Involvement of Human Multidrug and Toxic Compound Extrusion (MATE) Transporters in Testosterone Transport.

Multidrug and toxic compound extrusion (MATE) transporters are primarily expressed in the kidneys and liver, where they contribute to the excretion of organic cations. Our previous study suggested that pig MATE2 (class III) participates in testosterone secretion from Leydig cells. In humans, it is unclear which MATE class is involved in testosterone transport. In this study, we aimed to clarify whether human MATE1 (hMATE1) or human MATE2K (hMATE2K) mediates testosterone transport. To confirm that testosterone inhibits transporter-mediated tetraethylammonium (TEA) uptake, a cis-inhibition assay was performed using cells that stably expressed hMATE1 or hMATE2K. Docking simulations were performed to characterize differences in the binding of hMATE1 and hMATE2K to testosterone. Transport experiments in LLC-PK1 cells that stably expressed hMATE1 were used to test whether hMATE1 mediates testosterone transport. We detected differences between the amino acid sequences of the substrate-binding sites of hMATE1 and hMATE2K that could potentially be involved in testosterone binding. Testosterone and estradiol inhibited TEA uptake mediated by hMATE1 but not that mediated by hMATE2K. Transport experiments in LLC-PK1 cells indicated that testosterone might be transported via hMATE1. This study suggested that hMATE1, but not hMATE2K, is involved in human testosterone transport.

Carbon nanoparticles enhance potassium uptake via upregulating potassium channel expression and imitating biological ion channels in BY-2 cells.

BACKGROUND: Carbon nanoparticles (CNPs) have been reported to boost plant growth, while the mechanism that CNPs enhanced potassium uptake for plant growth has not been reported so far. RESULTS: In this study, the function that CNPs promoted potassium uptake in BY-2 cells was established and the potassium accumulated in cells had a significant correlation with the fresh biomass of BY-2 cells. The K+ accumulation in cells increased with the increasing concentration of CNPs. The K+ influx reached high level after treatment with CNPs and was significantly higher than that of the control group and the negative group treated with K+ channels blocker, tetraethylammonium chloride (TEA+). The K+ accumulation was not reduced in the presence of CNPs inhibitors. In the presence of potassium channel blocker TEA+ or CNPs inhibitors, the NKT1 gene expression was changed compared with the control group. The CNPs were found to preferentially transport K+ than other cations determined by rectification of ion current assay (RIC) in a conical nanocapillary. CONCLUSIONS: These results indicated that CNPs upregulated potassium gene expression to enhance K+ accumulation in BY-2 cells. Moreover, it was speculated that the CNPs simulated protein of ion channels via bulk of carboxyl for K+ permeating. These findings will provide support for improving plant growth by carbon nanoparticles.

Plasmodium falciparum chloroquine resistance transporter is a H+-coupled polyspecific nutrient and drug exporter.

Extrusion of chloroquine (CQ) from digestive vacuoles through the Plasmodium falciparum CQ resistance transporter (PfCRT) is essential to establish CQ resistance of the malaria parasite. However, the physiological relevance of PfCRT and how CQ-resistant PfCRT gains the ability to transport CQ remain unknown. We prepared proteoliposomes containing purified CQ-sensitive and CQ-resistant PfCRTs and measured their transport activities. All PfCRTs tested actively took up tetraethylammonium, verapamil, CQ, basic amino acids, polypeptides, and polyamines at the expense of an electrochemical proton gradient. CQ-resistant PfCRT exhibited decreased affinity for CQ, resulting in increased CQ uptake. Furthermore, CQ competitively inhibited amino acid transport. Thus, PfCRT is a H(+)-coupled polyspecific nutrient and drug exporter.

Bacterial production and reconstitution in proteoliposomes of Solanum lycopersicum CAT2: a transporter of basic amino acids and organic cations.

