Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity

Pelis, Ryan M.; Dangprapai, Yodying; Wunz, Theresa M.; Wright, Stephen H.

doi:10.1152/ajprenal.00496.2006

Cited by 17 publications

(21 citation statements)

References 30 publications

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“…We routinely use CHO cells as an expression system to study OCT2 transport (e.g., Suhre et al, 2005;Pelis et al, 2007Pelis et al, , 2012Harper and Wright, 2013). However, it is more common to use HEK293 cells as an expression for studying OCT-mediated transport (Nies et al, 2011).…”

Section: Resultsmentioning

confidence: 99%

“…HEK293 cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum with 100 mg/ml blasticidin. Cells that stably expressed OCT2 were prepared using methods described previously (Pelis et al, 2007). The OCT2-expressing cells were grown and maintained under hygromycin pressure (100 mg/ml) in plastic cell culture flasks at 37°C in a humidified atmosphere with 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

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Substrate-Dependent Ligand Inhibition of the Human Organic Cation Transporter OCT2

Belzer

Morales

Jagadish

et al. 2013

J Pharmacol Exp Ther

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Organic cation transporter 2 (OCT2) mediates the initial step in renal secretion of organic cations: uptake from the blood, across the basolateral membrane, and into the renal proximal tubule cells. Because of its potential as a target for unwanted drug-drug interactions (DDIs), considerable attention has been directed toward understanding the basis of OCT2 selectivity. These studies typically assess selectivity based on ligand inhibition profiles for OCT2-mediated transport of a probe substrate. However, little attention has been given to the potential influence of the substrate on the profile of ligand inhibition. Here we compared the IC 50 values obtained for a set of structurally distinct inhibitors against OCT2-mediated transport of three structurally distinct substrates: 1-methyl-4-phenylpyridinium (MPP); metformin; and a novel fluorescent substrate, N,N,Ntrimethyl-2-[methyl (7-nitrobenzo[c][l,2,5]oxadiazol-4-yl)amino] ethanaminium iodide (NBD-MTMA). The median IC 50 value for inhibition of MPP transport was 9-fold higher than that for inhibition of metformin transport. Similarly, the median IC 50 value for inhibition of MPP transport was 5-fold higher than that for NBD-MTMA transport. However, this was not a systematic difference in inhibitory efficacy; the ratio of IC 50 values, MPP versus NBD-MTMA, ranged from 88-fold (ipratropium) to 0.3-fold (metformin). These data show that 1) the choice of OCT2 substrate significantly influences both quantitative and qualitative inhibitory interactions with cationic drugs; and 2) ligand interactions with OCT2 are not restricted to competition for a common ligand binding site, consistent with a binding surface characterized by multiple, possibly overlapping interaction sites. Development of predictive models of DDIs with OCT2 must take into account the substrate dependence of ligand interaction with this protein.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Substrate-Dependent Ligand Inhibition of the Human Organic Cation Transporter OCT2

Belzer

Morales

Jagadish

et al. 2013

J Pharmacol Exp Ther

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“…In addition to PAH, MeHg also markedly decreased uptake of an organic cation, TEA, by kidney slices in all dietary groups. Although the exact mechanism behind this is not clear, inorganic mercury ion has been shown to interact with cysteine residues of human organic cation transporter 2 (hOCT2), and reduce hOCT2-mediated transport of TEA into Chinese hamster ovary cells stably expressing hOCT2 (Pelis et al 2007).…”

