Amino acid residues in transmembrane domain 10 of organic anion transporting polypeptide 1B3 are critical for cholecystokinin octapeptide transport

Human organic anion transporting polypeptides (OATP) 1B1 and 1B3 are multispecific transporters that mediate uptake of amphipathic organic compounds into hepatocytes. The two OATPs contain 12 transmembrane domains (TMs) and share 80% amino acid sequence identity. Besides common substrates with OATP1B1, OATP1B3 specifically transports cholecystokinin octapeptide (CCK-8). To determine which structural domains and/or residues are important for the substrate selectivity of OATP1B3, we constructed a series of chimeric proteins between OATP1B3 and 1B1, expressed them in HEK293 cells, and determined rates of uptake of CCK-8 along with surface expression of the proteins. Replacing TM10 in OATP1B3 with TM10 of OATP1B1 resulted in a dramatically reduced degree of CCK-8 transport, indicating that TM10 is crucial for recognition and/or translocation of CCK-8. Using site-directed mutagenesis, we identified three key residues within TM10, namely, Y537, S545, and T550. When we replaced these residues with the corresponding amino acid residues found in OATP1B1, the level of CCK-8 transport was similarly low as for the replacement of the whole TM10. Kinetic experiments showed that the K m values for CCK-8 transport in the TM10 replacement and triple mutant were only 1.3 and 1.1 microM, respectively, as compared to 16.3 microM for wild-type OATP1B3. Similarly, the V max values dropped from 495.5 pmol (normalized mg) (-1) min (-1) for wild-type OATP1B3 to 13.3 and 19.0 pmol (normalized mg) (-1) min (-1) for the TM10 replacement and triple mutant, respectively. Molecular modeling indicated that two of the three identified residues might form hydrogen bonds with CCK-8. In conclusion, we have identified three amino acid residues (Y537, S545, and T550) in TM10 of OATP1B3 that are important for CCK-8 transport.     

Human organic anion transporter 1B1 and 1B3 function as bidirectional carriers and do not mediate GSH-bile acid cotransport

Organic anion transporting polypeptides (OATP/SLCO) are generally believed to function as electroneutral anion exchangers, but direct evidence for this contention has only been provided for one member of this large family of genes, rat Oatp1a1/Oatp1 (Slco1a1). In contrast, a recent study has indicated that human OATP1B3/OATP-8 (SLCO1B3) functions as a GSH-bile acid cotransporter. The present study examined the transport mechanism and possible GSH requirement of the two members of this protein family that are expressed in relatively high levels in the human liver, OATP1B3/OATP-8 and OATP1B1/OATP-C (SLCO1B1). Uptake of taurocholate in Xenopus laevis oocytes expressing either OATP1B1/OATP-C, OATP1B3/OATP-8, or polymorphic forms of OATP1B3/OATP-8 (namely, S112A and/or M233I) was cis-inhibited by taurocholate and estrone sulfate but was unaffected by GSH. Likewise, taurocholate and estrone sulfate transport were trans-stimulated by estrone sulfate and taurocholate but were unaffected by GSH. OATP1B3/OATP-8 also did not mediate GSH efflux or GSH-taurocholate cotransport out of cells, indicating that GSH is not required for transport activity. In addition, estrone sulfate uptake in oocytes microinjected with OATP1B3/OATP-8 or OATP1B1/OATP-C cRNA was unaffected by depolarization of the membrane potential or by changes in pH, suggesting an electroneutral transport mechanism. Overall, these results indicate that OATP1B3/OATP-8 and OATP1B1/OATP-C most likely function as bidirectional facilitated diffusion transporters and that GSH is not a substrate or activator of their transport activity.     