KEY MESSAGE: The vacuolar SlCAT2 was cloned, over-produced in E. coli and reconstituted in proteoliposomes. Arg, Ornithine and Lys were identified as substrates. Unexpectedly, also the organic cations Tetraethylammonium and Acetylcholine were transported indicating involvement of SlCAT2 in signaling. In land plants several transporters are involved in ion and metabolite flux across membranes of cells or intracellular organelles. The vacuolar amino acid transporter CAT2 from Solanum lycopersicum was investigated in this work. SlCAT2 was cloned from tomato flower cDNA, over-produced in Escherichia coli and purified by Nichel-chelating chromatography. For functional studies, the transporter was reconstituted in proteoliposomes. Competence of SlCAT2 for Arg transport was demonstrated measuring uptake of [3H]Arg in proteoliposomes which was trans-stimulated by internal Arg or ornithine. Uptake of [3H]Ornithine and [3H]Lys was also detected at lower efficiency with respect to [3H]Arg. Transport was activated by the presence of intraliposomal ATP suggesting regulation by the nucleotide. The prototype for organic cations tetraethylammonium (TEA) was also transported by SlCAT2. However, scarce reciprocal inhibition between TEA and Arg was found, while the biguanide metformin was able to strongly inhibit uptake of both substrates. These findings suggest that amino acids and organic cations may interact with the transporter through different functional groups some of which are common for the two types of substrates. Interestingly, reconstituted SlCAT2 showed competence for acetylcholine transport, which was also inhibited by metformin. Kinetics of Arg and Ach transport were performed from which Km values of 0.29 and 0.79 mM were derived, respectively.

Unstirred Water Layers and the Kinetics of Organic Cation Transport.

PURPOSE: Unstirred water layers (UWLs) present an unavoidable complication to the measurement of transport kinetics in cultured cells, and the high rates of transport achieved by overexpressing heterologous transporters exacerbate the UWL effect. This study examined the correlation between measured Jmax and Kt values and the effect of manipulating UWL thickness or transport Jmax on the accuracy of experimentally determined kinetics of the multidrug transporters, OCT2 and MATE1. METHODS: Transport of TEA and MPP was measured in CHO cells that stably expressed human OCT2 or MATE1. UWL thickness was manipulated by vigorous reciprocal shaking. Several methods were used to manipulate maximal transport rates. RESULTS: Vigorous stirring stimulated uptake of OCT2-mediated transport by decreasing apparent Kt (Ktapp) values. Systematic reduction in transport rates was correlated with reduction in Ktapp values. The slope of these relationships indicated a 1500 µm UWL in multiwell plates. Reducing the influence of UWLs (by decreasing either their thickness or the Jmax of substrate transport) reduced Ktapp by 2-fold to >10-fold. CONCLUSIONS: Failure to take into account the presence of UWLs in experiments using cultured cells to measure transport kinetics can result in significant underestimates of the apparent affinity of multidrug transporters for substrates.

Components of foods inhibit a drug exporter, human multidrug and toxin extrusion transporter 1.

Human multidrug and toxic compounds extrusion transporter 1 (hMATE1/SLC47A1) is a H(+)-coupled organic cation exporter responsible for the final step of excretion of various xenobiotics at the kidney and liver. In this study, effects of dietary constituents on hMATE1 mediated drug transport were examined to evaluate possible food-drug interactions. Bergamottin inhibited hMATE1 mediated tetraethyl ammonium transport activity, with a Ki of 98.7 µM. Coumarins, flavonols, and catechin inhibited hMATE1 activity. Among 23 compounds tested, isorhamnetin was the strongest inhibitor of hMATE1 with the Ki of 0.32 µM in a competitive manner. Since isorhamnetin is abundant in Ginkgo biloba that is widely used for herbal supplements, the findings suggest the potential hMATE1 related food-drug interactions.

TREK-1 currents in smooth muscle cells from pregnant human myometrium.

