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PubMed1992

Molecular heterogeneity of the gamma-aminobutyric acid (GABA) transport system. Cloning of two novel high affinity GABA transporters from rat brain.

cDNA clones encoding two novel gamma-aminobutyric acid (GABA) transporters (designated GAT-2 and GAT-3) have been isolated from rat brain, and their functional properties have been examined in mammalian cells. The transporters display high affinity for GABA (Km approximately 10 microM) and exhibit pharmacological properties distinct from the previously cloned neuronal GABA transporter (GAT-1). Both transporters require sodium and chloride for transport activity. The nucleotide sequences of GAT-2 and GAT-3 predict proteins of 602 and 627 amino acids, respectively, which can be modeled with 12 transmembrane domains, similar to the topology proposed for other cloned neurotransmitter transporters. Localization studies indicate that both transporters are present in brain and retina, while GAT-2 is also present in peripheral tissues. The cloning of these transporter genes from rat brain reveals previously undescribed heterogeneity in GABA transporters.

Amino Acid Sequence↗
PubMed2003

Decreased activity of basolateral organic ion transports in hyperuricemic rat kidney: roles of organic ion transporters, rOAT1, rOAT3 and rOCT2.

We investigated organic anion and cation transport activity and the expression of several organic ion transporters in hyperuricemic rat kidney. Feeding oxonic acid, an inhibitor of uric acid metabolism, and uric acid for 10 days significantly increased plasma uric acid level. Plasma creatinine and blood urea nitrogen concentrations also increased in hyperuricemic rats, indicating impaired renal function. The accumulation of organic anions, p-aminohippurate (PAH) and methotrexate, and cations, tetraethylammonium (TEA) and cimetidine, into renal slices was markedly decreased, suggesting decreased transport activity for organic anions and cations at the basolateral membrane in the kidney. The expression levels of basolateral organic anion transporters rOAT1 and rOAT3, and organic cation transporter, rOCT2, significantly decreased in hyperuricemic rat kidney as assessed by mRNA and protein levels. In contrast, the expression of rOCT1 was unaltered by hyperuricemia at both mRNA and protein levels. Moreover, the mRNA expression of kidney-specific organic anion transporters, OAT-K1 and OAT-K2, and organic anion transporting polypeptide (oatp) 1, which localize at the brush-border membrane in the kidney, was unchanged in hyperuricemic rats. In conclusion, we showed decreased basolateral organic anion and cation transport activity, accompanied by a specific decrease in rOAT1, rOAT3 and rOCT2 expression in hyperuricemic rat kidney. These phenomena partly contribute to the changed renal disposition of organic anions and cations in hyperuricemia.

Animals↗
PubMed2006

Organic anion transporting polypeptide 2B1 and breast cancer resistance protein interact in the transepithelial transport of steroid sulfates in human placenta.

The human placenta has both protective and nurturing functions for the fetal organism. Uptake and elimination of xenobiotics and endogenous substances are facilitated by various transport proteins from the solute carrier (SLC) and ABC families, respectively. A functional interaction of uptake and elimination, which is a prerequisite for vectorial transport across cellular barriers, has not been described for placenta. In this study, we examined expression of organic anion transporter (OAT) 4 (SLC22A11), organic anion transporting polypeptide (OATP) 2B1 (SLCO2B1, OATP-B), and breast cancer resistance protein (BCRP) (ABCG2) in human placenta (n = 71) because all three proteins are involved in transmembranal transfer of estrone 3 sulfate (E3S; metabolic product) and dehydroepiandrosterone sulfate (DHEAS; precursor molecule). On the mRNA level, we found a significant correlation of OATP2B1 and BCRP (R(2) = 0.534; p < 0.01) but not between OAT4 and BCRP (R(2) = -0.104; p > 0.05). Localization studies confirmed basal expression of OATP2B1 and apical expression of BCRP. To study functional interactions between OATP2B1 and BCRP, we developed a Madin-Darby canine kidney cell model expressing both transport proteins simultaneously (OATP2B1 and BCRP in the basal and apical membrane, respectively). Using this cell model in a transwell system resulted in a significantly increased basal to apical transport of both E3S and DHEAS, when both transporters were expressed with no change of transfer in the apical to basal direction. Taken together, these data show the potential for a functional interaction of OATP2B1 and BCRP in transepithelial transport of steroid sulfates in human placenta.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed1999

Experience with the loading and transport of fuel assembly transport casks, including CASTOR casks, and the radiation exposure of personnel.

