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The kinetics of solute transport shed light on the roles these processes play in cellular physiology, and the absolute values of the kinetic parameters that quantitatively describe transport are increasingly used to model its impact on drug clearance. However, accurate assessment of transport kinetics is challenging. Although most carrier-mediated transport is adequately described by the Michaelis-Menten equation, its use presupposes that the rates of uptake used in the analysis of maximal rates of transport (Jmax) and half-saturation constants (Kt) reflect true unidirectional rates of influx from known concentrations of substrate. Most experimental protocols estimate the initial rate of transport from net substrate accumulation determined at a single time point (typically between 0.5 and 5 min) and assume it reflects unidirectional influx. However, this approach generally results in systematic underestimates of Jmax and overestimates of Kt; the former primarily due to the unaccounted impact of efflux of accumulated substrate, and the latter due to the influence of unstirred water layers. Here, we describe the bases of these timedependent effects and introduce a computational model that analyzes the time course of net substrate uptake at several concentrations to calculate Jmax and Kt for unidirectional influx, taking into account the influence of unstirred water layers and mediated efflux. This method was then applied to calculate the kinetics of transport of 1-methyl-4-phenylpryridinium and metformin by renal organic cation transporter 2 as expressed in cultured Chinese hamster ovary cells. NEW & NOTEWORTHY Here, we describe a mathematical model that uses the time course of net substrate uptake into cells from several increasing concentrations to calculate unique kinetic parameters [maximal rates of transport (Jmax) and half-saturation constants (Kt)] of the process. The method is the first to take into consideration the common complicating factors of unstirred layers and carrier-mediated efflux in the experimental determination of transport kinetics. [ABSTRACT FROM AUTHOR]

The fluorescent derivative of glucose, 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)-amino]-D-glucose (2NBDG), is a widely used surrogate reagent to visualize glucose uptake in live cells at single cell resolution. Using CRISPR-Cas9 gene editing in 5TGM1 myeloma cells, we demonstrate that ablation of the glucose transporter gene Slc2a1 abrogates radioactive glucose uptake but has no effect on the magnitude or kinetics of 2NBDG import. Extracellular 2NBDG, but not NBD-fructose was transported by primary plasma cells into the cytoplasm suggesting a specific mechanism that is unlinked from glucose import and that of chemically similar compounds. Neither excess glucose nor pharmacological inhibition of GLUT1 impacted 2NBDG uptake in myeloma cells or primary splenocytes. Genetic ablation of other expressed hexose transporters individually or in combination with one another also had no impact on 2NBDG uptake. Ablation of the genes in the Slc29 and Slc35 families of nucleoside and nucleoside sugar transporters also failed to impact 2NBDG import. Thus, cellular uptake of 2NBDG is not necessarily a faithful indicator of glucose transport and is promoted by an unknown mechanism. [ABSTRACT FROM AUTHOR]

