Critical amino acid residues in transmembrane domain 1 of the human organic anion transporter hOAT1

Human organic anion transporter 1 (hOAT1) belongs to a superfamily of organic anion transporters, which play critical roles in the body disposition of clinically important drugs, including anti-human immunodeficiency virus therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. Previously we suggested that the predicted transmembrane domain 1 (TM1) of hOAT1 might be important for its function. In the present study, we examined the role of each residue within TM1 of hOAT1 in substrate recognition and transport. Alanine scanning was used to construct mutants of hOAT1, and the uptake of model substrate para-aminohippurate was studied in COS-7 cells expressing the mutant transporters. This approach led to the discovery of two critical amino acid residues, Leu-30 and Thr-36. A substitution of Leu-30 or Thr-36 with alanine resulted in a complete loss of transport activities. We then further characterized Leu-30 and Thr-36 by mutagenizing these residues to amino acids with different physicochemical properties. Leu-30 was replaced with amino acids with varying sizes of side chains, including glycine, valine, and isoleucine. We showed that progressively smaller side chains at position 30 increasingly impaired hOAT1 function mainly because of the impaired surface expression of the transporter. Thr-36, another critical amino acid in TM1, was replaced by serine and cysteine. Similar to the substitution of Thr-36 by alanine, substitution by serine and cysteine at this position abolished transport activity without affecting the surface expression of the transporter. The fact that Thr-36 cannot be substituted with serine and that the side chains of alanine, serine, and cysteine are smaller than that of threonine by a methyl group indicate that both the methyl group and the hydroxyl group of Thr-36 could be critical for hOAT1 activity. Together we conclude that Leu-30 and Thr-36 play distinct roles in hOAT1 function. Leu-30 is important in targeting the transporter to the plasma membrane. In contrast, Thr-36 is critical for substrate recognition. The present study provided the first molecular evidence that transmembrane domain 1 is a critical determinant of hOAT1 function and may provide important insights into the structure-function relationships of the organic anion transporter family.     

The role of dileucine in the expression and function of human organic anion transporter 1 (hOAT1)

Human organic anion transporter hOAT1 plays a critical role in the body disposition of environmental toxins and clinically important drugs including anti-HIV therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. In the current study, we investigated the role of dileucine (L6L7) at the amino terminus of hOAT1 in the expression and function of the transporter. We substituted L6L7 with alanine (A) simultaneously. The resulting mutant transporter L6A/L7A showed no transport activity due to its complete loss of expression at the cell surface. Such loss of surface expression of L6A/L7A was consistent with a complete loss of an 80 kDa mature form and a dramatic decrease in a 60 kDa immature form of the mutant transporter in the total cell lysates. Treatment of L6A/L7A-expressing cells with proteasomal inhibitor resulted in a significant increase in the immature form of hOAT1, but not its mature form, whereas treatment of these cells with lysosomal inhibitor had no effect on the expression of the mutant transporters, suggesting that the mutant transporter was degraded through proteasomal pathway. The accumulation of mutant transporter in the endoplasmic reticulum (ER) was confirmed by coimmunolocalization of L6L7 with calnexin, an ER marker. Furthermore, treatment of L6A/L7A-expressing cells with sodium 4-phenylbutyrate (4PBA) and glycerol, two chemical chaperones, could not promote the exit of the immature form of the mutant transporter from the ER. Our data suggest that L6L7 are critical for the stability and ER export of hOAT1.     

Functional role of the C terminus of human organic anion transporter hOAT1

Human organic anion transporter hOAT1 plays critical roles in the body disposition of environmental toxins and clinically important drugs. In the present study, we examined the role of the C terminus of hOAT1 in its function. Combined approaches of cell surface biotinylation and transport analysis were employed for such purposes. It was found that deletion of the last 15 amino acids (residues 536-550) or the last 30 amino acids (residues 521-550) had no significant effect on transport activity. However, deletion of the entire C terminus (residues 506-550) completely abolished transport activity. Alanine scanning mutagenesis within the region of amino acids 506-520 led to the discovery of two critical amino acids: Glu-506 and Leu-512. Substitution of negatively charged Glu-506 with neutral amino acids alanine or glutamine resulted in complete loss of transport activity. However, such loss of transport activity could be rescued by substitution of Glu-506 with another negatively charged amino acid aspartic acid, suggesting the importance of negative charge at this position for maintaining the correct tertiary structure of the transporter, possibly by forming a salt bridge with a positively charged amino acid. Substitution of Leu-512 with amino acids carrying progressively smaller side chains including isoleucine, valine, and alanine resulted in mutants (L512I, L512V, and L512A) with increasingly impaired transport activity. However, the cell surface expression of these mutants was not affected. Kinetic analysis of mutant L512V revealed that the reduced transport activity of this mutant resulted mainly from a reduced maximum transport velocity Vmax without affecting the binding affinity (1/Km) of the transporter for its substrates, suggesting that the size of the side chain at position 512 critically affects transporter turnover number. Together, our results are the first to highlight the central role of the C terminus of hOAT1 in the function of this transporter.     