In 1997 and 1998, six spent fuel assembly transports started from the nuclear power plant Gemeinschaftskernkraftwerk Neckar (GKN), using CASTOR-V19 casks. Professor Kuni of Marburg University challenged the statement made by the German Federal Office for Radiation Protection (Bundesamt für Strahlenschutz (BfS)) based on accepted scientific knowledge, according to which so-called CASTOR transports present no risk, either to the population or to the escorting police units. This paper shows that the collective dose during the loading of the CASTOR casks amounted to 4.5 mSv (gamma and neutrons) per cask at the most, and that the maximum individual dose amounted to 0.26 mSv. In addition to these doses, the collective dose during handling and transport must be considered: this amounted to 0.35 mSv (gamma and neutrons). The dose to the police escort was <0.1 mSv (gamma and neutrons). In the light of these circumstances, this report is presented on contamination determined during the transport of CASTOR casks and of other spent fuel casks. The controls of spent fuel transports carried out since 1978, mainly with NTL 11 spent fuel casks, revealed that about one fifth of the transport casks which left the GKN with a surface contamination of <4 Bq cm(-2) (limit for surface contamination), presented degrees of contamination >4 Bq cm(-2) upon reaching the Valognes/Cogema terminal. However, transport casks coming from French plants also revealed degrees of contamination >4 Bq cm(-2), as well as 'hot spots'. No such contamination was found on NTL 11 casks transported from the GKN to Sellafield. Neither was any increased contamination found upon the arrival of CASTOR-V19 casks transported from GKN to Gorleben or Ahaus. The partially sensationalist media reports were inversely proportional to the actual radiological relevance of the matter. The German Commission on Radiation Protection (SSK) confirmed that the radiological effect of such contaminated spent fuel transports is negligible.

France↗
PubMed2005

The contribution of free water transport and small pore transport to the total fluid removal in peritoneal dialysis.

BACKGROUND: Water transport in peritoneal dialysis (PD) patients is across the small pores and water channels, the latter allowing free water transport. The objective of the study was to investigate the contribution of each transport route on transcapillary ultrafiltration (TCUF). METHODS: Standard peritoneal permeability analyses of 80 stable PD patients were analyzed. Twenty-nine patients were followed longitudinally. Fluid transport through small pores (SPT) was assessed by the amount of transported sodium. Free water transport (FWT) was calculated by subtracting SPT from TCUF. The contribution of FWT and SPT to the TCUF and water transport rates at any time point was computed. RESULTS: The ultrafiltered volume due to SPT increased gradually during the dwell, while FWT reached its maximum around 3 hours. The rate of FWT decreased continuously during the dwell. SPT decreased during the initial 2 hours and remained stable thereafter. At 60 minutes, the SPT (P < 0.05) and its contribution (P < 0.05) were positively related to the MTAC(creat). The contribution of FWT after 1 hour, but not the absolute amount, showed an inverse relationship. Peritoneal solute transport parameters (P < 0.01) and the contribution of SPT (P= 0.08), but none of the other fluid parameters showed a U-shape with the lowest values in the second year of PD (P < 0.01). CONCLUSION: The dwell courses of water transport suggest that the activity of water channels is dependent and limited by the crystalloid osmotic pressure gradient, while other determinants are important in SPT. The time-course of SPT paralleled that of peritoneal solute transport parameters.

Adult↗
PubMed2006

Girls' perception of physical environmental factors and transportation: reliability and association with physical activity and active transport to school.