Leucine heptad motifs within transmembrane domains affect function and oligomerization of human organic anion transporting polypeptide 1B1

Organic anion transporting polypeptides (OATPs) are transmembrane proteins responsible for the uptake of a wide range of endogenous compounds and clinically important drugs. The liver-specific OATP1B1 serves crucial roles in the removal of many orally administered drugs. The proper function of the transporter hence is essential for the pharmacokinetics of various therapeutic agents. Membrane proteins tend to form oligomers that are important for their stability, targeting and/or interactions with the substrates. Previous study in our laboratory revealed that OATP1B1 may form homo-oligomers and that a GXXXG motif localized at transmembrane domain 8 (TM8) may affect its oligomerization. In the current study, three short-form leucine heptad repeats within the transmembrane domains of OATP1B1 were investigated. It was found that the disruption of leucine heptad repeats within TM3 dramatically reduced the uptake function and protein-protein association of OATP1B1; while within TM8, only L378 is essential for the function of OATP1B1 and alanine replacement of L378 exhibited no effect on the oligomerization. The fragmental expression of TM3 interfered with the association of OATP1B1 homo-oligomers as well as its association with OATP1B3, which is also selectively expressed at human hepatocytes, suggesting that the region may be shared by both transporters for their protein-protein interactions.     

Fluorescence-based assays for the assessment of drug interaction with the human transporters OATP1B1 and OATP1B3

Hepatic disposition plays a significant role in the pharmacokinetics and pharmacodynamics of a variety of drugs. Sinusoidal membrane transporters have been shown to participate in the hepatic disposition of many pharmaceuticals. Two sinusoidal membrane transporters with an established role in hepatic disposition are OATP1B1 and OATP1B3 (organic anion-transporting polypeptides 1B1 and 1B3, respectively). OATP1B1 and OATP1B3 have been implicated in the hepatic uptake of statin drugs, and polymorphisms linked to OATP1B1 have been associated with deleterious patient endpoints. As a result, OATP1B1 and OATP1B3 represent sites for potential drug-drug interactions. Numerous methods exist for identifying potential drug-drug interactions with transporters. However, relatively few offer the convenience and speed of fluorescence-based assays. Here a fluorescence-based assay was developed for measuring the OATP1B1- and OATP1B3-mediated transport of 8-fluorescein-cAMP (8-FcA). The OATP1B1- and OATP1B3-mediated transport of 8-FcA was time dependent and saturable (K_m = 2.9 and 1.8 μM, V_max = 0.20 and 0.33 pmol/min/cm², respectively). Molecules known to interact with OATPs, including cyclosporin A, rifampicin, and glibenclamide, each demonstrated concentration-dependent inhibition of 8-FcA transport by OATP1B1 and OATP1B3. The in vitro fluorescence-based assays described here using 8-FcA as the substrate are convenient and rapid and have utility in screening drug candidates for potential drug-drug interactions with OATP1B1 and OATP1B3.     

Role of aromatic transmembrane residues of the organic anion transporter,rOAT3, in substrate recognition

Organic anion transporters (OATs, SLC21) are important in the excretion of endogenous and exogenous compounds in the kidney. The rat organic anion transporter, rOAT3, mediates the transport of organic anions such as p-aminohippurate (PAH) and estrone sulfate as well as the basic compound, cimetidine. In the present study, we examined the role of conserved transmembrane aromatic amino acid residues of rOAT3 in substrate recognition and transport. Alanine scanning followed by amino acid replacements was used to construct mutants of rOAT3. The uptake of model compounds was studied in Xenopus laevis oocytes expressing the mutant transporters. We observed that four mutants in transmembrane domain 7 (TMD 7), W334A, F335A, Y341A, and Y342Q, and one mutant in transmembrane domain 8 (TMD 8), F362S, exhibited a less than 2-fold enhanced uptake of PAH and cimetidine in comparison to wild-type rOAT3, which exhibited a 16-fold enhanced uptake of PAH and an 8-fold enhanced uptake of cimetidine. Estrone sulfate uptake in oocytes expressing any one of these five mutants remained at least 8-fold enhanced. The data suggest that the five residues, W334, F335, Y341, Y342, and F362, contribute differently to the transport of the small hydrophilic organic substrates PAH and cimetidine in comparison to the large hydrophobic organic substrate estrone sulfate. The effects of side chains of these five residues on transporter functions were also evaluated by constructing conservative mutations. We observed that the residues contribute to PAH and cimetidine transport in different ways: the -OH group of Y342, the indole ring of W334, and the aromatic rings of F335, Y341, and F362 are important for PAH and cimetidine transport by rOAT3. These data suggest that there is an aromatic pocket composed mainly of residues in TMD 7 in the translocation pathway of rOAT3, which is important for the transport of PAH and cimetidine. Aromatic residues in this pocket may interact directly with substrates of rOAT3 through hydrogen bonds and pi-pi interactions.     

