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Individual PKC-Phosphorylation Sites in Organic Cation Transporter 1 Determine Substrate Selectivity and Transport Regulation

Giuliano Ciarimboli^*, Hermann Koepsell, Mariya Iordanova^*, Valentin Gorboulev, Brigitte Dürner, Detlef Lang^*, Bayram Edemir^*, Rita Schröter^*, Truc Van Le^* and Eberhard Schlatter^*

^* Medizinische Klinik und Poliklinik D, Experimentelle Nephrologie, Universitätsklinikum Münster, Münster, Germany; and Institut für Anatomie und Zellbiologie, Universität Würzburg, Würzburg, Germany

Address correspondence to: Dr. Giuliano Ciarimboli, Medizinische Klinik und Poliklinik D, Experimentelle Nephrologie, Universitätsklinikum Münster, Domagkstrasse 3a, Münster, D-48149 Germany. Phone: +49-251-8356981; Fax: +49-251-8356973. E-mail: gciari{at}uni-muenster.de

Received for publication April 1, 2004. Accepted for publication February 23, 2005.

To elucidate the molecular mechanisms underlying stimulation of rat organic cation transporter type 1 (rOCT1) by protein kinase C (PKC) activation, functional properties and regulation of rOCT1 stably expressed in HEK293 cells after site-directed mutagenesis of putative PKC phosphorylation-sites were compared with wild-type (WT) rOCT1 using microfluorometric measurements with the fluorescence organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP⁺). Either substitutions of single (S286A, S292A, T296A, S328A, and T550A) or of all five PKC-sites (5x-PKC) with alanine suppressed PKC-induced stimulation of ASP⁺ uptake, whereas regulation by p56^lck tyrosine kinase was conserved in all mutants. Remarkably, the apparent affinities for TEA⁺, TPA⁺, and quinine were changed differently in each mutant (EC₅₀ in WT, S286A, S292A, T296A, S328A, T550A, and 5x-PKC in µmol: TEA⁺: 105, 153, 56, 1135, 484, 498, 518; TPA⁺: 0.1, 2.1, 0.3, 1.0, 43, 0.3, 2.2; quinine: 1.5, 3.0, 2.5, 4.8, 81, 7.6, 8.9, respectively). After mutations, no effects of PKC activation on apparent affinity of rOCT1 for these substrates could be detected, in contrast to what was observed in WT. PKC activation had no significant effect on rOCT1 trafficking from intracellular pools to the cell membrane. Substitution of all PKC sites suppressed PKC-induced phosphorylation of rOCT1. In conclusion, it was found that the presence of all five potential PKC phosphorylation sites is necessary for the PKC-induced stimulation of rOCT1. The different effects on the EC₅₀ values by the different mutations suggest that the large intracellular loop participates in building the substrate binding pocket of rOCT1 or specifically modulates its structure.

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