The mechanisms governing maintenance of quiescence during pregnancy remain largely unknown. The current study characterizes a stretch-activated, tetraethylammonium-insensitive K(+) current in smooth muscle cells isolated from pregnant human myometrium. This study hypothesizes that these K(+) currents can be attributed to TREK-1 and that upregulation of this channel during pregnancy assists with the maintenance of a negative cell membrane potential, conceivably contributing to uterine quiescence until full term. The results of this study demonstrate that, in pregnant human myometrial cells, outward currents at 80 mV increased from 4.8 ± 1.5 to 19.4 ± 7.5 pA/pF and from 3.0 ± 0.8 to 11.8 ± 2.7 pA/pF with application of arachidonic acid (AA) and NaHCO3, respectively, causing intracellular acidification. Similarly, outward currents were inhibited following application of 10 µM fluphenazine by 51.2 ± 9.8% after activation by AA and by 73.9 ± 4.2% after activation by NaHCO3. In human embryonic kidney (HEK-293) cells stably expressing TREK-1, outward currents at 80 mV increased from 91.0 ± 23.8 to 247.5 ± 73.3 pA/pF and from 34.8 ± 8.9 to 218.6 ± 45.0 pA/pF with application of AA and NaHCO3, respectively. Correspondingly, outward currents were inhibited 89.5 ± 2.3% by 10 µM fluphenazine following activation by AA and by 91.6 ± 3.4% following activation by NaHCO3. Moreover, currents in human myometrial cells were activated by stretch and were reduced by transfection with small interfering RNA or extracellular acidification. Understanding gestational regulation of expression and gating of TREK-1 channels could be important in determining appropriate maintenance of uterine quiescence during pregnancy.

Multiple mechanisms of ligand interaction with the human organic cation transporter, OCT2.

OCT2 is the entry step for organic cation (OC) secretion by renal proximal tubules. Although many drugs inhibit OCT2 activity, neither the mechanistic basis of their inhibition nor their transport status is generally known. Using representatives of several structural classes of OCT2-inhibitory ligands described recently (Kido Y, Matsson P, Giacomini KM. J Med Chem 54: 4548-4558, 2011), we determined the kinetic basis of their inhibition of 1-methyl-4-phenylpyridinium (MPP) transport into Chinese hamster ovary cells that stably expressed hOCT2. The "cluster II" inhibitors (which contain known OCT2 substrates) metformin and cimetidine interacted competitively with MPP. However, other cluster II compounds, including tetraethylammonium (TEA), diphenidol and phenyltoloxamine, were mixed-type inhibitors of MPP transport (i.e., decreasing J(max) and increasing K(t)). A cluster III (neutral steroid) representative, adrenosterone, and a cluster I (large, flexible cation) representative, carvedilol, displayed noncompetitive inhibitory profiles. Competitive counterflow (CCF) was used to determine whether the inhibitory ligands served as substrates of hOCT2. Carvedilol (cluster I) and adrenosterone (cluster III) did not support CCF, consistent with the prediction that members of these structural classes are likely to be nontransported inhibitors of OCT2. The cluster II representatives MPP, metformin, cimetidine, and TEA all supported CCF, consistent with independent assessments of their OCT2-mediated transport. However, the other cluster II representatives, diphenidol and phenyltoloxamine, failed to support CCF, suggesting that neither compound is transported by OCT2. An independent assessment of diphenidol transport (using liquid chromatography with tandem mass spectroscopy) confirmed this observation. The results underscore the caution required for development of predictive models of ligand interaction with multidrug transporters.

Functional significance of conserved cysteines in the human organic cation transporter 2.

