Uptake and desorption of hydrophilic compounds from human stratum corneum.

Small, polar compounds, both ionic and uncharged, partition into human stratum corneum immersed in aqueous solutions to an extent comparable to the water volume fraction of the tissue, then desorb in two phases. The fast phase has a time constant on the order of a few minutes, whereas the slow phase occurs over many hours. A physical model for this behavior involving a combination of tranverse diffusion through the tissue and lateral diffusion and exchange with skin appendages is presented. This concept is probed using excised human stratum corneum exposed to aqueous solutions of radiolabeled sodium chloride, tetraethyl ammonium bromide and mannitol, plus previously published data on six other compounds of varying molecular size and polarity. The fast phase desorption process becomes unimportant for lipophilic compounds. Slow phase desorption rates are size-selective, with larger species desorbing much more slowly than smaller ones. Interpreting the size-selectivity in terms of smooth cylindrical pores using the centerline approximation leads to an optimum pore radius of about 8-12Å, depending on the model chosen.

Role of organic cation transporters in the ocular disposition of its intravenously injected substrate in rabbits: implications for ocular drug therapy.

The present study was conducted to test the hypothesis; OCT may be active from blood-to-vitreous for the uptake of its substrates. Ocular uptake of Tetraethylammonium (TEA) across blood ocular barriers and the tissue distribution was evaluated in vivo in New Zealand albino rabbits after intravenous administration. Quinidine (blocker) pretreatment resulted in a significant (p < 0.05) reduction in the Area Under the Curve (AUC) of TEA in vitreous (4.2 fold) and aqueous humor (1.8 fold) as compared to the control group which supports the role of OCT in uptake transport of its substrate across Blood ocular barrier. The blockade of OCT also affected the elimination of its substrate resulting in increased plasma levels. In most of the tissues, OCT are functionally present from apical to basolateral. The gene expression studies also showed the presence of OCT1, OCTN1 and OCTN2 in various ocular tissues studied. The present findings suggest that OCT are functionally active in blood ocular barriers and involved in the transport of its substrate from blood-to-vitreous humor.

Potential pharmacokinetic role of organic cation transporters in modulating the transcorneal penetration of its substrates administered topically.

PURPOSE: We hypothesize organic cation transporters (OCT) may have a potential role in determining the pharmacokinetics and toxicity of organic cation drugs applied topically. Hence, in the present in vivo study, we attempted to evaluate the role of OCT in modulating the transport of its substrates after topical application. METHODS: New Zealand albino rabbits of either sex were used. Transcorneal penetration of OCT substrates tetraethylammonium and metformin after single instillation was evaluated in the absence and presence of OCT blockers (quinidine and atropine). Aqueous humor (AH) samples were collected through paracentesis amounting to 70-100 µl under topical anesthesia at various time intervals. The samples were subjected for estimation of both substrate as well as blocker concentrations using liquid chromatography mass spectrometry. RESULTS: Topical pre-treatment (30 min before substrate) of OCT blockers significantly decreased the transcorneal penetration of OCT substrates after single topical administration. The levels of blockers reaching AH in the presence of substrates were also modulated at 60 min after its administration as compared with its control. CONCLUSION: OCT are functionally active in the uptake of their substrates from tear to AH. Therefore, OCT in the corneal epithelium may be positioned from apical to basolateral. When administering their substrates/blockers topically, both may be competing for OCT for their uptake across the cornea, thereby decreasing the corneal penetration. Hence OCT can have a potential pharmacokinetic role in modulating the ocular bioavailability of their substrates administered topically, which are used as ocular therapeutics.

Human organic cation transporter 2 (hOCT2): Inhibitor studies using S2-hOCT2 cells.