Mutational analysis of the role of GXXXG motif in the function of human organic anion transporter 1 (hOAT1)

Human organic anion transporter hOAT1 plays a critical role in the body disposition of environmental toxins and clinically important drugs including anti-HIV therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. hOAT1 has two GXXXG motifs in its transmembrane domains 2 and 5, a motif linked to the protein processing and oligomerization of other proteins. In the current study, we substituted glycine of these GXXXG motifs with alanine and evaluated the effect of such mutations on the expression and function of hOAT1. Mutations of GXXXG motif in the transmembrane domain 2 resulted in mutants G144A and G148A, both of which had no transport activity due to complete loss in the surface and total cell expression of the transporter protein. Treatment of G144A- and G148A-expressing cells with proteasomal inhibitor resulted in the recovery of ER-resident immature form of hOAT1, but not its surface-resident mature form, whereas treatment of these cells with lysosomal inhibitor had no effect on the expression of the mutant transporters. Mutations of GXXXG motif in the transmembrane domain 5 resulted in mutants G223A and G227A, among which only G227 had dramatic reduction of transport activity due to dramatic loss in the surface and total cell expression of the transporter. The reduction in the surface expression of G227 was consistent with the decrease in maximum transport velocity Vmax. Treatment of G227A-expressing cells with proteasomal inhibitor or lysosomal inhibitor resulted in partial recovery of both the immature form and the mature form of hOAT1 in the total cell extracts. However, such partial recovery of the mature form in total cell extracts did not lead to the partial recovery of surface expression and function of the transporter. Our data suggest that the GXXXG motifs in transmembrane domains 2 and 5 play critical roles in the stability of hOAT1.     

Molecular cloning and characterization of two novel human renal organic anion transporters (hOAT1 and hOAT3)

The cloned organic anion transporters from rat, mouse, and winter flounder (rOAT1, mOAT1, fROAT) mediate the coupled exchange of alpha-ketoglutarate with multiple organic anions, including p-aminohippurate (PAH). We have isolated two novel gene products from human kidney which bear significant homology to the known OATs and belong to the amphiphilic solute facilitator (ASF) family. The cDNAs, hOAT1 and hOAT3, encode for 550- and 568-amino-acid residue proteins, respectively. hOAT1 and hOAT3 mRNAs are expressed strongly in kidney and weakly in brain. Both genes map to chromosome 11 region q11.7. PAH uptake by Xenopus laevis oocytes injected with hOAT1 mRNA is increased 100-fold compared to water-injected oocytes. PAH uptake is chloride dependent and is not further increased by preincubation of oocytes in 5 mM glutarate. Uptake of PAH is inhibited by probenicid, alpha-ketoglutarate, bumetanide, furosemide, and losartan, but not by salicylate, urate, choline, amilioride, and hydrochlorothiazide.     

Human renal organic anion transporter 1 (hOAT1) and its role in the nephrotoxicity of antiviral nucleotide analogs

hOAT1 is a renal membrane protein able to efficiently transport acyclic nucleoside phosphonates (ANPs). When expressed in CHO cells, hOAT1 mediates the uptake and cytotoxicity of ANPs suggesting that it plays an active role in the nephrotoxicity associated with cidofovir CMV therapy and high-dose adefovir HIV therapy. Although efficiently transported by hOAT1, tenofovir did not show any significant cytotoxicity in isolated human proximal tubular cells, which correlates with the lack of nephrotoxicity observed in HIV-infected patients on prolonged tenofovir therapy.     

