Biermann Jürgen, Lang Detlef, Gorboulev Valentin, Koepsell Hermann, Sindic Aleksandra, Schröter Rita, Zvirbliene Aurelija, Pavenstädt Hermann, Schlatter Eberhard, Ciarimboli Giuliano
Medizinische Klinik und Poliklinik D, Experimentelle Nephrologie, Universitätsklinikum Münster, Domagkstrasse 3a, D-48149 Münster, Germany.
Am J Physiol Cell Physiol. 2006 Jun;290(6):C1521-31. doi: 10.1152/ajpcell.00622.2005. Epub 2006 Jan 4.
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.
多特异性有机阳离子转运体（OCTs）具有一个带有不同相互作用结构域的大底物结合口袋。为了确定OCT的调节是否具有底物特异性，通过比较野生型（WT）人胚肾（HEK）-293细胞和稳定转染hOCT2的HEK-293细胞对合适的荧光有机阳离子的摄取情况来进行选择。N-脒基-3,5-二氨基-6-氯吡嗪甲酰胺（氨氯地平）和4-[4-(二甲氨基)-苯乙烯基]-N-甲基吡啶鎓（ASP）在37℃时在hOCT2中呈现浓度依赖性摄取。在减去37℃时WT细胞或8℃时hOCT2细胞中测定的非特异性摄取后，测量了两种底物的可饱和特异性摄取。计算出hOCT2介导的95μM氨氯地平和24μM ASP摄取的Km值。还测量了几种有机阳离子对氨氯地平和ASP摄取的抑制作用[氨氯地平和ASP的IC50（以μM计），四乙铵（TEA）分别为98和30，西咪替丁分别为14和26，四戊铵（TPA）分别为7和2]。抑制Ca2+/钙调蛋白复合物（氨氯地平和ASP分别为-55±5%，n = 10和-63±2%，n = 15）、刺激蛋白激酶C（PKC）（分别为-54±5%，n = 14和-31±6%，n = 26）和蛋白激酶A（PKA）（分别为-16±5%，n = 16和-
18±4%，n = 40）可显著降低氨氯地平和ASP的摄取，而抑制磷脂酰肌醇3-激酶则可增加摄取（分别为+28±6%，n = 8和+55±17%，n = 其结果是Vmax显著降低（从160降至99个光子/秒），这部分可以通过使用FACScan流式细胞术测定的膜相关hOCT2减少来解释（-22±6%，n = 9）。数据表明，hOCT2的可饱和转运可以通过荧光底物氨氯地平和ASP来测量，并且两种底物的转运活性受到类似的调节。抑制Ca2+/钙调蛋白复合物会通过hOCT2的转运导致转运能力发生变化。
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