Highly expressed in kidney and located on the basolateral membrane, human organic cation transporter 2 (hOCT2) can transport various compounds (i.e. drugs and toxins) into the proximal tubular cell. Using cultured proximal tubule cells stably expressing hOCT2 (i.e. S2-hOCT2 cells), we sought to probe different compound classes (e.g. analgesics, anti-depressants, anti-psychotics, disinfectant, herbicides, insecticides, local anesthetic, muscarinic acetylcholine receptor antagonist, sedatives, steroid hormone, stimulants and toxins) for their ability to inhibit (14)C-TEA uptake, a prototypical OCT2 substrate. Aconitine, amitriptyline, atropine, chlorpyrifos, diazepam, fenitrothion, haloperidol, lidocaine, malathion, mianserin, nicotine and triazolam significantly inhibited (14)C-TEA uptake; IC50 values were 59.2, 2.4, 2.0, 20.7, 32.3, 13.2, 32.5, 104.6, 71.1, 17.7, 52.8 and 65.5µM, respectively. In addition, aconitine, amitriptyline, atropine, chlorpyrifos, fenitrothion, haloperidol, lidocaine, and nicotine displayed competitive inhibition with Ki values of 145.6, 2.5, 2.4, 24.8, 16.9, 51.6, 86.8 and 57.7µM, respectively. These in vitro data support the notion that compounds pertaining to a wide variety of different drug classes have the potential to decrease renal clearance of drugs transported via hOCT2. Consequently, these data warrant additional studies to probe hOCT2 and its role to influence drug pharmacokinetics.

Evaluation of the functional importance of organic cation transporters on the ocular disposition of its intravitreally injected substrate in rabbits.

PURPOSE: To evaluate the functional role of organic cation transporters (OCT) and ocular tissue distribution of intravitreally injected OCT substrate tetraethylammonium (TEA) in presence of OCT blocker (quinidine). METHODS: New Zealand albino rabbits of either sex were used. Intravitreal quinidine pretreatment was made 30 min before the administration of TEA. Modulation of vitreous and ocular tissue kinetics of OCT substrate was evaluated with or without blocker pretreatment. Gamma scintigraphy was also performed to visualize the vitreous residence of (99m)Tc-labelled TEA in the presence and absence of blocker. RESULTS: Intravitreally injected quinidine did not significantly alter the ocular disposition of TEA. TEA showed less significant posterior elimination kinetics and slow anterior elimination which resulted in longer residence time of TEA in eye after intravitreal administration. CONCLUSIONS: Intravitreally injected OCT substrates may follow an anterior elimination pathway and prolonged residence time in vitreous humor. The present study shows that OCT may not be active from vitreous-to-blood route in the blood-retinal barrier.

Competitive inhibition of the luminal efflux by multidrug and toxin extrusions, but not basolateral uptake by organic cation transporter 2, is the likely mechanism underlying the pharmacokinetic drug-drug interactions caused by cimetidine in the kidney.

Cimetidine, an H2 receptor antagonist, has been used to investigate the tubular secretion of organic cations in human kidney. We report a systematic comprehensive analysis of the inhibition potency of cimetidine for the influx and efflux transporters of organic cations [human organic cation transporter 1 (hOCT1) and hOCT2 and human multidrug and toxin extrusion 1 (hMATE1) and hMATE2-K, respectively]. Inhibition constants (K(i)) of cimetidine were determined by using five substrates [tetraethylammonium (TEA), metformin, 1-methyl-4-phenylpyridinium, 4-(4-(dimethylamino)styryl)-N-methylpyridinium, and m-iodobenzylguanidine]. They were 95 to 146 µM for hOCT2, providing at most 10% inhibition based on its clinically reported plasma unbound concentrations (3.6-7.8 µM). In contrast, cimetidine is a potent inhibitor of MATE1 and MATE2-K with K(i) values (µM) of 1.1 to 3.8 and 2.1 to 6.9, respectively. The same tendency was observed for mouse Oct1 (mOct1), mOct2, and mouse Mate1. Cimetidine showed a negligible effect on the uptake of metformin by mouse kidney slices at 20 µM. Cimetidine was administered to mice by a constant infusion to achieve a plasma unbound concentration of 21.6 µM to examine its effect on the renal disposition of Mate1 probes (metformin, TEA, and cephalexin) in vivo. The kidney- and liver-to-plasma ratios of metformin both were increased 2.4-fold by cimetidine, whereas the renal clearance was not changed. Cimetidine also increased the kidney-to-plasma ratio of TEA and cephalexin 8.0- and 3.3-fold compared with a control and decreased the renal clearance from 49 to 23 and 11 to 6.6 ml/min/kg, respectively. These results suggest that the inhibition of MATEs, but not OCT2, is a likely mechanism underlying the drug-drug interactions with cimetidine in renal elimination.