The significance of conserved cysteines in the human organic cation transporter 2 (hOCT2), namely the six cysteines in the long extracellular loop (loop cysteines) and C474 in transmembrane helix 11, was examined. Uptake of tetraethylammonium (TEA) and 1-methyl-4-phenypyridinium (MPP) into Chinese hamster ovary cells was stimulated >20-fold by hOCT2 expression. Both cell surface expression and transport activity were reduced considerably following mutation of individual loop cysteines (C51, C63, C89, C103, and C143), and the C89 and C103 mutants had reduced Michaelis constants (K(t)) for MPP. The loop cysteines were refractory to interaction with thiol-reactive biotinylation reagents, except after pretreatment of intact cells with dithiothreitol or following cell membrane solubilization. Reduction of disulfide bridge(s) did not affect transport, but labeling the resulting free thiols with maleimide-PEO(2)-biotin did. Mutation of C474 to an alanine or phenylalanine did not affect the K(t) value for MPP. In contrast, the K(t) value associated with TEA transport was reduced sevenfold in the C474A mutant, and the C474F mutant failed to transport TEA. This study shows that some but not all of the six extracellular loop cysteines exist within disulfide bridge(s). Each loop cysteine is important for plasma membrane targeting, and their mutation can influence substrate binding. The effect of C474 mutation on TEA transport suggests that it contributes to a TEA binding surface. Given that TEA and MPP are competitive inhibitors, the differential effects of C474 modification on TEA and MPP binding suggest that the binding surfaces for each are distinct, but overlapping in area.

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.

Reconstitution in liposomes of the functionally active human OCTN1 (SLC22A4) transporter overexpressed in Escherichia coli.

The hOCTN1 (human organic cation transporter 1) overexpressed in Escherichia coli and purified by Ni-chelating chromatography has been reconstituted in liposomes by detergent removal with a batch-wise procedure. The reconstitution was optimized with respect to the protein concentration, the detergent/phospholipid ratio and the time of incubation with Amberlite XAD-4 resin. Time-dependent [(14)C]tetraethylammonium, [(3)H]carnitine or [(3)H]ergothioneine uptake was measured in proteoliposomes with activities ratios of 8:1.3:1 respectively. Optimal activity was found at pH 8.0. The transport depended on intraliposomal ATP. [(14)C]tetraethylammonium transport was inhibited by several compounds. The most effective were acetyl-choline and γ-butyrobetaine, followed by acetylcarnitine and tetramethylammonium. Reagents such as pyridoxal 5-phosphate, MTSES [sodium (2-sulfonatoethyl) methanethiosulfonate] and mercurials strongly inhibited the transport. From kinetic analysis of tetraethylammonium transport a K(m) of 0.77 mM was calculated. Acetylcholine and γ-butyrobetaine behaved as competitive inhibitors of TEA (tetraethylammonium) transport with K(i) values of 0.44 and 0.63 mM respectively.

Genetic variants of organic cation transporter 1 (OCT1) and OCT2 significantly reduce lamivudine uptake.

The study sought to investigate the effect of genetic variants of OCT1 (OCT1-P283L and -P341L) and OCT2 (OCT2-T199I, -T201M and -A270S), which were identified in a Korean population, on the transport of lamivudine in vitro and to compare the substrate dependent effects of OCT1 and OCT2 variants with 1-methyl-4-phenylpyridinium (MPP+), tetraethyl ammonium (TEA), metformin and lamivudine as substrates for these transporters. When the transport kinetics of lamivudine uptake in oocytes overexpressing OCT1 and OCT2 wild-type (WT) and variant proteins were measured, lamivudine uptake mediated by OCT1-WT was saturable, and uptake was decreased in oocytes expressing OCT1-P283L and -P341L variants compared with that in OCT1-WT. The Clint of lamivudine in oocytes expressing OCT1-P283L was decreased by 85.1% compared with OCT1-WT, whereas it was decreased by 48.7% in oocytes expressing OCT1-P341L. The Clint of lamivudine in oocytes expressing OCT2-T199I, -T201M and -A270S was decreased by 86.2%, 88.9% and 73.6%, respectively, compared with OCT2-WT. When comparing various substrates such as MPP+, TEA, metformin and lamivudine, the effects of the OCT1 genetic polymorphisms on their uptake were not identical. However, contrary to the case of OCT1, the uptake of MPP+, TEA, metformin and lamivudine in oocytes expressing OCT2-T199I, -T201M and -A270S variants was decreased significantly compared with that in oocytes expressing OCT2-WT. In conclusion, the effect of genetic variations of OCT1 and OCT2 on the uptake of MPP+, TEA, metformin and lamivudine was substrate-dependent.