BACKGROUND: Preliminary evidence suggests that the physical environment and transportation are associated with youth physical activity levels. Only a few studies have examined the association of physical environmental factors on walking and bicycling to school. Therefore, the purpose of this study was (1) to examine the test-retest reliability of a survey designed for youth to assess perceptions of physical environmental factors (e.g. safety, aesthetics, facilities near the home) and transportation, and (2) to describe the associations of these perceptions with both physical activity and active transport to school. METHODS: Test and retest surveys, administered a median of 12 days later, were conducted with 480 sixth- and eighth-grade girls in or near six U.S. communities. The instrument consisted of 24 questions on safety and aesthetics of the perceived environment and transportation and related facilities. Additionally, girls were asked if they were aware of 14 different recreational facilities offering structured and unstructured activities, and if so, whether they would visit these facilities and the ease with which they could access them. Test-retest reliability was determined using kappa coefficients, overall and separately by grade. Associations with physical activity and active transport to school were examined using mixed model logistic regression (n = 610), adjusting for grade, race/ethnicity, and site. RESULTS: Item-specific reliabilities for questions assessing perceived safety and aesthetics of the neighborhood ranged from 0.31 to 0.52. Reliabilities of items assessing awareness of and interest in going to the 14 recreational facilities ranged from 0.47 to 0.64. Reliabilities of items assessing transportation ranged from 0.34 to 0.58. Some items on girls' perceptions of perceived safety, aesthetics of the environment, facilities, and transportation were important correlates of physical activity and, in some cases, active transport to school. CONCLUSION: This study provides some psychometric support for the use of the questionnaire on physical environmental factors and transportation for studying physical activity and active transport to school among adolescent girls. Further work can continue to improve reliability of these self-report items and examine their association of these factors with objectively measured physical activity.

Journal Article↗
PubMed2004

Characterization of the effectiveness of hexose transporters for transporting xylose during glucose and xylose co-fermentation by a recombinant Saccharomyces yeast.

We have developed recombinant Saccharomyces yeasts that can effectively co-ferment glucose and xylose to ethanol. However, these yeasts still ferment glucose more efficiently than xylose. The transport of xylose could be one of the steps limiting the fermentation of xylose. In this study, we characterized the changes in the expression pattern of the hexose transporter and related genes during co-fermentation of glucose and xylose using one of our recombinant yeasts, Saccharomyces cerevisiae 424A(LNH-ST). The transcription of the hexose transporter and related genes was strongly influenced by the presence of glucose; HXT1, HXT2 and HXT3 were greatly activated by glucose and HXT5, HXT7 and AGT1 were significantly repressed by glucose. We also examined the effectiveness of individual transporters encoded by HXT1, HXT2, HXT4, HXT5, HXT7 and GAL2 genes for transporting xylose during co-fermentation of glucose and xylose in a Saccharomyces hxt degrees mutant (RE700A). We compared these hxt degrees derivatives to RE700A wild-type strain (S. cerevisiae MC996A) where all of them contained the same xylose metabolizing genes present in our xylose-fermenting yeasts such as 424A(LNH-ST). Our results showed that recombinant RE700A containing the cloned HXT7 or HXT5 were substantially more effective for fermenting xylose to ethanol. In addition, we found that the efficiency of transporters for intracellular accumulation of xylose was as follows: HXT7 > HXT5 > GAL2 > WT > HXT1 > HXT4 > > > RE700A. Furthermore, we provided evidence that the Saccharomyces galactose transporter system could be a highly effective xylose transporter. The information reported here should be of great importance for improving the Saccharomyces yeast transport of xylose.

Fermentation↗
PubMed2000

Cloning and characterization of human NTT5 and v7-3: two orphan transporters of the Na+/Cl- -dependent neurotransmitter transporter gene family.