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.     

Role of GSH in estrone sulfate binding and translocation by the multidrug resistance protein 1 (MRP1/ABCC1)

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent efflux pump that can confer resistance to multiple anticancer drugs and transport conjugated organic anions. Unusually, transport of several MRP1 substrates requires glutathione (GSH). For example, estrone sulfate transport by MRP1 is stimulated by GSH, vincristine is co-transported with GSH, or GSH can be transported alone. In the present study, radioligand binding assays were developed to investigate the mechanistic details of GSH-stimulated transport of estrone sulfate by MRP1. We have established that estrone sulfate binding to MRP1 requires GSH, or its non-reducing analogue S-methyl GSH (S-mGSH), and further that the affinity (Kd) of MRP1 for estrone sulfate is 2.5-fold higher in the presence of S-mGSH than GSH itself. Association kinetics show that GSH binds to MRP1 first, and we propose that GSH binding induces a conformational change, which makes the estrone sulfate binding site accessible. Binding of non-hydrolyzable ATP analogues to MRP1 decreases the affinity for estrone sulfate. However, GSH (or S-mGSH) is still required for estrone sulfate binding, and the affinity for GSH is unchanged. Estrone sulfate affinity remains low following hydrolysis of ATP. The affinity for GSH also appears to decrease in the post-hydrolytic state. Our results indicate ATP binding is sufficient for reconfiguration of the estrone sulfate binding site to lower affinity and argue for the presence of a modulatory GSH binding site not associated with transport of this tripeptide. A model for the mechanism of GSH-stimulated estrone sulfate transport is proposed.     

Polymorphisms in human organic anion-transporting polypeptide 1A2 (OATP1A2): implications for altered drug disposition and central nervous system drug entry

Organic anion-transporting polypeptide 1A2 (OATP1A2) is a drug uptake transporter known for broad substrate specificity, including many drugs in clinical use. Therefore, genetic variation in SLCO1A2 may have important implications to the disposition and tissue penetration of substrate drugs. In the present study, we demonstrate OATP1A2 protein expression in human brain capillary and renal distal nephron using immunohistochemistry. We also determined the extent of single nucleotide polymorphisms in SLCO1A2 upon analyses of ethnically defined genomic DNA samples (n = 95 each for African-, Chinese-, European-, and Hispanic-Americans). We identified six nonsynonymous polymorphisms within the coding region of SLCO1A2 (T38C (I13T), A516C (E172D), G559A (A187T), A382T (N128Y), A404T (N135I), and C2003G (T668S)), the allelic frequencies of which appeared to be ethnicity-dependent. In vitro functional assessment revealed that the A516C and A404T variants had markedly reduced capacity for mediating the cellular uptake of OATP1A2 substrates, estrone 3-sulfate and two delta-opioid receptor agonists, deltorphin II, and [D-penicillamine(2,5)]-enkephalin. On the other hand, the G559A and C2003G variants appeared to have substrate-dependent changes in transport activity. Cell surface biotinylation and immunofluorescence confocal microscopy suggested that altered plasma membrane expression of the transporter may contribute to reduced transport activity associated with the A516C, A404T, and C2003G variants. The A404T (N135I) variant also showed a shift in the apparent molecular size, indicative of alterations in glycosylation status. Taken together, these data suggest that SLCO1A2 polymorphisms may be an important yet unrecognized contributor to inter-individual variability in drug disposition and central nervous system entry of substrate drugs.     

