake %U https://www.ncbi.nlm.nih.gov/pubmed/29339398https://molpharm.aspetjournals.org/content/molpharm/93/4/402.full.pdf %V 93 %X The effects of mutations in the modeled outward-open cleft of rat organic cation transporter 1 (rOCT1) on affinities of substrates and inhibitors were investigated. Human embryonic kidney 293 cells were stably transfected with rOCT1 or rOCT1 mutants, and uptake of the substrates 1-methyl-4-phenylpyridinium(+) (MPP(+)) and tetraethylammonium(+) (TEA(+)) or inhibition of MPP(+) uptake by the nontransported inhibitors tetrabutylammonium(+) (TBuA(+)), tetrapentylammonium(+) (TPeA(+)), and corticosterone was measured. Uptake measurements were performed on confluent cell layers using a 2-minute incubation or in dissociated cells using incubation times of 1, 5, or 10 seconds. With both methods, different apparent Michaelis-Menten constant (K(m)) values, different IC(50) values, and varying effects of mutations were determined. In addition, varying IC(50) values for the inhibition of MPP(+) uptake and varying effects of mutations were obtained when different MPP(+) concentrations far below the apparent K(m) value were used for uptake measurements. Eleven mutations were investigated by measuring initial uptake in dissociated cells and employing 0.1 microM MPP(+) for uptake during inhibition experiments. Altered affinities for substrates and/or inhibitors were observed when Phe160, Trp218, Arg440, Leu447, and Asp475 were mutated. The mutations resulted in changes of apparent K(m) values for TEA(+) and/or MPP(+) Mutation of Trp218 and Asp475 led to altered IC(50) values for TBuA(+), TPeA(+), and corticosterone, whereas the mutation of Phe160 and Leu447 changed the IC(50) values for two inhibitors. Thereby amino acids in the outward-facing conformation of rOCT1 could be identified that interact with structurally different inhibitors and probably also with different substrates.

@article{gorboulev2018assay, abstract = {The effects of mutations in the modeled outward-open cleft of rat organic cation transporter 1 (rOCT1) on affinities of substrates and inhibitors were investigated. Human embryonic kidney 293 cells were stably transfected with rOCT1 or rOCT1 mutants, and uptake of the substrates 1-methyl-4-phenylpyridinium(+) (MPP(+)) and tetraethylammonium(+) (TEA(+)) or inhibition of MPP(+) uptake by the nontransported inhibitors tetrabutylammonium(+) (TBuA(+)), tetrapentylammonium(+) (TPeA(+)), and corticosterone was measured. Uptake measurements were performed on confluent cell layers using a 2-minute incubation or in dissociated cells using incubation times of 1, 5, or 10 seconds. With both methods, different apparent Michaelis-Menten constant (K(m)) values, different IC(50) values, and varying effects of mutations were determined. In addition, varying IC(50) values for the inhibition of MPP(+) uptake and varying effects of mutations were obtained when different MPP(+) concentrations far below the apparent K(m) value were used for uptake measurements. Eleven mutations were investigated by measuring initial uptake in dissociated cells and employing 0.1 microM MPP(+) for uptake during inhibition experiments. Altered affinities for substrates and/or inhibitors were observed when Phe160, Trp218, Arg440, Leu447, and Asp475 were mutated. The mutations resulted in changes of apparent K(m) values for TEA(+) and/or MPP(+) Mutation of Trp218 and Asp475 led to altered IC(50) values for TBuA(+), TPeA(+), and corticosterone, whereas the mutation of Phe160 and Leu447 changed the IC(50) values for two inhibitors. Thereby amino acids in the outward-facing conformation of rOCT1 could be identified that interact with structurally different inhibitors and probably also with different substrates.}, added-at = {2024-02-15T15:08:22.000+0100}, author = {Gorboulev, V. and Rehman, S. and Albert, C. M. and Roth, U. and Meyer, M. J. and Tzvetkov, M. V. and Mueller, T. D. and Koepsell, H.}, biburl = {https://www.bibsonomy.org/bibtex/20be69aeca34d438442315462cf44aa08/jvsi_all}, doi = {10.1124/mol.117.110767}, interhash = {38c6d1d280890de085ff95cb01e563ad}, intrahash = {0be69aeca34d438442315462cf44aa08}, issn = {1521-0111 (Electronic) 0026-895X (Linking)}, journal = {Mol Pharmacol}, keywords = {1-Methyl-4-phenylpyridinium/metabolism/pharmacology myOwn uni_network}, note = {Gorboulev, Valentin Rehman, Saba Albert, Christoph M Roth, Ursula Meyer, Marleen J Tzvetkov, Mladen V Mueller, Thomas D Koepsell, Hermann eng Research Support, Non-U.S. Gov't 2018/01/18 Mol Pharmacol. 2018 Apr;93(4):402-415. doi: 10.1124/mol.117.110767. Epub 2018 Jan 16.}, number = 4, pages = {402-415}, timestamp = {2024-02-15T15:11:55.000+0100}, title = {Assay Conditions Influence Affinities of Rat Organic Cation Transporter 1: Analysis of Mutagenesis in the Modeled Outward-Facing Cleft by Measuring Effects of Substrates and Inhibitors on Initial Uptake}, type = {Journal Article}, url = {https://www.ncbi.nlm.nih.gov/pubmed/29339398https://molpharm.aspetjournals.org/content/molpharm/93/4/402.full.pdf}, volume = 93, year = 2018 }