Recent investigations involving intact rabbit renal proximal tubules indicated that organic anion transporter 3 (OAT3) may be involved in the transport of 2,3-dimercapto-1-propanesulfonic acid (DMPS). Therefore, we evaluated the interaction of OAT3 with DMPS to determine the effect of OAT3 on basolateral DMPS uptake. We used stably transfected HEK293 cells expressing human and rabbit orthologs of the exchanger OAT1 and OAT3. Using 6-carboxyfluorescein (6-CF) as a substrate, the [IC.sub.50] determinations for reduced DMPS (DMPSH) revealed a stronger interaction with OAT1 than with OAT3 (rbOAT1, 123.3 ± 13.7; hOAT1, 85.1 ± 8.8; rbOAT3, 171.7 ± 22.3; and hOAT3, 172.2 ± 36.4 µmol/L). However, inhibition of 6-CF uptake by the oxidized form of DMPS (DMPSS), the main form of DMPS in the blood, showed a greater affinity for OAT3 (rbOAT1, 237.4 ± 23; hOAT1, 104.6 ± 13.1; rbOAT3, 52.4 ± 7.6; and hOAT3, 31.6 ± 6.6 µmol/L). To determine whether DMPSH and DMPSS are substrates for OAT3, we performed efflux studies with [[sup.14]C]glutarate and inwardly directed gradients of glutarate. The inhibitors trans-stimulated the efflux of [[sup.14]C]glutarate, suggesting that OAT3 may be able to transport both forms of DMPS. On the basis of the substantial interaction of OAT3 with DMPSS, we conclude that OAT3 represents the dominant basolateral player in renal detoxification processes resulting from use of DMPS. Key words: OAT1, mercury, kidney, nephrotoxicity, heavy metal, secretion, detoxification. De recentes etudes impliquant des experiences sur des tubules contournes proximaux de lapins intacts ont indique que le transporteur d'anions organiques 3 (OAT3) pourrait jouer un role dans le transport du DMPS. Nous avons donc evalue l'interaction entre l'OAT3 et le DMPS pour determiner l'impact de l'OAT3 sur la capture basolaterale de DMPS. Nous avons utilise; des cellules HEK293 transfectes de maniere stable, exprimant les orthologues des echangeurs OAT1 et OAT3 chez les humains et les lapins. Nous avons utilise: la 6-carboxyfluoresceine (6-CF) comme substrat. Les valeurs d'[IC.sub.50] pour le DMPS reduit (DMPSH) ont revele une interaction plus forte avec OAT1 qu'avec OAT3 (rbOAT1, 123,3 ± 13,7 µmol/L; hOAT1, 85,1 ± 8 µmol/L; rbOAT3, 171,7 ± 22,3 µmol/L; et hOAT3, 172,2 ± 36,4 µmol/L). Toutefois, l'inhibition de la capture de 6-CF par la forme oxyde de DMPS (DMPSS), la principale forme de DMPS dans le sang, a montre une preference pour OAT3 (rbOAT1, 237,4 ± 23 µmol/L; hOAT1, 104,6 ± 13,1 µmol/L; rbOAT3, 52,4 ± 7,6 µmol/L; et hOAT3, 31,6 ± 6,6 µmol/L). Pour determiner si le DMPSH et le DMPSS sont des substrats d'OAT3, nous avons examine; l'efflux avec du [[sup.14]C]glutarate et des gradients du glutarate diriges vers l'interieur. Les inhibiteurs ont frans-stimule l'efflux de [[sup.14]C]glutarate, ce qui laisse supposer que l'OAT3 pourrait transporter les deux formes de DMPS. Compte tenu de la forte interaction entre l'OAT3 et le DMPS, nous concluons que l'OAT3 represente le principal joueur basolateral dans les processus de detoxication renale associesa l'utilisation de DMPS. Mots-cles: OAT1, mercure, rein, nephrotoxocite, metal lourd, secretion, detoxication. [Traduit par la Redaction], Introduction Some heavy metals, including lead, cadmium, and mercury, are accumulated in renal proximal tubule (RPT) cells during urinary excretion, causing chronic renal diseases (Sabolic 2006; Bridges and Zalups 2005). [...]

The mammalian members of the Multidrug And Toxin Extruder family, i.e., MATE1 and MATE2-K, are suspected of mediating the luminal step in renal secretion of organic cations. The 1,000+ prokaryotic/fungal/plant MATE family members are predicted to have 12 transmembrane helices (TMHs), whereas MATE1/2-K appear to have an additional (13th) COOH-terminal helix. Here, we determined whether rabbit MATE 1 has an external COOH terminus, consistent with the presence of 13 TMHs. A V5 epitope tag at the COOH terminus of MATE1 was freely accessible to external V5 antibody, whereas tags at the [NH.sub.2] terminus, or at sites of truncation within the long cytoplasmic loop between predicted TMHs 12 and 13, were only accessible to the V5 antibody following permeabilization of the membrane. The truncated mutants that lacked TMH13 still retained transport activity, indicating that the terminal helix was not necessary for transport function. Cells that expressed a mutant lacking only TMH13 displayed similar [K.sub.t] and [J.sub.max] values to those of the full-length protein, although when normalized to protein expressed at the plasma membrane, the transport rate of the mutant was organic cation; transport; proximal tubule; kidney; luminal

