Individual PKC-phosphorylation sites in organic cation transporter 1 determine substrate selectivity and transport regulation

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 mu mol: 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.