Characterization of regulatory mechanisms and states of human organic cation transporter 2

Abstract

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.

The hOCT1 SNPs M420del and M408V alter imatinib uptake and M420del modifies clinical outcome in imatinib-treated chronic myeloid leukemia

“…2,20 To investigate whether the hOCT1 variants were functionally active, the hOCT1 substrates 14 C-TEA ϩ (Moravek Biochemicals) at 5M and ASP ϩ (Sigma-Aldrich) at 500M were used as described previously. 9,10,21 hOCT1 transcript levels For hOCT1 mRNA level analysis, 13 "test" and 53 "validation" samples were taken from newly diagnosed, treatment-naive chronic-phase patients who subsequently received imatinib. hOCT1 transcript levels were quantified by real-time quantitative RT-PCR on a LightCycler (Roche Diagnostics).…”

Fluorescent organic cations for human OCT2 transporters screening: uptake in CHO cells stably expressing hOCT2

“…As Km is a measure of transporter-substrate affinity [22][23], higher K m implies poorer binding/affinity. Biermann et al, [2] used Km to compare the affinities of different fluorescent compounds to hOCT2 in transfected HEK-293. According to the authors, amiloride and 4-Di-1-ASP had significantly higher uptake rates in hOCT2 cells compared to wild type cells.…”

“…cimetidine, procainamide, quinidine, salbutamol) and endogenous bioactive amines (e.g. dopamine, histamine, Nmethylnicotinamide, choline) are organic cations [1][2]. These charged molecules are transported across cell epithelia by carrier-mediated transport proteins, mainly organic cation transporters (OCTs) and proton/cation antiporters (MATEs) [3][4][5][6].…”

“…Based on this fact, the US Food and Drugs Administration agency (FDA) and European Medicines Agency (EMA) require that new drug candidates be screened for OCT2 activity during early stages of drug development if active renal excretion is envisaged [12][13]. The transporter is mainly expressed in the basolateral membrane of renal proximal tubules and is also expressed in small intestine, lung, placenta, thymus, brain and the inner ear [2,[14][15]. Although the role of the transporter in other tissues than the kidney is still under investigation, the OCT2 is critical for the detoxification and elimination of drugs and endogenous compounds from the systemic circulation and thus a significant determinant of drug response and sensitivity [2,4].…”

Characterization of the Basal Promoter Element of Human Organic Cation Transporter 2 Gene

“…So far, the pharmacokinetic significance of hOCT2 has been mainly demonstrated by expression and functional transport analyses. Regarding the regulatory aspects, transport activity of hOCT2 is controlled by protein phosphorylation, which is caused by protein kinase C, protein kinase A, phosphatidylinositol 3-kinase, and calcium/calmodulin complex, and the substrate affinity, plasma membrane expression of hOCT2, or both were altered (Ç etinkaya et al, 2003;Biermann et al, 2006). In contrast, transcriptional mechanisms of the hOCT2 gene have not been elucidated.…”