Development and validation of a highly sensitive LC-MS/MS method for organic cation transporter (OCT) substrate tetraethylammonium (TEA) in rabbits.

Tetraethylammonium is widely used as a probe in organic cation transporters studies. A simple, highly sensitive, and specific method using direct protein precipitation was developed using Hydrophilic Interaction Liquid Chromatography coupled with positive electrospray ionization tandem mass spectrometry for the determination of tetraethylammonium (TEA) in rabbit plasma. Isocratic separation was achieved using a ZIC-HILIC column with acetonitrile and 5mM ammonium acetate in the ratio of 8:2 containing 0.1% formic acid. Acquisition was performed in multiple reaction monitoring mode with the transitions: m/z 130→100 and 130→86 for TEA and m/z 276.1→142.2 for internal standard (homatropine). This method was validated to determine selectivity, linearity, sensitivity, precision, accuracy, recovery and stability. A good linearity was found within a range of 1.53-784.6 ng/mL. The above method has been demonstrated for its capability to estimate the plasma levels of TEA after its topical instillation in rabbit eyes. This method provides an accurate, precise and sensitive tool for determining TEA levels for transporter studies.

A pore residue of the KCNQ3 potassium M-channel subunit controls surface expression.

KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) are the principal subunits underlying the potassium M-current, which exerts a strong control on neuronal excitability. KCNQ3 subunits coassemble with KCNQ2 to form functional heteromeric channels that are specifically transported to the axonal initial segment and nodes of Ranvier. In contrast, there is no evidence for functional homomeric KCNQ3 channels in neurons, and it appears that these are inefficiently trafficked to the plasma membrane. Among eukaryotic potassium channels, the KCNQ3 subunit is unusual because it has an alanine in place of a threonine at the pore inner vestibule, three residues upstream of the GYG signature sequence of the selectivity filter. This residue is critical for the potentiation of the current after heteromerization, but the mechanism is unknown. We report that the presence of this uncommon residue at position 315 has a strong impact on the stability of the homotetramers and on channel trafficking. Wild-type KCNQ3 expressed alone is retained within the endoplasmic reticulum, and this mechanism is overcome by the substitution of threonine for Ala315. KCNQ3 subunits require assembly with KCNQ2 to exit this compartment, whereas KCNQ3-A315T is no longer dependent on KCNQ2 to form channels that are efficiently trafficked to the plasma membrane. The presence of this alanine, therefore, plays an important role in regulating the subunit composition of functional M-channels expressed at the surface of neurons.

Potent and specific inhibition of mMate1-mediated efflux of type I organic cations in the liver and kidney by pyrimethamine.

This report describes a potent and selective inhibitor of multidrug and toxin extrusion (MATE) protein, pyrimethamine (PYR), and examines its effect on the urinary and biliary excretion of typical Mate1 substrates in mice. In vitro inhibition studies demonstrated that PYR is a potent inhibitor of mouse (m)Mate1 (K(i) = 145 nM) among renal organic cation transporters mOctn1 and mOctn2 (K(i) > 30 microM), mOct1 (K(i) = 3.6 microM), and mOct2 (K(i) = 6.0 microM). PYR inhibited the uptake of metformin by kidney brush-border membrane vesicles (BBMVs) (K(i) = 41 nM) and canalicular membrane vesicles in the presence of outward gradient of H+. PYR treatment significantly increased the kidney-to-plasma ratio of tetraethylammonium, and both the liver- and kidney-to-plasma ratios of metformin in mice, whereas it did not affect their plasma concentrations and urinary excretion rates. Furthermore, the plasma lactate concentration, a biomarker for inhibition of gluconeogenesis by metformin, was significantly higher in the PYR-treated group than in the control group. These results not only suggest the importance of mMate1 in the efflux of organic cations into the urine and bile in mice but also the importance of canalicular efflux mediated by MATE proteins for the therapeutic efficacy of metformin. PYR is a potent inhibitor of human (h)MATE1 and hMATE2-K (K(i) = 77 and 46 nM, respectively) and H+ and organic cation exchanger in human kidney BBMVs (K(i) = 31 nM) in the presence of outward gradient of H+. Taken together, PYR can be used as a potent probe inhibitor of human MATE transporters.

