Valentin Gorboulev

Na+-d-glucose Cotransporter SGLT1 is Pivotal for Intestinal Glucose Absorption and Glucose-Dependent Incretin Secretion

To clarify the physiological role of Na+-d-glucose cotransporter SGLT1 in small intestine and kidney, Sglt1−/− mice were generated and characterized phenotypically. After gavage of d-glucose, small intestinal glucose absorption across the brush-border membrane (BBM) via SGLT1 and GLUT2 were analyzed. Glucose-induced secretion of insulinotropic hormone (GIP) and glucagon-like peptide 1 (GLP-1) in wild-type and Sglt1−/− mice were compared. The impact of SGLT1 on renal glucose handling was investigated by micropuncture studies. It was observed that Sglt1−/− mice developed a glucose-galactose malabsorption syndrome but thrive normally when fed a glucose-galactose–free diet. In wild-type mice, passage of d-glucose across the intestinal BBM was predominantly mediated by SGLT1, independent the glucose load. High glucose concentrations increased the amounts of SGLT1 and GLUT2 in the BBM, and SGLT1 was required for upregulation of GLUT2. SGLT1 was located in luminal membranes of cells immunopositive for GIP and GLP-1, and Sglt1−/− mice exhibited reduced glucose-triggered GIP and GLP-1 levels. In the kidney, SGLT1 reabsorbed ∼3% of the filtered glucose under normoglycemic conditions. The data indicate that SGLT1 is 1) pivotal for intestinal mass absorption of d-glucose, 2) triggers the glucose-induced secretion of GIP and GLP-1, and 3) triggers the upregulation of GLUT2.

A glucose sensor hiding in a family of transporters

We have examined the expression and function of a previously undescribed human member (SGLT3/SLC5A4) of the sodium/glucose cotransporter gene family (SLC5) that was first identified by the chromosome 22 genome project. The cDNA was cloned and sequenced, confirming that the gene coded for a 659-residue protein with 70% amino acid identity to the human SGLT1. RT-PCR and Western blotting showed that the gene was transcribed and mRNA was translated in human skeletal muscle and small intestine. Immunofluorescence microscopy indicated that in the small intestine the protein was expressed in cholinergic neurons in the submucosal and myenteric plexuses, but not in enterocytes. In skeletal muscle SGLT3 immunoreactivity colocalized with the nicotinic acetylcholine receptor. Functional studies using the Xenopus laevis oocyte expression system showed that hSGLT3 was incapable of sugar transport, even though SGLT3 was efficiently inserted into the plasma membrane. Electrophysiological assays revealed that glucose caused a specific, phlorizin-sensitive, Na+-dependent depolarization of the membrane potential. Uptake assays under voltage clamp showed that the glucose-induced inward currents were not accompanied by glucose transport. We suggest that SGLT3 is not a Na+/glucose cotransporter but instead a glucose sensor in the plasma membrane of cholinergic neurons, skeletal muscle, and other tissues. This points to an unexpected role of glucose and SLC5 proteins in physiology, and highlights the importance of determining the tissue expression and function of new members of gene families.

Electrogenic Properties and Substrate Specificity of the Polyspecific Rat Cation Transporter rOCT1

The previously cloned rat cation transporter rOCT1 detected in renal proximal tubules and hepatocytes (Gründemann, D., Gorboulev, V., Gambaryan, S., Veyhl, M., and Koepsell, H. (1994) Nature 372, 549-552) was expressed in Xenopus oocytes, and transport properties were analyzed using tracer uptake studies and electrophysiological measurements. rOCT1 induced highly active transport of a variety of cations, including the classical substrates for cation transport, such as N-1-methylnicotinamide, 1-methyl-4-phenylpyridinium (MPP), and tetraethylammonium (TEA), but also the physiologically important choline. In oocytes rOCT1 also mediated efflux of MPP, which could be trans-stimulated by MPP and TEA. Cation transport via rOCT1 was electrogenic. In voltage-clamped oocytes, transport of TEA and choline via rOCT1 produced inwardly directed currents, which were independent of extracellular ion composition or pH. The choline- and TEA-induced currents were voltage-dependent at nonsaturating concentrations, and the apparent affinity of these cations was decreased at depolarized voltages. Other substrates transported by rOCT1 were the polyamines spermine and spermidine. Interestingly, the previously described potent inhibitors of rOCT1, cyanine 863, quinine, and D-tubocurarine were substrates themselves. The data indicate that rOCT1 is an effective transport system that is responsible for electrogenic uptake of a wide variety of organic cations into epithelial cells of renal proximal tubules and hepatocytes.