Identification of amino acids essential for estrone-3-sulfate transport within transmembrane domain 2 of organic anion transporting polypeptide 1B1

As an important structure in membrane proteins, transmembrane domains have been found to be crucial for properly targeting the protein to cell membrane as well as carrying out transport functions in transporters. Computer analysis of OATP sequences revealed transmembrane domain 2 (TM2) is among those transmembrane domains that have high amino acid identities within different family members. In the present study, we identify four amino acids (Asp70, Phe73, Glu74, and Gly76) that are essential for the transport function of OATP1B1, an OATP member that is specifically expressed in the human liver. A substitution of these four amino acids with alanine resulted in significantly reduced transport activity. Further mutagenesis showed the charged property of Asp70 and Glu74 is critical for proper function of the transporter protein. Comparison of the kinetic parameters indicated that Asp70 is likely to interact with the substrate while Glu74 may be involved in stabilizing the binding site through formation of a salt-bridge. The aromatic ring structure of Phe73 seems to play an important role because substitution of Phe73 with tyrosine, another amino acid with a similar structure, led to partially restored transport function. On the other hand, replacement of Gly76 with either alanine or valine could not recover the function of the transporter. Considering the nature of a transmembrane helix, we proposed that Gly76 may be important for maintaining the proper structure of the protein. Interestingly, when subjected to transport function analysis of higher concentration of esteone-3-sulfate (50 μM) that corresponds to the low affinity binding site of OATP1B1, mutants of Phe73, Glu74, and Gly76 all showed a transport function that is comparable to that of the wild-type, suggesting these amino acids may have less impact on the low affinity component of esteone-3-sulfate within OATP1B1, while Asp 70 seems to be involved in the interaction of both sites.     

Suppressor mutations in the transmembrane segments of P-glycoprotein promote maturation of processing mutants and disrupt a subset of drug-binding sites

Defective folding of cystic fibrosis transmembrane conductance regulator protein missing Phe508 (DeltaF508) is the major cause of cystic fibrosis. The folding defect in DeltaF508 cystic fibrosis transmembrane conductance regulator might be correctable because misfolding of a P-glycoprotein (P-gp; ABCB1) mutant lacking the equivalent residue (DeltaY490) could be corrected with drug substrates or by introduction of an arginine residue into transmembrane (TM) segments 5 (I306R) or 6 (F343R). Possible mechanisms of arginine rescue were that they mimicked some of the effects of drug substrate interactions with P-gp or that they affected global folding such that all drug substrate/modulator interactions with P-gp were altered. To distinguish between these mechanisms, we tested whether arginines introduced into other TMs predicted to line the drug-binding pocket (TM1 or TM3) would affect folding. It was found that mutation of L65R(TM1) or T199R(TM3) promoted maturation of processing mutants. We then tested whether arginine suppressor mutations had local or global effects on P-gp interactions with drug substrates and modulators. The L65R(TM1), T199R(TM3), I306R(TM5), or F343R(TM6) mutations were introduced into the P-gp mutant L339C(TM6)/F728C(TM7), and thiol cross-linking was carried out in the presence of various concentrations of vinblastine, cyclosporin A, or rhodamine B. The presence of arginine residues reduced the apparent affinity of P-gp for vinblastine (L65R, T199R, and I306R), cyclosporin (I306R and F343R), or rhodamine B (F343R) by 4-60-fold. These results show that the arginine mutations affect a subset of drug-binding sites and suggest that they rescue processing mutants by mimicking drug substrate interactions with P-gp.     