An electroneutral organic cation (OC)/proton exchanger in the apical membrane of proximal tubules mediates the final step of renal OC excretion. Two members of the multidrug and toxin extrusion family, MATE1 and MATE2-K, were recently identified in human and rodent kidney and proposed to be the molecular basis of renal OC/[H.sup.+] exchange. To take advantage of the comparative value of the large database on the kinetic and selectivity characteristics of OC/[H.sup.+] exchange that exists for rabbit kidney, we cloned rbMATE1 and rbMATE2-K. The rabbit homologs have 75% (MATE1) and 74% (MATE2-K) amino acid identity to their human counterparts (and 51% identity with each other), rbMATE1 and rbMATE2-K exhibited [H.sup.+] gradient-dependent uptake and efflux of tetraethylammonium (TEA) when expressed in Chinese hamster ovary cells. Both transporters displayed similar affinities for selected compounds [[IC.sub.50] values within 2-fold for TEA, l-methyl-4-phenylpyridinium, and quinidine] and very different affinities for others ([IC.sub.50] values differing by 8- to 80-fold for choline and cimetidine, respectively). These results indicate that rbMATE1 and rbMATE2-K are multispecific OC/[H.sup.+] exchangers with similar, but distinct, functional characteristics. Overall, the selectivity of MATE 1 and MATE2-K correlated closely with that observed in rabbit renal brush-border membrane vesicles. organic cation; transport; tetraethylammonium; proximal tubule; kidney doi:10.1152/ajprenal.00102.2007

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-PE[O.sub.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.sup.20] (as Hg[Cl.sub.2]) would interact with C474 to reduce hOCT2-mediated transport. Uptake of [[sup.3]H]tetraethyl-ammonium (TEA) into Chinese hamster ovary cells stably expressing hOCT2 was reduced in a concentration-dependent manner by Hg[Cl.sub.2], with an I[C.sub.50] of 3.9 [+ or -] 0.11 [micro]M. Treatment with 10 [micro]M Hg[Cl.sub.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.sup.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 Hg[Cl.sub.2] than wild-type, whereas the C451- and C474-containing mutants were more sensitive than the quadruple mutant. Consistent with the Hg[Cl.sub.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.sup.2+] with these residues causes reduced TEA transport activity. Hg[Cl.sub.2]; organic cation transport; renal proximal tubule; cysteine accessibility; tetraethylammonium doi: 10.1152/ajprenal.00496.2006.

OCT2, an organic cation transporter critical for removal of many drugs and toxins from the body, contains consensus sites for N-glycosylation at amino acid position 71, 96, and 112. However, the extent to which these sites are glycosylated by the cell, and the influence glycosylation has on OCT2 function, remains unknown. To address these issues, the acquisition of N-glycosylation was disrupted by mutating the amino acid asparagine (N) to glutamine (Q) at these sites in the rabbit ortholog of OCT2, which was expressed in Chinese hamster ovary cells. Disruption of N-glycosylation followed by Western blotting indicated that each site is indeed glycosylated and that OCT2 contains no other sites of Nglycosylation. Plasma membrane expression (determined by surface biotinylation) of the N112Q mutant, but not N71Q or N96Q mutants, was fourfold lower than that of wild-type OCT2, and unglycosylated OCT2 (NT1Q/N96Q/N112Q) was sequestered in an unidentified intracellular compartment. The N71Q, N96Q, and N112Q mutants had a higher affinity (~2-fold) for tetraethylammonium (TEA). Maximum transport rate was reduced in the N96Q (3-fold) and N112Q (5-fold) mutants, but not the N71Q mutant, and unglycosylated OCT2 failed to transport TEA (associated with its absence in the plasma membrane). Whereas the reduction in maximum transport rate of the N112Q mutant is consistent with its reduced plasma membrane expression, the lower rate of the N96Q mutant, which appeared to traffic properly, suggests that glycosylation at N96 increases the transporter turnover number. SLC22A2; organic cation; rabbit

Relative contribution of OAT and OCT transporters to organic electrolyte transport in rabbit proximal tubule. Am J Physiol Renal Physiol 287: F999-F1010, 2004. First published July 13, 2004; doi:10.1152/ajprenal. 00156.2004.--We compared the characteristics of several cloned rabbit organic electrolyte (OE) transporters expressed in cultured cells with their behavior in intact rabbit renal proximal tubules (RPT) to determine the contribution of each to basolateral uptake of the weak acid ochratoxin A (OTA) and the weak base cimetidine (CIM). The activity of organic anion transporters OAT1 and OAT3 proved to be distinguishable because OATI had a high affinity for PAH ([K.sub.t] of 20 [micro]M) and did not support estrone sulfate (ES) transport, whereas OAT3 had a high affinity for ES ([K.sub.t] of 4.5 [micro]M) and a weak interaction with PAH (I[C.sub.50] > 1 mM). In contrast, both transporters robustly accumulated OTA. Intact RPT also accumulated OTA, with OAT1 and OAT3 each responsible for ~50%: ES and PAH each reduced uptake by ~50%, and the combination of the two eliminated mediated OTA uptake. The weak base CIM was transported by OAT3 ([K.sub.1] of 80 [micro]M) and OCT2 ([K.sub.t] of 2 [micro]M); OCT1 had a comparatively low affinity for CIM, and CIM uptake by OATI was equivocal. Intact RPT accumulated CIM, with TEA and ES reducing CIM uptake by 20 and 75%, respectively, suggesting that OAT3 plays a quantitatively more significant role in CIM uptake in the early proximal tubule than OCT1/2. In single S2 segments of RPT, ES and TEA each blocked ~50% of CIM uptake. Thus the fractional contribution of different OE transporters to renal secretion is influenced by their affinity for substrate and relative expression level in RPT. kidney