Orphan transporters form a growing subfamily of genes related by sequence similarity to the Na+/Cl- -dependent neurotransmitter superfamily. Using a combination of database similarity searching and cloning methods, we have identified and characterized two novel human orphan transporter genes, v7-3 and NTT5. Similar to other known orphan transporters, v7-3 and NTT5 contain 12 predicted transmembrane domains, intracellular N- and C-terminal domains, and large extracellular loops between transmembrane (TM) domains 3 and 4 and between TM domains 7 and 8. Residues within the extracellular loops are also predicted to contain sites for N-linked glycosylation. Human v7-3, the species orthologue of rat v7-3, contains an open reading frame (ORF) of 730 amino acids. Human NTT5 is a new member of the orphan transporter family and has an ORF of 736 amino acids. The amino acid sequences of human v7-3 and NTT5 are greater than 50% similar to other known orphan neurotransmitter transporters and also show sequence similarity to the human serotonin and dopamine transporters. Radiation hybrid mapping located the human v7-3 and NTT5 genes on chromosomes 12q21.3-q21.4 and 19q13.1-q13.4, respectively. Human mRNA distribution analysis by TaqMan reverse transcription-polymerase chain reaction showed that v7-3 mRNA is predominantly expressed in neuronal tissues, particularly amygdala, putamen, and corpus callosum, with low-level expression in peripheral tissues. In contrast, NTT5 mRNA was highly expressed in peripheral tissues, particularly in testis, pancreas, and prostate. Transient transfection with epitope-tagged transporter constructs demonstrated v7-3 to be expressed at the cell surface, whereas NTT5 was predominantly intracellular, suggestive of a vesicular location. Although the substrates transported by these transporters remain unknown, their specific but widespread distribution suggests that they may mediate distinct and important functions within the brain and the periphery.

AMP-Activated Protein Kinases↗
PubMed1998

Lactate/H+ transport kinetics in rat skeletal muscle related to fibre type and changes in transport capacity.

Lactate/H+ transport kinetics were determined by means of the pH-sensitive probe BCECF in sarcolemmal giant vesicles, obtained from rat skeletal muscle, and related to variations in lactate/H+ transport capacity. Vesicle preparations were made from red and white muscles, mixed muscles, denervated muscles, muscles of old rats and rats that had been subjected to high-intensity training, endurance training, repeated exposure to hypoxia, and hypothyroid or hyperthyroid treatments. The lactate/H+ transport capacity of red muscles was greater than that of white muscles, and this difference was associated with a higher maximal transport rate (V max) in red muscles, whereas the K m was similar in the two muscle types. High-intensity training and hyperthyroidism increased the lactate/H+ transport capacity by enhancing V max without affecting K m. Similarly, a reduced transport capacity with old age and hypothyroidism was due to a decrease in V max. The denervation-induced decline in lactate/H+ transport capacity resulted from both an increased K m and a reduced V max. The present data show that muscle type differences and most changes in the lactate/H+ transport capacity are mediated by modifications in V max, which is expected to represent the number of membrane transporter molecules. K m is unaffected by most treatments and appears to be independent of fibre type.

Animals↗
PubMed1987

Nucleoside and nucleobase transport and metabolism in wild type and nucleoside transport-deficient Aedes albopictus cells.

Nucleoside and nucleobase transport and metabolism were measured in ATP-depleted and normal Aedes albopictus mosquito cells (line C-7-10) by rapid kinetic techniques. The cells possess a facilitated diffusion system for nucleosides, which in its broad substrate specificity and kinetic properties resembles that present in many types of mammalian cells. The Michaelis-Menten constant for uridine transport at 28 degrees C is about 180 microM. However, the nucleoside transporter of the mosquito cells is resistant to inhibition by nmolar concentrations of nitrobenzylthioinosine and the cells lack high affinity nitrobenzylthioinosine binding sites. The cells also possess an adenine transporter, which is distinct from the nucleoside transporter. They lack, however, a hypoxanthine transport system and are deficient in hypoxanthine phosphoribosyltransferase activity, which explains their failure to efficiently salvage hypoxanthine from the medium. The cells possess uridine and thymidine phosphorylase activities and, in contrast to cultured mammalian cells, efficiently convert uracil to nucleotides. An adenosine-resistant variant (CAE-3-6) of the C-7-10 cell line is devoid of significant nucleoside transport activity but transports adenine normally. Residual entry of various nucleosides into these cells and of hypoxanthine and cytosine into wild type and mutant cells is strictly non-mediated. The rate of permeation of various nucleosides and of hypoxanthine into the CAE-3-6 cells is related to their hydrophobicity. Uridine permeation into CAE-3-6 cells exhibits an activation energy of about 20 kcal/mol. At high uridine concentrations permeation is sufficiently rapid to partly overcome the limitation in nucleoside salvage imposed by the nucleoside transport defect in these cells.