Iontophoretic transport of charged macromolecules across human sclera.

The mechanisms of transscleral iontophoresis have been investigated previously with small molecules in rabbit sclera. The objective of the present study was to examine transscleral iontophoretic transport of charged macromolecules across excised human sclera. Passive and 2mA iontophoretic transport experiments were conducted in side-by-side diffusion cells with human sclera. The effects of iontophoresis upon transscleral transport of model permeants bovine serum albumin (BSA) and polystyrene sulfonic acid (PSS) as well as a model drug bevacizumab (BEV) were determined. Passive and iontophoretic transport experiments of tetraethylammonium (TEA) and salicylic acid (SA) and passive transport experiments of the macromolecules served as the controls. The results of iontophoresis enhanced transport of TEA and SA across human sclera were consistent with those in a previous rabbit sclera study. For the iontophoretic transport of macromolecules BSA and BEV, higher iontophoretic fluxes were observed in anodal iontophoresis as compared to passive and cathodal iontophoresis. This suggests the importance of electroosmosis. For the polyelectrolyte PSS, higher iontophoretic flux was observed in cathodal iontophoresis compared to anodal iontophoresis. Both electroosmosis and electrophoresis affected iontophoretic fluxes of the macromolecules; the relative contributions of electroosmosis and electrophoresis were a function of molecular size and charge of the macromolecules.

Role of estrogen in renal handling of organic cation, tetraethylammonium: in vivo and in vitro studies.

This study examined the effect of estrogen (17beta-estradiol) on renal handling of organic cation, tetraethylammonium (TEA), both in vivo and in vitro. Clearance of TEA in ovariectomized (OVX) mice was increased by 38% above intact animals, which was able to be returned to control level by estrogen supplementation. The mechanism of this effect was examined in isolated mouse renal proximal tubules (mRPT), showing that [(3)H]-TEA uptake was higher in OVX mice than control, and estrogen supplementation returned uptake to normal level. Kinetics analysis of [(3)H]-TEA uptake indicated an increase in numbers of organic cation transporters in OVX mice but no change in substrate affinity. However, mRNA levels determined by quantitative real time polymerase chain reaction of the relevant transporters at basolateral (organic cation transporter (OCT)1, OCT2 and OCT3) and apical (organic cation/carnitine transporter (OCTN)1, OCTN2 and multidrug and toxin extrusion (MATE)1) membranes of OVX mice were not significantly changed, with only MATE2 mRNA of OVX mice being significantly decreased. The realization that estrogen status affects renal clearance of organic cations will be of importance when assessing the susceptibility of an individual to drug-induced toxicity.

Regulatory role of testosterone in organic cation transport: in vivo and in vitro studies.