Development and characterization of immobilized human organic anion transporter-based liquid chromatographic stationary phase: hOAT1 and hOAT2

This paper reports the development of liquid chromatographic columns containing immobilized organic anion transporters (hOAT1 and hOAT2). Cellular membrane fragments from MDCK cells expressing hOAT1 and S2 cells expressing hOAT2 were immobilized on the surface of the immobilized artificial membrane (IAM) liquid chromatographic stationary phase. The resulting stationary phases were characterized by frontal affinity chromatography, using the marker ligand [3H]-adefovir for the hOAT1 and [14C]-p-aminohippurate for the hOAT2 in the presence of multiple displacers. The determined binding affinities (Kd) for eight OAT1 ligands and eight OAT2 ligands were correlated with literature values and a statistically significant correlation was obtained for both the hOAT1 and hOAT2 columns: r2=0.688 (p<0.05) and r2=0.9967 (p<0.0001), respectively. The results indicate that the OAT1 and OAT2 have been successfully immobilized with retention of their binding activity. The use of these columns to identify ligands to the respective transporters will be presented.     

Determination of the external loops and the cellular orientation of the N- and the C-termini of the human organic anion transporter hOAT1

The OAT (organic anion transporter) family mediates the absorption, distribution and excretion of a diverse array of environmental toxins and clinically important drugs. OAT dysfunction significantly contributes to renal, hepatic, neurological and fetal toxicity and disease. As a first step to establish the topological model of hOAT1 (human OAT1), we investigated the external loops and the cellular orientation of the N- and the C-termini of this transporter. Combined approaches of immunofluorescence studies and site-directed chemical labelling were used for such purpose. Immunofluorescence microscopy of Myc-tagged hOAT1 expressed in cultured cells identified that both the N- and the C-termini of the transporter were located in the cytoplasm. Replacement of Lys59 in the predicted extracellular loop I with arginine resulted in a mutant (K59R), which was largely inaccessible for labelling by membrane-impermeable NHS (N-hydroxysuccinimido)-SS (dithio)-biotin present in the extracellular medium. This result suggests that loop I faces outside of the cell membrane. A single lysine residue introduced into putative extracellular loops III, V and VI of mutant K59R, which is devoid of extracellular lysine, reacted readily with membrane-impermeable NHS-SS-biotin, suggesting that these putative extracellular loops are in the extracellular domains of the protein. These studies provided the first experimental evidence on the extracellular loops and the cellular orientation of the N- and the C-termini of hOAT1.     

Inhibition of the human organic anion transporter 1 by the caffeine metabolite 1-methylxanthine

Caffeine (1,3,7-trimethylxanthine) is daily and widely consumed in beverages and food and is mainly metabolized to 1,7-dimethylxanthine and 1-methylxanthine. Indirect clinical evidence suggests that 1-methylxanthine interacts with the organic anion transport system in the human kidney. In this study the effect of caffeine and its main metabolites on the human organic anion transporter 1 (hOAT1) was investigated using CHO cells overexpressing hOAT1. The uptake of 6-carboxyfluorescein into CHO(hOAT) cells was significantly inhibited by > or = 100 microM of 1-methylxanthine. Five hundred micromolar 1-methylxanthine was equieffective to 100 microM probenecid. In contrast, caffeine and 1,7-dimethylxanthine did not inhibit the transport of 6-carboxyfluorescein at concentrations up to 500 microM. In conclusion, the caffeine metabolite 1-methylxanthine inhibits the transport activity of hOAT1 in vitro. The central involvement of hOAT1 in the renal excretion of numerous drugs suggests that this inhibition may alter the pharmacokinetics of a series of clinically important drugs in humans.     

Cloning of the pig renal organic anion transporter 1 (pOAT1)

A pig kidney cDNA library was screened for the porcine ortholog of the multispecific organic anion transporter 1 (pOAT1). Several positive clones were isolated resulting in two alternatively spliced cDNA clones of pOAT1 (pOAT1 and pOAT1A). pOAT1-cDNAs consist of 2126 or 1895 base pairs (EMBL Acc. No. AJ308234 and AJ308235) encoding 547 or 533 amino acid residue proteins with 89, 87, 83 and 81% homology to the human, rabbit, rat, and mouse OAT1, respectively. Heterologous expression of pOAT1 in Xenopus laevis oocytes revealed an apparent K(m) for [3H]PAH of 3.75 +/- 1.6 microM. [3H]PAH uptake mediated by pOAT1 was abolished by 0.5 mM glutarate or 1 mM probenecid. Functional characterization of pOAT1A did not show any affinity for [3H]PAH. In summary, we cloned two alternative splice variants of the pig ortholog of organic anion transporter 1. One splice form (pOAT1) showed typical functional characteristics of organic anion transporter 1, whereas the second form appears not to transport PAH.     