Ethanol inhibition of N-methyl-D-aspartate receptors is reduced by site-directed mutagenesis of a transmembrane domain phenylalanine residue

N-Methyl-D-aspartate (NMDA) receptors (NRs) are ionotropic receptors activated by glutamate and the co-agonist glycine. Ethanol inhibits NMDA receptor function, although its site of action is undefined. We hypothesized that ethanol acts at specific amino acids contained within the transmembrane (TM) domains of the receptor. In this study, NR1 and NR2A subunits were altered by mutagenesis and tested for sensitivity to ethanol. Three NR1 mutants (W636A, F817A, and L819A) and one NR2A mutant (F637A) failed to generate functional receptors. Pre-TM1 (I546A, L551A, F554A, and F558A), TM1 (W563A), and TM2 (W611A) NR1 mutations did not affect ethanol sensitivity of heteromeric receptors. In contrast, altering a TM3 phenylalanine to alanine (F639A) reduced the ethanol inhibition of NMDA receptors expressed in oocytes and human embryonic kidney 293 cells. Mutation of the nearby methionine (M641) to alanine did not affect ethanol sensitivity, whereas changing Phe(639) to tryptophan slightly enhanced ethanol inhibition. NR1(F639A) did not alter the agonist potency of glutamate but did produce a leftward shift in the glycine concentration response for receptors containing NR2A and NR2B subunits. NR1(F639A) also reduced the potency of the competitive glycine antagonist 5,7-dichlorokynurenic acid and increased the efficacy of the glycine partial agonist 3-amino-1-hydroxy-2-pyrrolidinone ((+)-HA-966). These results suggest that ethanol may interact with amino acids contained in the TM3 domain of NMDA subunits that are involved in transducing agonist binding to channel opening.     

Charged amino acids in the sixth transmembrane helix of multidrug resistance protein 1 (MRP1/ABCC1) are critical determinants of transport activity

The multidrug resistance protein, MRP1 (ABCC1), is an ATP-binding cassette transporter that confers resistance to chemotherapeutic agents. MRP1 also mediates transport of organic anions such as leukotriene C(4) (LTC(4)), 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG), estrone 3-sulfate, methotrexate (MTX), and GSH. We replaced three charged amino acids, Lys(332), His(335), and Asp(336), predicted to be in the sixth transmembrane (TM6) helix of MRP1 with neutral and oppositely charged amino acids and determined the effect on substrate specificity and transport activity. All mutants were expressed in transfected human embryonic kidney cells at levels comparable with wild-type MRP1, and confocal microscopy showed that they were correctly routed to the plasma membrane. Vesicular transport studies revealed that the MRP1-Lys(332) mutants had lost the ability to transport LTC(4), and GSH transport was reduced; whereas E(2)17betaG, estrone 3-sulfate, and MTX transport were unaffected. E(2)17betaG transport was not inhibited by LTC(4) and could not be photolabeled with [(3)H]LTC(4), indicating that the MRP1-Lys(332) mutants no longer bound this substrate. Substitutions of MRP1-His(335) also selectively diminished LTC(4) transport and photolabeling but to a lesser extent. Kinetic analyses showed that V(max) (LTC(4)) of these mutants was decreased but K(m) was unchanged. In contrast to the selective loss of LTC(4) transport in the Lys(332) and His(335) mutants, the MRP1-Asp(336) mutants no longer transported LTC(4), E(2)17betaG, estrone 3-sulfate, or GSH, and transport of MTX was reduced by >50%. Lys(332), His(335), and Asp(336) of TM6 are predicted to be in the outer leaflet of the membrane and are all capable of forming intrahelical and interhelical ion pairs and hydrogen bonds. The importance of Lys(332) and His(335) in determining substrate specificity and of Asp(336) in overall transport activity suggests that such interactions are critical for the binding and transport of LTC(4) and other substrates of MRP1.     