The renal proximal tubule (RPT) plays a crucial role in organic cation (OC) secretion and has a major impact on pharmacokinetics of OC drugs. Secretory transport is vectorial. Thus, it involves transporters located at both basolateral and apical membranes. Although sex hormones have been shown to regulate OC transport, there is little data on the effect of testosterone on OC secretion in a whole animal. Therefore, we determined the clearance of tetraethylammonium (TEA), a model OC substrate, in intact and castrated male mice. Castration significantly decreased renal TEA secretion by 30%, and testosterone supplementation returned TEA secretion to control levels in castrated mice. The mechanism of this effect was further examined in isolated mouse renal proximal tubules (mRPT). TEA uptake in isolated mRPT from castrated mice was reduced by 36%. This effect was reversed in tubules from castrated mice supplemented with testosterone. Kinetic analysis of [(3)H]-TEA uptake in isolated mRPT showed a decreased V(max) with no change in K(m), implying that the decrease in transport rate was caused by lowering in the number of transporters in castrated mice rather than a change in transporter affinity. Quantitative real time polymerase chain reaction (real time PCR) revealed that organic cation transporter (OCT)2 is the major TEA transporter in male mice. Moreover, OCT2 mRNA level was significantly reduced after castration. Castrated mice also showed a modest increase in organic cation/carnitine transporter 1 (OCTN1) mRNA level, indicating that testosterone may also regulate apical OCTN1 expression. These data suggest that testosterone regulates transepithelial transport of OC through modulation of OCT2 expression in male mice.

The influence of porosity changes in human epidermal membrane during iontophoresis on the permeability enhancement of a model peptide.

PURPOSE: We tested the hypothesis that the increases in the porosity of the skin during iontophoresis would not significantly increase the transport of peptides due to the small size of electrically induced pores. To investigate this mechanistically, we used human epidermal membrane under constant voltage conditions, applying the Nernst-Planck equation to the transport of a small ionic solute, tetraethylammonium bromide (TEAB), and a model peptide, luteinizing hormone releasing hormone. METHODS: Steady-state flux of the drugs was determined under passive conditions and also during iontophoresis using constant DC voltages applied across side-by-side diffusion cells. Electrical conductance measurements were used to monitor the porosity changes that occur during electrical field application. RESULTS: Porosity increases observed in the membrane substantially increased the permeability enhancement of the small ionic solute TEAB. The permeability enhancement was well described by Nernst-Planck model predictions after porosity changes in the membrane were taken into account. Enhancement of luteinizing hormone releasing hormone under identical conditions was much less than TEAB. The porosity increases induced by iontophoresis had little or no effect on the permeability enhancement of the larger molecule. CONCLUSIONS: These findings closely parallel those reports that have found electrically induced pores to be significantly smaller than preexisting pores in the human epidermal membrane. The data obtained also support the view that iontophoresis-induced pores, alone, may provide limited benefit for macromolecule transport across the skin.

Effects of ionic strength on passive and iontophoretic transport of cationic permeant across human nail.

PURPOSE: Transport across the human nail under hydration can be modeled as hindered transport across aqueous pore pathways. As such, nail permselectivity to charged species can be manipulated by changing the ionic strength of the system in transungual delivery to treat nail diseases. The present study investigated the effects of ionic strength upon transungual passive and iontophoretic transport. METHODS: Transungual passive and anodal iontophoretic transport experiments of tetraethylammonium ion (TEA) were conducted under symmetric conditions in which the donor and receiver had the same ionic strength in vitro. Experiments under asymmetric conditions were performed to mimic the in vivo conditions. Prior to the transport studies, TEA uptake studies were performed to assess the partitioning of TEA into the nail. RESULTS: Permselectivity towards TEA was inversely related to ionic strength in both passive and iontophoretic transport. The permeability and transference number of TEA were higher at lower ionic strengths under the symmetric conditions due to increased partitioning of TEA into the nail. Transference numbers were smaller under the asymmetric conditions compared with their symmetric counterparts. CONCLUSIONS: The results demonstrate significant ionic strength effects upon the partitioning and transport of a cationic permeant in transungual transport, which may be instrumental in the development of transungual delivery systems.

Testosterone-independent down-regulation of Oct2 in the kidney medulla from a uranyl nitrate-induced rat model of acute renal failure: effects on distribution of a model organic cation, tetraethylammonium.