A three-dimensional model of human organic anion transporter 1: aromatic amino acids required for substrate transport

Organic anion transporters (OATs) play a critical role in the handling of endogenous and exogenous organic anions by excretory and barrier tissues. Little is known about the OAT three-dimensional structure or substrate/protein interactions involved in transport. In this investigation, a theoretical three-dimensional model was generated for human OAT1 (hOAT1) based on fold recognition to the crystal structure of the glycerol 3-phosphate transporter (GlpT) from Escherichia coli. GlpT and hOAT1 share several sequence motifs as major facilitator superfamily members. The structural hOAT1 model shows that helices 5, 7, 8, 10, and 11 surround an electronegative putative active site ( approximately 830A(3)). The site opens to the cytoplasm and is surrounded by three residues not previously examined for function (Tyr(230) (domain 5) and Lys(431) and Phe(438) (domain 10)). Effects of these residues on p-aminohippurate (PAH) and cidofovir transport were assessed by point mutations in a Xenopus oocyte expression system. Membrane protein expression was severely limited for the Y230A mutant. For the K431A and F438A mutants, [(3)H]PAH uptake was less than 30% of wild-type hOAT1 uptake after protein expression correction. Reduced V(max) values for the F438A mutant confirmed lower protein expression. In addition, the F438A mutant exhibited an increased affinity for cidofovir but was not significantly different for PAH. Differences in handling of PAH and cidofovir were also observed for the Y230F mutant. Little uptake was determined for cidofovir, whereas PAH uptake was similar to wild-type hOAT1. Therefore, the hOAT1 structural model has identified two new residues, Tyr(230) and Phe(438), which are important for substrate/protein interactions.     

Functional invalidation of the autotaxin gene by a single amino acid mutation in mouse is lethal

Autotaxin is a member of the phosphodiesterase family of enzymes, (NPP2). It is an important secreted protein found in conditioned medium from adipocytes. It also has a putative role in the metastatic process. Based on these observation, further validation of this potential target was necessary, apart from the classical biochemical ones. The construction of a knock out mouse strain for ATX was started. In this paper, we report the generation of a mouse line displaying an inactivated ATX gene product. The KO line was designed in order to generate a functional inactivation of the protein. In this respect, the threonine residue T210 was replaced by an alanine (T210A) leading to a catalytically inactive enzyme. If the experimental work was straight forward, we disappointedly discovered at the final stage that the breeding of heterozygous animals, ATX -/+, led to the generation of a Mendelian repartition of wild-type and heterozygous, but no homozygous were found, strongly suggesting that the ATX deletion is lethal at an early stage of the development. This was confirmed by statistical analysis. Although other reported the same lethality for attempted ATX-/- mice generation [van Meeteren, L.A., Ruurs, P., Stortelers, C., Bouwman, P., van Rooijen, M.A., Pradère, J.P., Pettit, T.R., Wakelam, M.J.O., Saulnier-Blache, J.S., Mummery, C.L., Moolenar, W.H. and Jonkers, J. (2006) Autotaxin, a secreted lysophospholipase D, is essential for blood vessel formation during development, Mol. Cell. Biol. 26, 5015-5022; Tanaka, M., Okudaira, S., Kishi, Y., Ohkawa, R., Isei, S., Ota, M., Noji, S., Yatomi, Y., Aoki, J., and Arai, H. (2006) Autotaxin stabilizes blood vessels and is required for embryonic vasculature by producing lysophosphatidic acid, J. Biol. Chem. 281, 25822-25830], they used more drastic multiple exon deletions in the ATX gene, while we chose a single point mutation. To our knowledge, the present work is the first showing such a lethality in any gene after a point mutation in an enzyme catalytic site.     

Regulation by protein kinase C of organic anion transport driven by rat organic anion transporter 3 (rOAT3)

The organic anion transporter 3 (rOAT3) is a multispecific OAT localized at the basolateral membrane of the proximal tubule. The purpose of this study was to elucidate the role of protein kinase C (PKC) in the regulation of organic anion transport driven by rOAT3 and its mechanism of action. For this purpose, we established and utilized cells derived from the second segment of proximal tubule from mice stably expressing rOAT3 (S2 rOAT3). Phorbol 12-myristate 13-acetate (PMA), a PKC stimulator, attenuated the cellular uptake of estrone sulfate (ES), a prototype organic anion for rOAT3, in a dose- and time-dependent manner. PMA treatment resulted in a decrease in the Vmax, but not the Km of uptake of ES in S2 rOAT3. Treatment of S2 rOAT3 with other PKC stimulators or diacylglycerols also inhibited the uptake of ES, whereas that with an inactive phorbol ester did not. Chelerythrine chloride, a PKC inhibitor, reversed the PMA-induced decrease in uptake of ES in S2 rOAT3. These results suggest that PKC activation downregulates rOAT3-mediated organic anion transport. This down-regulation may be due to the inhibition of translocation or internalization of the rOAT3 protein, resulting in the decrease in the Vmax of rOAT3-mediated organic anion transport.     