The organic cation (OC) transporters OCT1 and OCT2 are suspected of mediating substrate entry from the blood into proximal tubule cells as the first step in renal secretion of OCs. We examined the contribution of each process in different rabbit renal proximal tubule (RPT) segments, taking advantage of the fact that rabbit orthologs of OCT1 and OCT2 can be distinguished by the high affinity of the former for tyramine (TYR) and of the latter for cimetidine (CIM). We verified that TEA uptake, for which both transporters share a similar affinity, is relatively constant in all three segments (apparent inhibitory constant of 33, 74, and 30 [micro]M and maximal rate of mediated TEA uptake of 0.8, 1.0, and 1.2 pmol*[mm.sup.-1]*[min.sup.-1] in S1, S2, and S3, respectively). In the S1 segment, TYR was a more effective inhibitor of TEA uptake than CIM (I[C.sub.50] values of 39 and 328 [micro]M, respectively), implicating OCT1 as the predominant pathway for TEA transport. The opposite profiles were noted in the S2 segment (I[C.sub.50] values of 302 and 20 [micro]M for TYR and CIM, respectively) and S3 segment (I[C.sub.50] values of 2,900 and 54 [micro]M for TYR and CIM, respectively), suggesting that OCT2 is the predominant TEA transporter in the later portion of RPT. TEA sufficient to saturate OCT1 and OCT2 blocked only 37% of mediated amantadine transport in the S2 segment, confirming the functional presence of at least one additional OC transporter (perhaps OCT3). These data indicate that renal OC transport involves the concerted activity of a suite of transport processes. organic cation; cimetidine; kidney; organic cation transporter 1; organic cation transporter 2; tetraethylammonium

I. Introduction II. Renal Organic Cation Transport A. Overview of the physiological characteristics of renal OC transport B. Molecular characteristics of renal OC transporters C. Physiological integration of renal OC [...]

A strategy was developed to determine the distribution of activity mediated by the organic cation (OC) transporters OCT1 and OCT2 in rabbit renal proximal tubule (RPT). Both transporters displayed similar affinities for tetraethylammonium (TEA; in CHO-K1 cells, TEA concentrations that resulted in half-maximal transport were 19.9 and 34.5 [micro]M for OCT1 and OCT2, respectively). Similarly, some OCs showed little capacity to discriminate between the two processes (I[C.sub.50] values for ephedrine of 13.6 and 24.2 [micro]M for OCT1 and OCT2, respectively). However, OCT2 had a higher affinity for cimetidine and [2-(4-nitro-2,1,3-benzoxadiazol-7-yl) aminoethyl]trimethylammonium (NBD-TMA; 1.3 and 1.4 [micro]M, respectively) than did OCT1 (97.3 and 108 [micro]M, respectively). Conversely, OCT1 had a higher affinity for tyramine and pindolol than did OCT2 (21.2 and 2.4 vs. 361 and 50 [micro]M, respectively). We designated these as 'discriminatory inhibitors' and used them to determine the relative contribution of OCT1 and OCT2 for TEA transport in single S2 segments of rabbit RPT. Cimetidine and NBD-TMA were high-affinity inhibitors of TEA transport in S2 segments (median I[C.sub.50] values of 12.3 and 1.4 [micro]M, respectively); in comparison, tyramine and pindolol were low-affinity inhibitors (265 and 69.3 [micro]M, respectivelyt. These I[C.sub.50] values were sufficiently close to those for OCT2 to support the conclusion that TEA transport in the S2 segment of rabbit RPT is dominated by OCT2. However, the profile of inhibition of tyramine (an OCT1-selective substrate) transport in single S2 segments indicated that, despite a comparatively low level of expression, OCT1 can play a dominant role in the uptake of selected OC substrates. organic cation; transport; tetraethylammonium; cimetidine; kidney