Although acute renal failure (ARF) has been an area of extensive research in recent decades, our understanding of ARF is far from complete. Organic cations (OCs) are primarily excreted via vectorial transport by various renal organic cation transporters (OCTs). It is reasonable to assume that ARF may alter the expression profiles of these transporters. In a rat ARF model, induction of ARF by uranyl nitrate (UN) treatment significantly decreased the levels of Oct2 (slc22a2) mRNA and protein in the kidney medulla. mRNA expression of the other OCTs was not appreciably altered. The plasma level of testosterone, a well-known regulator of Oct2, was not changed, suggesting that the Oct2 down-regulation is testosterone-independent. The effect of reduced Oct2 expression on the distribution of a model OC, tetraethylammonium (TEA), in various rat tissues including kidney cortex and kidney medulla was investigated during steady state plasma TEA concentrations. The steady state tissue-to-plasma (T/P) TEA ratio was decreased in the kidney medulla (approximately 15-fold) during ARF. These results indicate that, in a rat model of ARF, reduced Oct2 expression in the kidney medulla results in decreased distribution of TEA to the kidney medulla, thereby reducing renal clearance of TEA in UN-ARF rats.

Directional transcellular transport of bisoprolol in P-glycoprotein-expressed LLC-GA5-COL150 cells, but not in renal epithelial LLC-PK1 Cells.

To evaluate the mechanism responsible for the tubular secretion of bisoprolol, we compared transcellular transport of bisoprolol with that of tetraethylammonium (TEA), cimetidine, and quinidine across LLC-PK1 cell monolayers grown on porous membrane filters. TEA and cimetidine were actively transported in the basolateral-to-apical direction by the specific transport system. Pharmacokinetic analysis indicated that basolateral influx and apical efflux were cooperatively responsible for the directional transport of TEA and cimetidine. Lipophilic cationic drugs, quinidine, S-nicotine, and bisoprolol, significantly diminished basolateral influx and apical efflux clearance of cimetidine. However, transcellular transport of quinidine in the basolateral-to-apical direction was similar to that in the opposite direction in LLC-PK1 cells. In contrast, quinidine was transported actively in the basolateral-to-apical direction in P-glycoprotein-expressed LLC-GA5-COL150 cells. Pharmacokinetic analysis indicated that P-glycoprotein increased the apical efflux of quinidine and also decreased the apical influx of the drug. Basolateral-to-apical transport of bisoprolol was also similar to apical-to-basolateral transport in LLC-PK1 cells, whereas the drug was directionally transported from the basolateral to the apical side in LLC-GA5-COL150 cells. These results suggested that bisoprolol was not significantly transported via transport systems involved in the directional transport of TEA and cimetidine, but that P-glycoprotein was responsible for the directional transport of bisoprolol as well as quinidine in renal epithelial cells.

A novel variant of mouse MATE-1 H+/organic cation antiporter with a long hydrophobic tail.

Mammalian multidrug and toxic compound extrusion 1 (MATE1) are polyspecific H+-coupled exporters of organic cations (OCs) and responsible for excretion of metabolic waste products and xenobiotics. Here, we report a novel variant of mouse MATE1, mMATE1b, that has a long carboxyl terminal hydrophobic tail homologous to other MATE1 transporter proteins. Mouse MATE1b mediates tetraethylammonium (TEA) uptake with properties similar to that of mMATE1 and is localized in renal brush border membranes. Thus, mMATE1b is a functional variant of mMATE1 and seems to be the true counterpart to other MATE1 transporters.

Scintillation proximity assay for measuring uptake by the human drug transporters hOCT1, hOAT3, and hOATP1B1.