Identification of genetic variations of the human organic cation transporter hOCT1 and their functional consequences

By systematic mutation screening of the polyspecific organic cation transporter hOCT1 (SLC22A1) in 57 Caucasians, 25 genetic variations were identified and further analysed for population frequency. Five mutations resulting in the amino acid changes Arg61Cys, Cys88Arg, Phe160Leu, Gly401Ser, and Met420del, with respective allele frequencies of 9.1, 0.6, 22, 3.2, and 16%, were functionally characterized upon expression in Xenopus oocytes. Phe160Leu and Met420del exhibited substrate affinities and selectivites identical to hOCT1 wild-type. In contrast, uptake of 0.1 microm [3H]1-methyl-4-phenylpyridinium ([3H]MPP) by Arg61Cys, Cys88Arg and Gly401Ser were reduced to 30, 1.4 and 0.9% compared to wild-type, respectively. Since transport of 1 microm [3H]serotonin by Cys88Arg and Gly401Ser was reduced to only 13 and 12% of wild-type, these mutants exhibit a changed substrate selectivity. The data show that the mutants Arg61Cys, Cys88Arg and Gly401Ser could affect the disposition of OCT1 substrates and as a consequence may alter the duration and intensity of effects of drugs and neurotransmitters which are substrates for hOCT1.

Molecular pharmacology of organic cation transporters in kidney.

The homeostasis of endogenous organic cations, such as choline and N P-methylnicotinamide, monoamine neurotransmitters, cationic drugs, such as cimetidine, morphine, quinine and amantadine, and of cationic xenobiotics is controlled by reabsorption and excretion in the small intestine, by metabolic conversion and excretion in the liver, and by reabsorption and excretion in the kidney [1–6]. In the kidney organic cations may be ultrafiltrated in the glomeruli and reabsorbed or secreted in renal tubules. Hydrophilic cations are readily ultrafiltrated, however, more hydrophobic cations are bound to plasma membrane proteins that may not permeate the filtration barrier. Secretion and reabsorption of organic cations have been described in renal proximal tubules but may also occur in distal tubules or collecting ducts [7–11]. These processes have been functionally characterized by measurements using intact kidneys or tissue slices [10, 12–14], by microperfusion experiments using proximal tubules [7, 9, 15], by uptake measurements using isolated tubules or cells [8, 11, 16, 17], and by uptake measurements using membrane vesicles from proximal tubules [18–28]. In luminal and basolateral membranes of proximal tubules different transport processes have been demonstrated, although all the involved transport systems may not have been identified by these measurements because a variety of different polyspecific organic cation transporters with overlapping substrate specificity are expressed [5]. After the cloning of the first organic cation transporter rOCT1 from a rat kidney library [29], an increasing number of homologous cation transporters have been identified and it has been shown that the renal anion transporters also belong to this new protein family. The functional characterization of the expressed transporters and their immunohistochemical localization in the kidney has begun [30–35]. This review describes the molecular structure of renal organic cation transporters and homologous gene products. The transport properties and renal localization of the cloned cation transporters are summarized and their presumed role in renal cation handling of endogenous and exogenous cations is discussed.

Transport of Lamivudine [(-)-β-l-2′,3′-Dideoxy-3′-thiacytidine] and High-Affinity Interaction of Nucleoside Reverse Transcriptase Inhibitors with Human Organic Cation Transporters 1, 2, and 3