Functional expression of the rat organic anion transporter 1 (rOAT1) in Saccharomyces cerevisiae

Organic anion transporter 1 (OAT1) is localized in the basolateral membrane of the proximal tubule in the kidney and plays an essential role in eliminating a wide range of organic anions, preventing their toxic effects on the body. Structural and functional studies of the transporter would be greatly assisted by inexpensive and rapid expression in the yeast Saccharomyces cerevisiae. The gene encoding rat OAT1 (rOAT1) contains many yeast non-preferred codons at the N-terminus and so was modified by fusion of the favored codon sequence of a hemagglutinin (HA) epitope preceding the start codon. The modified gene was cloned into several yeast expression plasmids, both integrative and multicopy, with either ADH1 promoter or GAL1 promoter in order to find a suitable expression system. Compared with the wild type gene, a substantial increase in rOAT1 expression was achieved by modification in the translational initiation region, suggesting that the codon chosen at the N-terminus influenced its expression. The highest inducible expression of rOAT1 was obtained under GAL1 promoter in 2 mu plasmid. A large fraction of rOAT1 was glycosylated in yeast, unaffected by growth temperature. The recombinant yeast expressing rOAT1 showed an increase in the uptake of p-aminohippurate (PAH) and this showed a positive correlation with rOAT1 expression level. Location of rOAT1 predominantly in the yeast plasma membrane confirmed correct processing. The importance of glycosylation for rOAT1 targeting was also shown. To our knowledge, this is the first successful functional expression of rOAT1 in the yeast S. cerevisiae.     

The Purkinje cell degeneration 5J mutation is a single amino acid insertion that destabilizes Nna1 protein

In the mouse, Purkinje cell degeneration (pcd) is a recessive mutation characterized by degeneration of cerebellar Purkinje cells, retinal photoreceptors, olfactory bulb mitral neurons, and certain thalamic neurons, and is accompanied by defective spermatogenesis. Previous studies of pcd have led to the identification of Nna1 as the causal gene; however, how loss of Nna1 function results in neurodegeneration remains unresolved. One useful approach for establishing which functional domains of a protein underlie a recessive phenotype has been to determine the genetic basis of the various alleles at the locus of interest. Because none of the pcd alleles analyzed at the time of the identification of Nna1 provided insight into the molecular basis of Nna1 loss-of-function, we obtained a recent pcd remutation—pcd^5J, and after determining that its phenotype is comparable to existing pcd severe alleles, we sought its genetic basis by sequencing Nna1. In this article we report that pcd^5J results from the insertion of a single GAC triplet encoding an aspartic acid residue at position 775 of Nna1. Although this insertion does not affect Nna1 expression at the RNA level, Nna1^pcd-5J protein expression is markedly decreased. Pulse-chase experiments reveal that the aspartic acid insertion dramatically destabilizes Nna1^pcd-5J protein, accounting for the observation that pcd^5J is a severe allele. The presence of a readily detectable genetic mutation in pcd^5J confirms that Nna1 loss-of-function alone underlies the broad pcd phenotype and will facilitate further studies of how Nna1 loss-of-function produces neurodegeneration and defective spermatogenesis in pcd mice.     

Molecular characterization of the renal organic anion transporter 1

Organic anions of diverse chemical structures are secreted in renal proximal tubules. The first step in secretion, uptake of organic anions across the basolateral membrane of tubule cells, is mediated for the polyspecific organic anion transporter 1 (OAT1), which exchanges extracellular organic anions for intracellular α-ketoglutarate or glutarate. OAT1 orthologs cloned from various species show 12 putative transmembrane domains and possess several sites for potential post-translational modification. The gene for the human OAT1 is located on chromosome 11q13.1 and is composed of 10 exons. Alternative splicing within exon 9 gives rise to four variants, two of which (OAT1-1 and OAT1-2) are functional. Following heterologous expression in Xenopus laevis oocytes, flounder renal OAT1 transported p-aminohippurate, glutarate, several diuretics, and the nephrotoxic agent ochratoxin A. Two cationic amino acid residues, lysine 394 and arginine 478, were found to be important for interaction with glutarate. Anionic neurotransmitter metabolites and the heavy-metal chelator, 2,3-dimercaptopropane sulfonate, interacted with the rabbit renal OAT1, which is expressed in kidneys and the retina.     