Adenine↗
PubMed2004

The synthesis of SNAT2 transporters is required for the hypertonic stimulation of system A transport activity.

In cultured human fibroblasts incubated under hypertonic conditions, the stimulation of system A for neutral amino acid transport, associated to the increased expression of the mRNA for SNAT2 transporter, leads to an expanded intracellular amino acid pool and to the recovery of cell volume. A protein of nearly 60 kDa, recognized by an antiserum against SNAT2, is increased both in the pool of biotinylated membrane proteins and in the total cell lysate of hypertonically stressed cells. The increased level of SNAT2 transporters in hypertonically stressed cells is confirmed by immunocytochemistry. DRB, an inhibitor of transcription, substantially inhibits the increase of SNAT2 proteins on the plasma membrane, completely suppresses the stimulation of system A transport activity, and markedly delays the cell volume recovery observed during the hypertonic treatment. On the contrary, if the transport activity of system A is adaptively increased by amino acid starvation in the presence of DRB, the increase of SNAT2 transporters on the plasma membrane is still clearly detectable and the transport change only partially inhibited. It is concluded that the synthesis of new SNAT2 transporters is essential for the hypertonic stimulation of transport system A, but accounts only in part for the adaptive increase of the system.

Amino Acid Transport System A↗
PubMed2004

Insulin and glucose induced changes in expression level of nucleoside transporters and adenosine transport in rat T lymphocytes.

Adenosine is an endogenous agent exerting potent action on the immune system including regulation of lymphocyte functioning. Impaired T lymphocyte functioning is a common feature of diabetes. The aims of this study were to examine the effects of glucose and insulin on nucleoside transporters (NT) expression level and adenosine (Ado) transport in rat T lymphocytes cultured under the defined concentrations of glucose and insulin. Performed experiments revealed that rat T lymphocytes expressed the equilibrative nucleoside transporter type 1 and 2 (rENT1, rENT2) and concentrative nucleoside transporter type 2 (rCNT2). The mRNA levels of rENT2 and rCNT2 were highly dependent on insulin but were not affected by changes in extracellular glucose concentration. Exposition of T cells to 10nM insulin resulted in 73% increase in rENT2 mRNA and 50% decrease in the rCNT2 mRNA level. The level of rENT1 mRNA was sensitive to extracellular glucose concentration but not to insulin. The highest differences among cells cultured in high (20mM) and low (5mM) glucose were observed in equilibrative nitrobenzylthioinosine sensitive adenosine transport, which was lowered by 65% in cells cultured at high glucose. Alterations in adenosine transport were accompanied by changes in adenosine accumulation in the cell. These results indicate that adenosine transport in rat T lymphocytes is independently and differentially regulated by glucose and insulin by means of changes in the nucleoside transporters expression level. Altered adenosine transport has a great impact on its intracellular level. This suggests that under diabetic conditions adenosine action on T lymphocytes might be altered.

Adenosine↗
PubMed2003

Regulation of reduced-folate transporter-1 (RFT-1) by homocysteine and identity of transport systems for homocysteine uptake in retinal pigment epithelial (RPE) cells.

