Molecular interaction of organic cation transporters with the tetraspanin CD63 modulates transporter trafficking and their trafficking‐associated regulation

Abstract

Human organic cation transporters (hOCT1 to 3) are involved in the transport of endogenous and exogenous organic cations in the liver, kidney and brain. Here we analyse the regulation of hOCT1 to hOCT3 by direct protein‐protein interaction using a mating based Split‐Ubiquitin‐Yeast‐Two‐Hybrid System and pull‐down assays. Specifically, we found that the tetraspanin CD63 is an interacting partner of hOCT2 and hOCT3. A strong co‐localisation of CD63 and hOCT2 was shown on the plasma membrane and in vesicular compartments of HEK293 cells by TIRF‐microscopy and in tissue sections of human kidneys by immunofluorescence. Further immunofluorescence and surface biotinylation experiments after CD63 overexpression revealed a loss of cell surface hOCT2 and its relocalisation into intracellular vesicles. In HEK293 cells stably transfected with hOCT2, CD63‐overexpression caused a decrease, CD63‐knockdown an increase of cation‐uptake. As functional consequences of CD63 knockout in mice we observed that OCT2 lost its specific basolateral localization in the kidney, appearing to be widespread distributed in the cell and that basophil‐mediated histamine secretion, which apparently involves OCT3, was increased. Our data strongly suggest that CD63 regulates the function of hOCT2 and −3 by influencing their trafficking to/from the cell membrane and processing via the intracellular sorting machinery. Supported by the Deutsche Forschungsgemeinschaft (DFG CI 107/4‐1 to 2).