Nucleoside reverse transcriptase inhibitors (NRTIs) need to enter cells to act against the HIV-1. Human organic cation transporters (hOCT1–3) are expressed and active in CD4+ T cells, the main target of HIV-1, and have been associated with antiviral uptake in different tissues. In this study, we examined whether NRTIs interact and are substrates of hOCT in cells stably expressing these transporters. Using [3H]N-methyl-4-phenylpyridinium, we found a high-affinity interaction among abacavir [[(1S,4R)-4-[2-amino-6-(cyclopropylamino)purin-9-yl]-cyclopent-2-enyl]methanol sulfate] (ABC); <0.08 nM], azidothymidine [3′-azido-3′-deoxythymidine (AZT); <0.4 nM], tenofovir disoproxil fumarate (<1.0 nM), and emtricitabine (<2.5 nM) and hOCTs. Using a wide range of concentrations of lamivudine [(-)-β-l-2′,3′-dideoxy-3′-thiacyitidine (3TC)], we determined two different binding sites for hOCTs: a high-affinity site (Kd1 = 12.3–15.4 pM) and a low-affinity site (Kd2 = 1.9–3.4 mM). Measuring direct uptake of [3H]3TC and inhibition with hOCT substrates, we identified 3TC as a novel substrate for hOCT1, 2, and 3, with hOCT1 as the most efficient transporter (Km = 1.25 ± 0.1 mM; Vmax = 10.40 ± 0.32 nmol/mg protein/min; Vmax/Km = 8.32 ± 0.40 μl/mg protein/min). In drug-drug interaction experiments, we analyzed cis-inhibition of [3H]3TC uptake by ABC and AZT and found that 40 to 50% was inhibited at low concentrations of the drugs (Ki = 22–500 pM). These data reveal that NRTIs experience a high-affinity interaction with hOCTs, suggesting a putative role for these drugs as modulators of hOCT activity. Finally, 3TC is a novel substrate for hOCTs and the inhibition of its uptake at low concentrations of ABC and AZT could have implications for the pharmacokinetics of 3TC.

Transport of Lamivudine (3TC) and High-Affinity Interaction of Nucleoside Reverse Transcriptase Inhibitors With Human Organic Cation Transporters 1, 2, and 3

Nucleoside reverse transcriptase inhibitors (NRTIs) need to enter cells to act against the HIV-1. Human organic cation transporters (hOCT1–3) are expressed and active in CD4+ T cells, the main target of HIV-1, and have been associated with antiviral uptake in different tissues. In this study, we examined whether NRTIs interact and are substrates of hOCT in cells stably expressing these transporters. Using [3H]N-methyl-4-phenylpyridinium, we found a high-affinity interaction among abacavir [[(1S,4R)-4-[2-amino-6-(cyclopropylamino)purin-9-yl]-cyclopent-2-enyl]methanol sulfate] (ABC); <0.08 nM], azidothymidine [3′-azido-3′-deoxythymidine (AZT); <0.4 nM], tenofovir disoproxil fumarate (<1.0 nM), and emtricitabine (<2.5 nM) and hOCTs. Using a wide range of concentrations of lamivudine [(-)-β-l-2′,3′-dideoxy-3′-thiacyitidine (3TC)], we determined two different binding sites for hOCTs: a high-affinity site (Kd1 = 12.3–15.4 pM) and a low-affinity site (Kd2 = 1.9–3.4 mM). Measuring direct uptake of [3H]3TC and inhibition with hOCT substrates, we identified 3TC as a novel substrate for hOCT1, 2, and 3, with hOCT1 as the most efficient transporter (Km = 1.25 ± 0.1 mM; Vmax = 10.40 ± 0.32 nmol/mg protein/min; Vmax/Km = 8.32 ± 0.40 μl/mg protein/min). In drug-drug interaction experiments, we analyzed cis-inhibition of [3H]3TC uptake by ABC and AZT and found that 40 to 50% was inhibited at low concentrations of the drugs (Ki = 22–500 pM). These data reveal that NRTIs experience a high-affinity interaction with hOCTs, suggesting a putative role for these drugs as modulators of hOCT activity. Finally, 3TC is a novel substrate for hOCTs and the inhibition of its uptake at low concentrations of ABC and AZT could have implications for the pharmacokinetics of 3TC.

Individual PKC-Phosphorylation Sites in Organic Cation Transporter 1 Determine Substrate Selectivity and Transport Regulation

To elucidate the molecular mechanisms underlying stimulation of rat organic cation transporter type 1 (rOCT1) by protein kinase C (PKC) activation, functional properties and regulation of rOCT1 stably expressed in HEK293 cells after site-directed mutagenesis of putative PKC phosphorylation-sites were compared with wild-type (WT) rOCT1 using microfluorometric measurements with the fluorescence organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP(+)). Either substitutions of single (S286A, S292A, T296A, S328A, and T550A) or of all five PKC-sites (5x-PKC) with alanine suppressed PKC-induced stimulation of ASP(+) uptake, whereas regulation by p56(lck) tyrosine kinase was conserved in all mutants. Remarkably, the apparent affinities for TEA(+), TPA(+), and quinine were changed differently in each mutant (EC(50) in WT, S286A, S292A, T296A, S328A, T550A, and 5x-PKC in mumol: TEA(+): 105, 153, 56, 1135, 484, 498, 518; TPA(+): 0.1, 2.1, 0.3, 1.0, 43, 0.3, 2.2; quinine: 1.5, 3.0, 2.5, 4.8, 81, 7.6, 8.9, respectively). After mutations, no effects of PKC activation on apparent affinity of rOCT1 for these substrates could be detected, in contrast to what was observed in WT. PKC activation had no significant effect on rOCT1 trafficking from intracellular pools to the cell membrane. Substitution of all PKC sites suppressed PKC-induced phosphorylation of rOCT1. In conclusion, it was found that the presence of all five potential PKC phosphorylation sites is necessary for the PKC-induced stimulation of rOCT1. The different effects on the EC(50) values by the different mutations suggest that the large intracellular loop participates in building the substrate binding pocket of rOCT1 or specifically modulates its structure.