Active Pharmaceutical Ingredient Uptake by Zebrafish (Danio rerio) Oct2 (slc22a2) Transporter Expressed in Xenopus laevis Oocytes.

Uptake of active pharmaceutical ingredients (APIs) across the gill epithelium of fish is via either a passive or facilitated transport process, with the latter being more important at the lower concentrations more readily observed in the environment. The solute carrier (SLC) 22A family, which includes the organic cation transporter OCT2 (SLC22A2), has been shown in mammals to transport several endogenous chemicals and APIs. Zebrafish oct2 was expressed in Xenopus oocytes and the uptake of ranitidine, propranolol, and tetraethylammonium characterized. Uptake of ranitidine and propranolol was time- and concentration-dependent with a km and Vmax for ranitidine of 246 µM and 45 pmol/(oocyte × min) and for propranolol of 409 µM and 190 pmol/(oocyte × min), respectively. Uptake of tetraethylammonium (TEA) was inhibited by propranolol, amantadine, and cimetidine, known to be human OCT2 substrates, but not quinidine or ranitidine. At external media pH 7 and 8 propranolol uptake was 100-fold greater than at pH 6; pH did not affect ranitidine or TEA uptake. It is likely that cation uptake is driven by the electrochemical gradient across the oocyte. Uptake kinetics parameters, such as those derived in the present study, coupled with knowledge of transporter localization and abundance and API metabolism, can help derive pharmacokinetic models. Environ Toxicol Chem 2022;41:2993-2998. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Brain natriuretic peptide modulates the delayed rectifier outward K(+) current and promotes the proliferation of mouse Schwann cells.

Brain natriuretic peptide (BNP) may act as a neuromodulator via its associated receptors (natriuretic peptide receptors, NPRs) in the central nervous system (CNS), but few studies have reported its activity in the peripheral nervous system (PNS). In this study, we observed that BNP increased the tetraethylammonium chloride (TEA)-sensitive delayed rectifier outward potassium current (I(K)) in mouse Schwann cells (SCs) using whole-cell recording techniques. At concentrations of 1-100 nM, BNP reversibly activated I(K) in a dose-dependent manner, with modulating its steady-state activation and inactivation properties. The effect of BNP on I(K) was abolished by preincubation with the specific antagonist of NPR-A, and could not be mimicked by application of NPR-C agonist. These results were supported by immunocytochemical findings indicating that NPR-A was expressed in SCs. The application of 8-Br-guanosine 3',5'-monophosphate (8-Br-cGMP) mimicked the effect of BNP on I(K), but BNP was unable to further increase I(K) after the application of cyclic guanosine monophosphate (cGMP). Genistein blocked I(K) and also completely eliminated the effects of BNP and cGMP on I(K). The selective K(V)2.1 subunit blocker, Jingzhaotoxin-III (JZTX-III), reduced I(K) amplitude by 30%, but did not abolish the increase effect of BNP on I(K) amplitude. In addition, BNP significantly stimulated SCs proliferation and this effect could be partly inhibited by TEA. Together these results suggest that BNP modulated I(K) probably via cGMP- and tyrosine kinase-dependent pathways by activation of NPR-A. This effect of BNP on I(K) in SCs might partly explain its effect on cell proliferation.

Sound stimulation modulates high-threshold K(+) currents in mouse auditory brainstem neurons.

The auditory system provides a valuable experimental model to investigate the role of sensory activity in regulating neuronal membrane properties. In this study, we have investigated the role of activity directly by measuring changes in medial nucleus of the trapezoid body (MNTB) neurons in normal hearing mice subjected to 1-h sound stimulation. Broadband (4-12 kHz) chirps were used to activate MNTB neurons tonotopically restricted to the lateral MNTB, as confirmed by c-Fos-immunoreactivity. Following 1-h sound stimulation a substantial increase in Kv3.1b-immunoreactivity was measured in the lateral region of the MNTB, which lasted for 2 h before returning to control levels. Electrophysiological patch-clamp recordings in brainstem slices revealed an increase in high-threshold potassium currents in the lateral MNTB of sound-stimulated mice. Current-clamp and dynamic-clamp experiments showed that MNTB cells from the sound-stimulated mice were able to maintain briefer action potentials during high-frequency firing than cells from control mice. These results provide evidence that acoustically driven auditory activity can selectively regulate high-threshold potassium currents in the MNTB of normal hearing mice, likely due to an increased membrane expression of Kv3.1b channels.