Functional differences in steroid sulfate uptake of organic anion transporter 4 (OAT4) and organic anion transporting polypeptide 2B1 (OATP2B1) in human placenta

Human trophoblasts depend on the supply of external precursors such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16alpha-OH-DHEA-S for synthesis of estrogens. Recently, we have characterized the uptake of DHEA-S by isolated mononucleated trophoblasts and identified different transporter polypeptides involved in this process. Immunohistochemistry of 1st and 3rd trimester placenta detected organic anion transporter 4 (OAT4) and organic anion transporting polypeptide 2B1 (OATP2B1, former name OATP-B) in cytotrophoblast membranes and at the basal surface of the syncytiotrophoblast, indicating that both transporter polypeptides are involved in placental uptake of foetal derived steroid sulfates. In the present study we have characterized and compared the kinetics of DHEA-S and estrone sulfate (E(1)S) uptake by these transporters stably expressed in FlpIn -HEK293 cells using the Flp recombinase-mediated site-specific recombination. Uptake of E(1)S by OAT4- and OATP2B1-transfected cells was highly increased compared to the non-transfected cells. In contrast, DHEA-S uptake was only highly increased in OAT4 (40 times), but only weakly enhanced in OATP2B1 cells. The uptake of DHEA-S and E(1)S by OAT4 was partly Na(+)-dependent (about 50%), whereas uptake of DHEA-S by OATP2B1 was Na(+)-independent. Kinetic analysis of the initial uptake rates of E(1)S by OAT4 and OATP2B1 gave very similar values for K(m) (about 20microM) and V(max) (about 600pmol/(minxmg protein)). In contrast, the affinity of DHEA-S towards OATP2B1 was about 10 times lower (K(m)>200microM) then for OAT4 (K(m)=29microM). Our results suggest different physiological roles of the two transporter polypeptides in placental uptake of foetal derived steroid sulfates. OATP2B1 seems not to be involved in de novo synthesis of placental estrogens but may contribute to the clearance of estrogen sulfates from foetal circulation.     

Identification of a novel gene family encoding human liver-specific organic anion transporter LST-1.

We have isolated a novel liver-specific organic anion transporter, LST-1, that is expressed exclusively in the human, rat, and mouse liver. LST-1 is a new gene family located between the organic anion transporter family and prostaglandin transporter. LST-1 transports taurocholate (Km = 13.6 microM) in a sodium-independent manner. LST-1 also shows broad substrate specificity. It transports conjugated steroids (dehydroepiandrosterone sulfate, estradiol-17beta-glucuronide, and estrone-3-sulfate), eicosanoids (prostaglandin E2, thromboxane B2, leukotriene C4, leukotriene E4), and thyroid hormones (thyroxine, Km = 3.0 microM and triiodothyronine, Km = 2.7 microM), reflecting hepatic multispecificity. LST-1 is probably the most important transporter in human liver for clearance of bile acids and organic anions because hepatic levels of another organic anion transporter, OATP, is very low. This is also the first report of the human molecule that transports thyroid hormones.     

Mutations in transmembrane domains 5 and 7 of the human excitatory amino acid transporter 1 affect the substrate-activated anion channel

L-Glutamate is the predominant excitatory neurotransmitter in the brain, and its extracellular concentration is tightly controlled by the excitatory amino acid transporters (EAATs). The transport of 1 glutamate molecule is coupled to the cotransport of 3 Na+ and 1 H+ and the countertransport of 1 K+. In addition to substrate transport, the binding of glutamate and Na+ activates an anion current which is thermodynamically uncoupled from the transport process. We have identified three amino acid residues in EAAT1 (D272 in TM5, K384 and R385 in TM7) that influence the amplitude of the anion channel current relative to the transport current. Transporters containing the mutations R268A, D272A, D272K, K384A, K384D, R385A, and R385D were expressed in Xenopus laevis oocytes and their transport and anion channel functions measured using the two-electrode voltage clamp techniques. The D272, K384, and R385 mutant transporters showed no change in transport properties but have increased levels of anion channel activity compared to wild-type transporters. These results identify additional residues of the EAAT1 transporter that may contribute to the gating mechanism of the anion channel of glutamate transporters and also provide hints as to how substrate binding leads to channel activation.