Transport of fluorescent chenodeoxycholic acid via the human organic anion transporters OATP1B1 and OATP1B3

This study sought to clarify the contributions of organic anion-transporting polypeptide (OATP) 1B1 and 1B3 to the liver uptake of chenodeoxycholic acid (CDCA). We synthesized a fluorescent version of CDCA, chenodeoxychilyl-(Nepsilon-NBD)-lysine (CDCA-NBD), to characterize transporter-mediated uptake. CDCA-NBD is efficiently transported by OATP1B1 and OATP1B3 with high affinities. The Michaelis-Menten constants for CDCA-NBD uptake by OATP1B1 and OATP1B3 were 1.45 +/- 0.39 microM and 0.54 +/- 0.09 microM, respectively. By confocal laser scanning microscopy, CDCA-NBD, which is taken up by OATP1B1 and OATP1B3, was observed to localize to the cytosol. We also examined the transport of newly synthesized fluorescent bile acids. NBD-labeled bile acids, including cholic acid, deoxycholic acid, lithocholic acid, and ursodeoxycholic acid, were all transported by OATP1B1 and OATP1B3. CDCA-NBD exhibited the highest rate of transport of the five NBD-labeled bile acids examined in OATP1B1- and OATP1B3-expressing cells. Our results suggest that OATP1B1 and OATP1B3 play important roles in CDCA uptake into the liver. Fluorescent bile acids are useful tools to characterize the uptake properties of membrane transporters.     

Transmembrane segment 7 of human P-glycoprotein forms part of the drug-binding pocket

P-gp (P-glycoprotein; ABCB1) protects us by transporting a broad range of structurally unrelated compounds out of the cell. Identifying the regions of P-gp that make up the drug-binding pocket is important for understanding the mechanism of transport. The common drug-binding pocket is at the interface between the transmembrane domains of the two homologous halves of P-gp. It has been shown in a previous study [Loo, Bartlett and Clarke (2006) Biochem. J. 396, 537-545] that the first transmembrane segment (TM1) contributed to the drug-binding pocket. In the present study, we used cysteine-scanning mutagenesis, reaction with an MTS (methanethiosulfonate) thiol-reactive analogue of verapamil (termed MTS-verapamil) and cross-linking analysis to test whether the equivalent transmembrane segment (TM7) in the C-terminal-half of P-gp also contributed to drug binding. Mutation of Phe728 to cysteine caused a 4-fold decrease in apparent affinity for the drug substrate verapamil. Mutant F728C also showed elevated ATPase activity (11.5-fold higher than untreated controls) after covalent modification with MTS-verapamil. The activity returned to basal levels after treatment with dithiothreitol. The substrates, verapamil and cyclosporin A, protected the mutant from labelling with MTS-verapamil. Mutant F728C could be cross-linked with a homobifunctional thiol-reactive cross-linker to cysteines I306C(TM5) and F343C(TM6) that are predicted to line the drug-binding pocket. Disulfide cross-linking was inhibited by some drug substrates such as Rhodamine B, calcein acetoxymethyl ester, cyclosporin, verapamil and vinblastine or by vanadate trapping of nucleotides. These results indicate that TM7 forms part of the drug-binding pocket of P-gp.     

Characterization of the role of polar amino acid residues within predicted transmembrane helix 17 in determining the substrate specificity of multidrug resistance protein 3