Selective transport of monoamine neurotransmitters by human plasma membrane monoamine transporter and organic cation transporter 3.

The plasma membrane monoamine transporter (PMAT) and organic cation transporter 3 (OCT3) are the two most prominent low-affinity, high-capacity (i.e., uptake(2)) transporters for endogenous biogenic amines. Using the Flp-in system, we expressed human PMAT (hPMAT) and human OCT3 (hOCT3) at similar levels in human embryonic kidney 293 cells. Parallel and detailed kinetics analysis revealed distinct and seemingly complementary patterns for the two transporters in transporting monoamine neurotransmitters. hPMAT is highly selective toward serotonin (5-HT) and dopamine, with the rank order of transport efficiency (V(max)/K(m)) being: dopamine, 5-HT ≫ histamine, norepinephrine, epinephrine. The substrate preference of hPMAT toward these amines is substantially driven by large (up to 15-fold) distinctions in its apparent binding affinities (K(m)). In contrast, hOCT3 is less selective than hPMAT toward the monoamines, and the V(max)/K(m) rank order for hOCT3 is: histamine > norepinephrine, epinephrine > dopamine >5-HT. It is noteworthy that hOCT3 demonstrated comparable (≤2-fold difference) K(m) toward all amines, and distinctions in V(max) played an important role in determining its differential transport efficiency toward the monoamines. Real-time reverse transcription-polymerase chain reaction revealed that hPMAT is expressed at much higher levels than hOCT3 in most human brain areas, whereas hOCT3 is selectively and highly expressed in adrenal gland and skeletal muscle. Our results suggest that hOCT3 represents a major uptake(2) transporter for histamine, epinephrine, and norepinephrine. hPMAT, on the other hand, is a major uptake(2) transporter for 5-HT and dopamine and may play a more important role in transporting these two neurotransmitters in the central nervous system.

The "flipped" state in E71A-K+-channel KcsA exclusively alters the channel gating properties by tetraethylammonium and phosphatidylglycerol.

Mutation E71A in the bacterial K(+)-channel KcsA has been shown to abolish the activation-coupled inactivation of KcsA via significant alterations of the peptide backbone in the vicinity of the selectivity filter. In the present study, we examined channel-blocking behavior of KcsA-E71A by tetraethylammonium (TEA) from both the extra- and the intracellular sides. First, we found that E71A is inserted either in cis or trans orientation in a planar lipid bilayer; however, it exhibits only one orientation in proteoliposomes as determined by extravesicular partial chymotrypsin digestion. Second, E71A exhibits a lower extracellular TEA affinity and is more sensitive to intracellular TEA compared to wild-type KcsA, which apparently has >50-fold higher affinity for extracellular TEA and approximately 2.5-fold lower affinity for intracellular TEA compared to E71A. In additional experiments, we investigated the influence of negatively charged phosphatidylglycerol (PG) on channel-gating properties in phosphatidylcholine lipid bilayers. It was found that high PG content decreases the single-channel conductance and increases the channel open time and open probability. Taken together, our data suggest that the "flipped" conformation of the selectivity filter present in E71A allows weaker extracellular and stronger intracellular TEA binding, whereas higher PG content decreases channel conductivity and stabilizes the channel open "flipped" state via electrostatic interaction in the proximity of the channel pore.

The effect of acyclovir on the tubular secretion of creatinine in vitro.