Reduced-folate transporter-1 (RFT-1) transports reduced-folates, such as N5-methyltetrahydrofolate (MTF), the predominant circulating form of folate. In RPE, RFT-1 is localized to the apical membrane and is thought to transport folate from RPE to photoreceptor cells. Folate is required for DNA, RNA, protein synthesis and the conversion of homocysteine (Hcy) to methionine. Decreased folate levels are associated with increased Hcy levels. In the present study, we asked whether RFT-1 activity in RPE is altered under high Hcy conditions and examined the transport mechanism for Hcy in RPE. Treatment of ARPE-19 cells, a human RPE cell line, with Hcy at concentrations higher than 50 microM led to a significant decrease in RFT-1 activity. This effect increased as the treatment time increased. The inhibitory effect of Hcy on RFT-1 activity was not non-specific, as the activities of several other nutrient transporters were not affected under identical conditions. The effect of Hcy on RFT-1 was associated primarily with a decrease in the maximal velocity with no detectable change in substrate affinity. The decrease in RFT-1 activity was accompanied by parallel changes in RFT-1 mRNA and protein. Uptake of Hcy in ARPE-19 cells occurred via several transport systems, including Na+-independent systems L and b(0,+) and the Na+-dependent systems B0, ATB(0,+) and A. Studies of the interaction of Hcy with one of the cloned transporters (ATB(0,+)) provided direct evidence for the translocation of Hcy across the membrane via the transporter. We conclude that several transport systems operate in ARPE-19 cells for the entry of Hcy and that high levels of Hcy have deleterious effects on the expression and activity of RFT-1 in these cells.

Biological Transport↗
PubMed1988

Insulin-mediated translocation of glucose transporters from intracellular membranes to plasma membranes: sole mechanism of stimulation of glucose transport in L6 muscle cells.

Plasma membranes and light microsomes were isolated from fused L6 muscle cells. Pre-treatment of cells with insulin did not affect marker enzyme or protein distribution in isolated membranes. The number of glucose transporters in the isolated membranes was calculated from the D-glucose-protectable binding of [3H]cytochalasin B. Glucose transporter number was higher in plasma membranes and lower in intracellular membranes derived from insulin-treated cells than in the corresponding fractions from untreated cells. The net increase in glucose transporters in plasma membranes was identical to the net decrease in glucose transporters in light microsomes (2 pmol/1.23 x 10(8) cells). The fold increase in glucose transporter number/mg protein in plasma membranes (2-fold) was similar to the fold increase in glucose transport caused by insulin. This suggests that recruitment of glucose transporters from intracellular membranes to the plasma membrane is the major mechanism of stimulation of hexose transport in L6 muscle cells. This is the first report of isolation of the two insulin-sensitive membrane elements from a cell line, and the results indicate that, in contrast to rat adipocytes, there is not change in the intrinsic activity of the transporters in response to insulin.

Biological Transport↗
PubMed1996

Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate.

Three glutamate transporters have been identified in rat, including astroglial transporters GLAST and GLT-1 and a neuronal transporter EAAC1. Here we demonstrate that inhibition of the synthesis of each glutamate transporter subtype using chronic antisense oligonucleotide administration, in vitro and in vivo, selectively and specifically reduced the protein expression and function of glutamate transporters. The loss of glial glutamate transporters GLAST or GLT-1 produced elevated extracellular glutamate levels, neurodegeneration characteristic of excitotoxicity, and a progressive paralysis. The loss of the neuronal glutamate transporter EAAC1 did not elevate extracellular glutamate in the striatum but did produce mild neurotoxicity and resulted in epilepsy. These studies suggest that glial glutamate transporters provide the majority of functional glutamate transport and are essential for maintaining low extracellular glutamate and for preventing chronic glutamate neurotoxicity.

ATP-Binding Cassette Transporters↗
PubMed2001

Transport-defective mutations alter the conformation of the energy-coupling motif of an outer membrane transporter.