Section: Discussionmentioning

confidence: 99%

Dietary fats altered nephrotoxicity profile of methylmercury in rats

Jin

Lok

Caldwell

et al. 2008

J of Applied Toxicology

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Weanling male Sprague-Dawley rats were administered semi-purified isocaloric diet containing soy oil (SO), seal oil (SE), docosahexaenoic acid (DHA), fish oil (FO) or lard (LA) for 28 days, and then gavaged with 0, 1 or 3 mg MeHg kg(-1) body weight per day and fed the same diet for 14 days. Serum and 24 h urine samples were collected on the day of necropsy, and analyzed for markers of kidney function and diseases. Kidney slices were analyzed for para-amino-hippurate (PAH) and tetraethylammonium (TEA) uptake, total mercury and MeHg content, and examined for pathological lesions. Total mercury and MeHg contents increased significantly and dose-dependently in all dietary groups. MeHg significantly increased relative kidney weight in all groups, serum creatinine in all except SO group, serum uric acid in the DHA and LA groups, serum Mg in all except the LA group, and urinary protein in the SO group. MeHg significantly decreased serum urea nitrogen in SE, FO and LA groups, urinary creatinine in the DHA group, PAH uptake in all except the SE group, and TEA uptake in all groups. MeHg caused nephrosis in all dietary groups. MeHg also significantly increased neutrophil counts in all except the SE group, decreased serum albumin and triglyceride in all except the DHA group, and increased serum total cholesterol in all groups, suggesting a nephrotic syndrome-like outcome. These results confirmed that kidney tubules are major targets of MeHg nephrotoxicity. Treatment with dietary fats did not prevent, but rather altered the profile of, nephrotoxicity of MeHg in rats.

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“…10). Recently, it was shown that cysteine 451 in human OCT2 was accessible to modification by HgCl 2 that was added to the extracellular side of the plasma membrane (25). The authors concluded that cysteine 451 resides in the aqueous milieu.…”

Section: Discussionmentioning

confidence: 99%

Identification of cysteines in rat organic cation transporters rOCT1 (C322, C451) and rOCT2 (C451) critical for transport activity and substrate affinity

Sturm¹,

Gorboulev²,

Gorbunov³

et al. 2007

American Journal of Physiology-Renal Physiology

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Effects of the sulfhydryl reagent methylmethanethiosulfonate (MMTS) on functions of organic cation transporters (OCTs) were investigated. Currents induced by 10 mM choline [I(max(choline))] in Xenopus laevis oocytes expressing rat OCT1 (rOCT1) were increased four- to ninefold after 30-s incubation with 5 mM MMTS whereas I(max(choline)) by rat OCT2 was 70% decreased. MMTS activated the rOCT1 transporter within the plasma membrane without changing stoichiometry between translocated charge and cation. After modification of oocytes expressing rOCT1 or rOCT2 with MMTS, I(0.5(choline)) values for choline-induced currents were increased. For rOCT1 it was shown that MMTS increased I(0.5) values for different cations by different degrees. Mutagenesis of individual cysteine residues in rOCT1 revealed that modification of cysteine 322 in the large intracellular loop, and of cysteine 451 at the transition of the transmembrane alpha-helix (TMH) 10 to the short intracellular loop between the TMH 10 and 11 is responsible for the observed effects of MMTS. After replacement of cysteine 451 by methionine, the IC(50(choline)) for choline to inhibit MPP uptake by rOCT1 was increased whereas the I(0.5(choline)) value for choline-induced current remained unchanged. At variance, in double mutant Cys322Ser, Cys451Met, I(0.5(choline)) was increased compared with rOCT1 wild-type whereas in the single mutant Cys322Ser I(0.5(choline)) was not changed. The data suggest that modification of rOCT1 at cysteines 322 and 451 leads to an increase in turnover. They indicate that cysteine 451 in rOCT1 interacts with the large intracellular loop and that cysteine 451 in both rOCT1 and rOCT2 is critical for the affinity of choline.

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Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity

Cited by 17 publications

References 30 publications

Substrate-Dependent Ligand Inhibition of the Human Organic Cation Transporter OCT2

Substrate-Dependent Ligand Inhibition of the Human Organic Cation Transporter OCT2

Dietary fats altered nephrotoxicity profile of methylmercury in rats

Identification of cysteines in rat organic cation transporters rOCT1 (C322, C451) and rOCT2 (C451) critical for transport activity and substrate affinity

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