Human multidrug resistance protein (MRP) 3 is the most closely related homologue of MRP1. Like MRP1, MRP3 confers resistance to etoposide (VP-16) and actively transports 17 beta-estradiol 17-(beta-D-glucuronide) (E(2)17 beta G), cysteinyl leukotriene 4 (LTC(4)), and methotrexate, although with generally lower affinity. Unlike MRP1, MRP3 also transports monovalent bile salts. We have previously demonstrated that hydrogen-bonding residues predicted to be in the inner-leaflet spanning segment of transmembrane (TM) 17 of MRP1 are important for drug resistance and E(2)17 beta G transport. We have now examined the importance of the hydrogen-bonding potential of residues in TM17 of MRP3 on both substrate specificity and overall activity. Mutation S1229A reduced only methotrexate transport. Mutations S1231A and N1241A decreased resistance to VP-16 and transport of E(2)17 beta G and methotrexate but not taurocholate. Mutation Q1235A also reduced resistance to VP-16 and transport of E(2)17beta G but increased taurocholate transport without affecting transport of methotrexate. Mutations Y1232F and S1233A reduced resistance to VP-16 and the transport of all three substrates tested. In contrast, mutation T1237A markedly increased VP-16 resistance and transport of all substrates. On the basis of the substrates analyzed, residues Ser(1229), Ser(1231), Gln(1235), and Asn(1241) play an important role in determining the specificity of MRP3, while mutation of Tyr(1232), Ser(1233), and Thr(1237) affects overall activity. Unlike MRP1, the involvement of polar residues in determining substrate specificity extends throughout the TM helix. Furthermore, elimination of the hydrogen-bonding potential of a single amino acid, Thr(1237), markedly enhanced the ability of the protein to confer drug resistance and to transport all substrates examined.     

Membrane dynamics of γ‐secretase with the anterior pharynx‐defective 1B subunit

The four‐subunit protease complex γ‐secretase cleaves many single‐pass transmembrane (TM) substrates, including Notch and β‐amyloid precursor protein to generate amyloid‐β (Aβ), central to Alzheimer's disease. Two of the subunits anterior pharynx‐defective 1 (APH‐1) and presenilin (PS) exist in two homologous forms APH1‐A and APH1‐B, and PS1 and PS2. The consequences of these variations are poorly understood and could affect Aβ production and γ‐secretase medicine. Here, we developed the first complete structural model of the APH‐1B subunit using the published cryo‐electron microscopy (cryo‐EM) structures of APH1‐A (Protein Data Bank: 5FN2, 5A63, and 6IYC). We then performed all‐atom molecular dynamics simulations at 303 K in a realistic bilayer system to understand both APH‐1B alone and in γ‐secretase without and with substrate C83‐bound. We show that APH‐1B adopts a 7TM topology with a water channel topology similar to APH‐1A. We demonstrate direct transport of water through this channel, mainly via Glu84, Arg87, His170, and His196. The apo and holo states closely resemble the experimental cryo‐EM structures with APH‐1A, however with subtle differences: The substrate‐bound APH‐1B γ‐secretase was quite stable, but some TM helices of PS1 and APH‐1B rearranged in the membrane consistent with the disorder seen in the cryo‐EM data. This produces different accessibility of water molecules for the catalytic aspartates of PS1, critical for Aβ production. In particular, we find that the typical distance between the catalytic aspartates of PS1 and the C83 cleavage sites are shorter in APH‐1B, that is, it represents a more closed state, due to interactions with the C‐terminal fragment of PS1. Our structural‐dynamic model of APH‐1B alone and in γ‐secretase suggests generally similar topology but some notable differences in water accessibility which may be relevant to the protein's existence in two forms and their specific function and location.     

Inhibition of human organic anion transporting polypeptide OATP 1B1 as a mechanism of drug-induced hyperbilirubinemia

OATP1B1 (a.k.a. OATP-C, OATP2, LST-1, or SLC21A6) is a liver-specific organic anion uptake transporter and has been shown to be a higher affinity bilirubin uptake transporter than OATP1B3. Using human embryonic kidney (HEK 293) cells stably transfected with OATP1B1, we have studied the effects of indinavir, saquinavir, cyclosporin A, and rifamycin SV on human OATP1B1 transport function. These drugs are potent inhibitors of OATP1B1 transport activity in vitro. We further provide evidence that the calculated fraction of OATP1B1 inhibited at the clinical exposure level correlated very well with the observed hyperbilirubinemia outcome for these drugs in humans. Our data support the hypothesis that inhibition of OATP1B1 is an important mechanism for drug-induced unconjugated hyperbilirubinemia. Inhibition of OATPs may be an important mechanism in drug-drug and drug-endogenous substance interactions.     