Increasing evidence suggests a key role of transport proteins in the pharmacokinetics of drugs. Within the solute carrier (SLC) family, various organic cation transporters (OCTs), organic anion transporters (OATs), and organic anion transporting polypeptides (OATPs) that interact with drug molecules have been identified. Traditionally, cellular uptake assays require multiple steps and provide low experimental throughput. We here demonstrate the use of a scintillation proximity approach to detect substrate uptake by human drug transporters in real time. HEK293 cells stably transfected with hOCT1, hOATP1B1, or hOAT3 were grown directly in Cytostar-T scintillating microplates. Confluent cell monolayers were incubated with 14C- or 3H-labeled transporter substrates. Cellular uptake brings the radioisotopes into proximity with the scintillation plate base. The resulting light emission signals were recorded on-line in a microplate scintillation counter. Results show time- and concentration-dependent uptake of 14C-tetraethylammonium, 3H-methylphenylpyridinium (HEK-hOCT1), 3H-estradiol-17beta-D-glucuronide (HEK-hOATP1B1), and 3H-estrone-3-sulfate (HEK-hOAT3), while no respective uptake was detected in empty vector-transfected cells. Km of 14C-tetraethylammonium and 3H-estrone-3-sulfate uptake and hOAT3 inhibition by ibuprofen and furosemide were similar to conventional dish uptake studies. The scintillation proximity approach is high throughput, amenable to automation and allows for identification of SLC transporter substrates and inhibitors in a convenient and reliable fashion, suggesting its broad applicability in drug discovery.

Effect of gramicidin on phospholipid-modified monolayers and on ion transfer at a liquid-liquid interface.

The interaction of hybrid lipid/gramicidin A (gA) monolayers with dextran sulfate (DS) and the effect of this interaction on ion transfer at a liquid-liquid interface is reported. The interfacial and physicochemical properties are studied with Langmuir-Blodgett (LB) and electrochemical techniques. The results obtained from compression isotherms demonstrate that the interactions between the different species in the hybrid monolayer vary according to the chemical nature of the lipid (hydrocarbon region and charge of the head group). Interfacial capacitance measured with AC voltammetry indicates that the DS chains form a rather flat and compact layer when adsorbed to either zwitterionic or negatively charged phospholipid monolayers, and that calcium, even at low concentrations, interacts with the monolayers. These results are successfully described by a model based on the solution of the Poisson-Boltzmann equation in the interfacial region. Ion transfer and interactions with the lipid/gA/DS-modified monolayers were also studied with electrochemical techniques. Admittance data show that although the studied ions are not using gA channels for the transfer through the lipid membranes, the incorporation of gA in the lipid domain and the adsorption of DS at the interface have a significant effect on ion transfer across the monolayers. This effect can be explained as a consequence of the modified surface charge and of the compactness of the lipid domain due to its interaction with gA and to calcium and DS adsorption at the interface. The ion-transfer rate, therefore, depends on the composition of the monolayer and the chemical nature of the ion.

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

Human organic cation transporter 2 (hOCT2) is essential for the renal tubular secretion of many toxic organic cations. Previously, of the cysteines (C437, C451, C470, and C474) that occur within transmembrane helices that comprise the hydrophilic cleft (proposed site of substrate binding), only C474 was accessible to maleimide-PEO(2)-biotin (hydrophilic thiol-reactive reagent), and covalent modification of this residue caused lower transport rates (Pelis RM, Zhang X, Dangprapai Y, Wright SH, J Biol Chem 281: 35272-35280, 2006). Thus it was hypothesized that the environmental contaminant Hg(2+) (as HgCl(2)) would interact with C474 to reduce hOCT2-mediated transport. Uptake of [(3)H]tetraethylammonium (TEA) into Chinese hamster ovary cells stably expressing hOCT2 was reduced in a concentration-dependent manner by HgCl(2), with an IC(50) of 3.9 +/- 0.11 microM. Treatment with 10 microM HgCl(2) caused a sixfold reduction in the maximal rate of TEA transport but did not alter the affinity of hOCT2 for TEA. To determine which cysteines interact with Hg(2+), a mutant with all four cleft cysteines converted to alanines (quadruple mutant), and four variants of this mutant, each with an individual cysteine restored, were created. The quadruple mutant was less sensitive to HgCl(2) than wild-type, whereas the C451- and C474-containing mutants were more sensitive than the quadruple mutant. Consistent with the HgCl(2) effect on transport, MTSEA-biotin only interacted with C451 and C474. These data indicate that C451 and C474 of hOCT2 reside in the aqueous milieu of the cleft and that interaction of Hg(2+) with these residues causes reduced TEA transport activity.