Polyspecific organic cation transporters (OCTs) have a large substrate binding pocket with different interaction domains. To determine whether OCT regulation is substrate specific, suitable fluorescent organic cations were selected by comparing their uptake in wild-type (WT) human embryonic kidney (HEK)-293 cells and in HEK-293 cells stably transfected with hOCT2. N-amidino-3,5-diamino-6-chloropyrazine-carboxamide (amiloride) and 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP) showed concentration-dependent uptake in hOCT2 at 37 degrees C. After subtraction of unspecific uptake determined in WT at 37 degrees C or in hOCT2 at 8 degrees C saturable specific uptake of both substrates was measured. Km values of hOCT2-mediated uptake of 95 microM amiloride and 24 microM ASP were calculated. Inhibition of amiloride and ASP uptake by several organic cations was also measured [IC50 (in microM) for amiloride and ASP, respectively, tetraethylammonium (TEA) 98 and 30, cimetidine 14 and 26, and tetrapentylammonium (TPA) 7 and 2]. Amiloride and ASP uptake were significantly reduced by inhibition of Ca2+/CaM complex (-55 +/- 5%, n = 10 and -63 +/- 2%, n = 15, for amiloride and ASP, respectively) and stimulation of PKC (-54 +/- 5%, n = 14, and -31 +/- 6%, n = 26) and PKA (-16 +/- 5%, n = 16, and -18 +/- 4%, n = 40), and they were increased by inhibition of phosphatidylinositol 3-kinase (+28 +/- 6%, n = 8, and +55 +/- 17%, n = 16). Inhibition of Ca2+/CaM complex resulted in a significant decrease of Vmax (160-99 photons/s) that can be explained in part by a reduction of the membrane-associated hOCT2 (-22 +/- 6%, n = 9) as determined using FACScan flow cytometry. The data indicate that saturable transport by hOCT2 can be measured by the fluorescent substrates amiloride and ASP and that transport activity for both substrates is regulated similarly. Inhibition of the Ca2+/CaM complex causes changes in transport capacity via hOCT2 trafficking.
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