Metabolism of dehydroepiandrosterone sulfate and estrone-sulfate by human platelets

The aim of the present research was to study the uptake of DHEAS, and to establish the intracrine capacity of human platelets to produce sex steroid hormones. The DHEAS transport was evaluated through the uptake of [(3)H]-DHEAS in the presence or absence of different substrates through the organic anion transporting polypeptide (OATP) family. The activity of sulfatase enzyme was evaluated, and the metabolism of DHEAS was measured by the conversion of [(3)H]-DHEAS to [(3)H]-androstenedione, [(3)H]-testosterone, [(3)H]-estrone and [(3)H]-17beta-estradiol. Results indicated the existence in the plasma membrane of an OATP with high affinity for DHEAS and estrone sulphate (E(1)S). The platelets showed the capacity to convert DHEAS to active DHEA by the steroid-sulfatase activity. The cells resulted to be a potential site for androgens production, since they have the capacity to produce androstenedione and testosterone; in addition, they reduced [(3)H]-estrone to [(3)H]-17beta-estradiol. This is the first demonstration that human platelets are able to import DHEAS and E(1)S using the OATP family and to convert DHEAS to active DHEA, and to transform E(1)S to 17beta-estradiol.     

Pharmacogenetics of Membrane Transporters: An Update on Current Approaches

This review provides an overview of the pharmacogenetics of membrane transporters including selected ABC transporters (ABCB1, ABCC1, ABCC2, and ABCG2) and OATPs (OATP1B1 and OATP1B3). Membrane transporters are heavily involved in drug clearance and alters drug disposition by actively transporting substrate drugs between organs and tissues. As such, polymorphisms in the genes encoding these proteins may have significant effects on the absorption, distribution, metabolism and excretion of compounds, and may alter pharmacodynamics of many agents. This review discusses the techniques used to identify substrates and inhibitors of these proteins and subsequently to assess the effect of genetic mutation on transport, both in vitro and in vivo. A comprehensive list of substrates for the major drug transporters is included. Finally, studies linking transporter genotype with clinical outcomes are discussed.     

Inhibitory Effects of Green Tea and (–)-Epigallocatechin Gallate on Transport by OATP1B1, OATP1B3, OCT1, OCT2, MATE1, MATE2-K and P-Glycoprotein

Green tea catechins inhibit the function of organic anion transporting polypeptides (OATPs) that mediate the uptake of a diverse group of drugs and endogenous compounds into cells. The present study was aimed at investigating the effect of green tea and its most abundant catechin epigallocatechin gallate (EGCG) on the transport activity of several drug transporters expressed in enterocytes, hepatocytes and renal proximal tubular cells such as OATPs, organic cation transporters (OCTs), multidrug and toxin extrusion proteins (MATEs), and P-glycoprotein (P-gp). Uptake of the typical substrates metformin for OCTs and MATEs and bromosulphophthalein (BSP) and atorvastatin for OATPs was measured in the absence and presence of a commercially available green tea and EGCG. Transcellular transport of digoxin, a typical substrate of P-gp, was measured over 4 hours in the absence and presence of green tea or EGCG in Caco-2 cell monolayers. OCT1-, OCT2-, MATE1- and MATE2-K-mediated metformin uptake was significantly reduced in the presence of green tea and EGCG (P < 0.05). BSP net uptake by OATP1B1 and OATP1B3 was inhibited by green tea [IC₅₀ 2.6% (v/v) and 0.39% (v/v), respectively]. Green tea also inhibited OATP1B1- and OATP1B3-mediated atorvastatin net uptake with IC₅₀ values of 1.9% (v/v) and 1.0% (v/v), respectively. Basolateral to apical transport of digoxin was significantly decreased in the presence of green tea and EGCG. These findings indicate that green tea and EGCG inhibit multiple drug transporters in vitro. Further studies are necessary to investigate the effects of green tea on prototoypical substrates of these transporters in humans, in particular on substrates of hepatic uptake transporters (e.g. statins) as well as on P-glycoprotein substrates.