BACKGROUND: While generally well tolerated, severe nephrotoxicity has been observed in some children receiving acyclovir. A pronounced elevation in plasma creatinine in the absence of other clinical manifestations of overt nephrotoxicity has been frequently documented. Several drugs have been shown to increase plasma creatinine by inhibiting its renal tubular secretion rather than by decreasing glomerular filtration rate (GFR). Creatinine and acyclovir may be transported by similar tubular transport mechanisms, thus, it is plausible that in some cases, the observed increase in plasma creatinine may be partially due to inhibition of tubular secretion of creatinine, and not solely due to decreased GFR. Our objective was to determine whether acyclovir inhibits the tubular secretion of creatinine. METHODS: Porcine (LLC-PK1) and human (HK-2) renal proximal tubular cell monolayers cultured on microporous membrane filters were exposed to [2-14C] creatinine (5 µM) in the absence or presence of quinidine (1E+03 µM), cimetidine (1E+03 µM) or acyclovir (22-89 µM) in incubation medium. RESULTS: Results illustrated that in evident contrast to quinidine, acyclovir did not inhibit creatinine transport in LLC-PK1 and HK-2 cell monolayers. CONCLUSIONS: The results suggest that acyclovir does not affect the renal tubular handling of creatinine, and hence, the pronounced, transient increase in plasma creatinine is due to decreased GFR, and not to a spurious increase in plasma creatinine.

Efficacy of external tetraethylammonium block of the KcsA potassium channel: molecular and Brownian dynamics studies.

Blockade of the KcsA potassium channel by externally applied tetraethylammonium is investigated using molecular dynamics calculations and Brownian dynamics simulations. In KcsA, the aromatic rings of four tyrosine residues located just external to the selectivity filter create an attractive energy well or a binding cage for a tetraethylammonium molecule. We first investigate the effects of re-orienting the four tyrosine residues such that the centers of the aromatic rings face the tetraethylammonium molecule directly. Then, we systematically move the residues inward in both orientations so that the radius of the binding cage formed by them becomes smaller. For each configuration, we construct a one-dimensional free energy profile by bringing in a tetraethylammonium molecule from the external reservoir toward the selectivity filter. The free energy profile is then converted to a one-dimensional potential energy profile, taking the available space between the tyrosine residues and the tetraethylammonium molecule into account. Incorporating this potential energy profile into the Brownian dynamics algorithm, we determine the conductance properties of the channel under various conditions, construct the current-tetraethylammonium-concentration curve and compare it with the experimentally determined inhibitory constant k(i) for externally applied tetraethylammonium. We show that the experimentally determined binding affinity for externally applied tetraethylammonium can be replicated when each of the four tyrosine residues is moved inward by about 0.7 angstroms, irrespective of orientation of their aromatic rings.

Genetic variants in multidrug and toxic compound extrusion-1, hMATE1, alter transport function.

hMATE1 (human multidrug and toxin compound extrusion-1; encoded by SLC47A1) is thought to have an important function in the renal and hepatic elimination of drugs, endogenous compounds and environmental toxins. The goals of this study were to identify genetic variants of hMATE1 and to determine their effects on hMATE1 transport function. We identified four synonymous and six nonsynonymous, coding region variants in DNA samples from 272 individuals (68 Caucasians, 68 African Americans, 68 Asian Americans and 68 Mexican Americans). The overall prevalence of hMATE1 nonsynonymous variants was relatively low with three singleton variants and three variants having allele frequencies > or =2% in a specific ethnic group. The nonsynonymous hMATE1 variants were constructed and stably transfected into HEK-293 cells. Uptake studies using four known hMATE1 substrates (paraquat, metformin, tetraethylammonium and oxaliplatin) were performed in cells transfected with hMATE1 reference or variants. We found that two singleton variants, G64D and V480M, produced a complete loss of function for all four tested substrates whereas three polymorphic variants (allele frequencies > or =2%), L125F, V338I and C497S, significantly altered the transport function in a substrate-dependent manner. Confocal microscopy studies were consistent with functional studies suggesting that the altered function of the variants was due to altered localization to the plasma membrane. These data suggest that nonsynonymous variants in hMATE1 may alter drug disposition and ultimately affect clinical drug response.