The bacterial outer membrane transporter for vitamin B(12), BtuB, derives its energy for transport by interacting with the trans-periplasmic membrane protein TonB. This interaction with TonB occurs in part through an N-terminal segment in the BtuB sequence called the Ton box. In the present study, site-directed spin labeling of intact outer membrane preparations was used to investigate the conformation of the Ton box in wild-type BtuB and in two transport-defective mutants, L8P and V10P. In the wild-type protein, the Ton box is folded into the barrel of the transporter. The conformation of this segment is dramatically different in the transport-defective mutants L8P and V10P, where the Ton box is found to be flexible, and undocked from the transporter barrel with a greater exposure to the periplasm. In the wild-type protein, vitamin B(12) induces an undocking of the Ton box, but its addition to these transport defective mutants produces little or no change in the conformation of the Ton box. Proline substitutions at positions that do not alter transport do not alter the wild-type conformation of the Ton box; thus, the effect of substituting proline at positions 8 and 10 on the docked state of the Ton box appears to be unique. The failure of these mutants to execute the B(12) transport cycle may be a result of the altered conformation of the Ton box.

Amino Acid Motifs↗
PubMed1997

Role of conserved arginine and glutamate residues on the cytosolic surface of glucose transporters for transporter function.

The role of conserved arginine and glutamic acid residues at the cytoplasmic surface of the GLUT4 for transporter function was investigated by site-directed mutagenesis and expression of the constructs in COS-7 cells. Reconstituted glucose transport activity, cytochalasin B binding, and photolabeling with the exofacial label 2-N4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1, 3-bis(d-mannosyloxy)-2-propylamine (ATB-BMPA) was assayed in membranes from transfected cells and corrected for immunoreactivity of expressed transporters. Exchange of Arg 92 (R92L amino acid residues are numbered according to the corresponding residues in the GLUT1) or Arg 333/334 (RR333/4LA) reduced or suppressed transport activity with no or very little effect on photolabeling with ATB-BMPA and cytochalasin B binding. It is suggested that the lack of these residues selectively disturbes the substrate-induced conformational change of the carrier during transport. Exchange of Glu 146 (E146D) or Arg 153 (R153L) markedly reduced transport activity, ATB-BMPA photolabeling, and cytochalasin B binding. Transport activity and ATB-BMPA labeling were abolished in the mutants E329Q, E393D, and R400L, whereas binding of cytochalasin B was normal. Thus, exchange of Glu 329, Glu 393, and Arg 400 appears to arrest the transporter in an inward facing conformation. It is concluded that the conserved arginine and glutamate residues at the cytoplasmic surface of the glucose transporter GLUT4 are essential for its appropriate conformation, and that it is the interaction of charged residues which mediates the oscillation between outward and inward facing states.

Affinity Labels↗
PubMed1999

Effects of glibenclamide on glycylsarcosine transport by the rat peptide transporters PEPT1 and PEPT2.

1 Glibenclamide is a widely used sulphonylurea for the treatment of non-insulin-dependent diabetes mellitus (NIDDM). This agent has been reported to inhibit the activities of various ion channels and transporters. In the present study, we examined the effects of glibenclamide on the function of the H+/peptide cotransporters PEPT1 and PEPT2 by using stable transfectants. 2 Uptake of [14C]-glycylsarcosine, a typical substrate for peptide transporters, by PEPT1- or PEPT2-expressing transfectant was inhibited by glibenclamide as well as other sulphonylureas including tolbutamide. 3 Kinetic analysis revealed that the inhibition by glibenclamide was noncompetitive. Dixon plot analyses showed that the Ki values of this agent were 25 and 7.8 microM for PEPT1 and PEPT2, respectively. 4 Glibenclamide did not inhibit Na+-coupled alanine and alpha-methyl-D-glucoside transport, suggesting that the inhibitory effects of glibenclamide on peptide transporters were not due to nonspecific interactions. 5 There was little uptake of [3H]-glibenclamide by PEPT-expressing transfectants as compared to mock-transfected cells, suggesting that glibenclamide was not a substrate for these peptide transporters. 6 In summary, glibenclamide inhibited the [14C]-glycylsarcosine transport by PEPT1 and PEPT2 in a noncompetitive fashion, although glibenclamide per se was not transported through these transporters. These findings would provide important information for clinical, physiological and biochemical aspects of peptide transporters.

Alanine↗