Regulation of the human organic cation transporter hOCT1

The human organic cation transporter type 1 (hOCT1) is an important transport system for small organic cations in the liver. Organic cation transporters are regulated by different signaling pathways, but the regulation of hOCT1 has not yet been studied. In this work, we have for the first time investigated the regulation of hOCT1. hOCT1 was expressed in Chinese hamster ovary cells (CHO‐hOCT1) and in human embryonic kidney cells (HEK293‐hOCT1). Its activity was monitored using microfluorimetry with the fluorescent organic cation 4‐(4‐(dimethylamino)styryl)‐N‐methylpyridinium (ASP+) as substrate. hOCT1 expressed in CHO‐cells was inhibited by protein kinase A (PKA) activation (1 µM forskolin, −58 ± 6%, n = 12), calmodulin inhibition (0.1 µM calmidazolium, −68 ± 3%, n = 6; 10 µM ophiobolin A, −48 ± 10%, n = 7), calmodulin‐dependent kinase II inhibition (1 µM KN62, −78 ± 4%, n = 12), and inhibition of p56lck tyrosine kinase (10 µM aminogenistein, −35 ± 7%, n = 12). The apparent affinities for TEA+ were lower in CHO‐hOCT1 than in HEK293‐hOCT1, while those for TPA+ and quinine were almost identical; the rank order of EC50 values (TPA+ > quinine > TEA+) was independent of the expression system. EC50 values for TEA+ in CHO‐hOCT1 or HEK293‐hOCT1 were increased under calmidazolium incubation (6.3 and 1.4 mM, respectively). hOCT1 was inhibited by PKA and endogenously activated by calmodulin, calmodulin‐dependent kinase II, and p56lck tyrosine kinase. Regulation pathways were the same in the two expression systems. Since apparent substrate affinities depend on activity of regulatory pathways, the expression system plays a role in determining the substrate affinities.

Cell Free Expression and Functional Reconstitution of Eukaryotic Drug Transporters

Polyspecific organic cation and anion transporters of the SLC22 protein family are critically involved in absorption and excretion of drugs. To elucidate transport mechanisms, functional and biophysical characterization of purified transporters is required and tertiary structures must be determined. Here, we synthesized rat organic cation transporters OCT1 and OCT2 and rat organic anion transporter OAT1 in a cell free system in the absence of detergent. We solubilized the precipitates with 2% 1-myristoyl-2-hydroxy- sn-glycero-3-[phospho- rac-(1-glycerol)] (LMPG), purified the transporters in the presence of 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or octyl glucoside, and reconstituted them into proteoliposomes. From 1 mL reaction vessels 0.13-0.36 mg of transporter proteins was purified. Thus, from five to ten 1 mL reaction vessels sufficient protein for crystallization was obtained. In the presence of 1% LMPG and 0.5% CHAPS, OCT1 and OAT1 formed homo-oligomers but no hetero-oligomers. After reconstitution of OCT1, OCT2, and OAT1 into proteoliposomes, similar Michaelis-Menten K m values were measured for uptake of 1-methyl-4-phenylpyridinium and p-aminohippurate (PAH (-)) by the organic cation and anion transporters, respectively, as after expression of the transporters in cells. Using the reconstituted system, evidence was obtained that OAT1 operates as obligatory and electroneutral PAH (-)/dicarboxylate antiporter and contains a low-affinity chloride binding site that stimulates turnover. PAH (-) uptake was observed only with alpha-ketoglutarate (KG (2-)) on the trans side, and trans-KG (2-) increased the PAH (-) concentration in voltage-clamped proteoliposomes transiently above equilibrium. The V max of PAH (-)/KG (2-) antiport was increased by Cl (-) in a manner independent of gradients, and PAH (-)/KG (2-) antiport was independent of membrane potential in the absence or presence of Cl (-).