ABCMdb

ABCC1 p.Lys332Leu

Predicted by SNAP2:	A: D (63%), C: D (59%), D: D (80%), E: D (75%), F: D (80%), G: D (71%), H: D (53%), I: D (63%), L: N (53%), M: D (53%), N: D (71%), P: D (85%), Q: D (59%), R: N (61%), S: D (59%), T: D (63%), V: D (71%), W: D (85%), Y: D (80%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, L: D, M: D, N: D, P: D, Q: D, R: N, S: D, T: D, V: D, W: D, Y: D,

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[hide] Charged amino acids in the sixth transmembrane hel... J Biol Chem. 2002 Nov 1;277(44):41326-33. Epub 2002 Aug 18. Haimeur A, Deeley RG, Cole SP
Charged amino acids in the sixth transmembrane helix of multidrug resistance protein 1 (MRP1/ABCC1) are critical determinants of transport activity.
J Biol Chem. 2002 Nov 1;277(44):41326-33. Epub 2002 Aug 18., 2002-11-01 [PMID:12186871]

Abstract [show]
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.

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47 The sequences of the individual sense strands, with the altered codons underlined and the corresponding changes in amino acids indicated in parentheses were as follows: (K332D) 5Ј-CTC ATG AGC TTC TTC TTC GAC GCC ATC CAC GAC CTG-3Ј; (K332L) 5Ј-CTC ATG AGC TTC TTC TTC CTG GCC ATC CAC GAC CTG-3Ј; (H335E) 5Ј-GC TTC TTC TTC AAG GCC ATC GAG GAC CTG ATG ATG-3Ј; (H335L) 5Ј-GC TTC TTC TTC AAG GCC ATC TTG GAC CTG ATG ATG-3Ј; (H335Q) 5Ј-GC TTC TTC TTC AAG GCC ATC CAG GAC CTG ATG ATG-3Ј; (D336R) 5Ј-C AAG GCC ATC CAC CGG CTG ATG ATG TTT TCG-3Ј; (D336L) 5Ј-C AAG GCC ATC CAC CTG CTG ATG ATG TTT TCG-3Ј. Login to comment
106 In the case of the MRP1-Lys332 mutants K332D and K332L (Fig. 3B) and the MRP1-Asp336 mutants D336L and D336R (Fig. 3D), LTC4 uptake was reduced to levels that were indistinguishable from those observed with vesicles prepared from empty vector-transfected control cells. Login to comment
131 In contrast, LTC4 had very little effect (Ͻ15%) on E217betaG uptake by MRP1 mutants K332D and K332L, indicating that loss of LTC4 transport in these mutants is associated with a loss of binding of this substrate. On the other hand, LTC4 was still able to inhibit E217betaG uptake by MRP1 mutants H335E, H335L, and H335Q, which is consistent with only a partial reduction in LTC4 transport activity observed with these mutants (Fig. 6B). Login to comment
140 B, wild-type MRP1 (f), MRP1 mutants K332D (Œ) and K332L (‚), and control empty pcDNA3.1(-) vector (E). Login to comment
152 However, this band is not detectable in [3 H]LTC4 photolabeled proteins from cells expressing comparable levels of the MRP1 mutants K332D and K332L or mutants D336L and D336R, indicating that these mutations abrogate photolabeling and hence binding of this compound. Login to comment
160 The relative levels of [3 H]GSH uptake by the MRP1 K332D and K332L mutants were less than 15% of wild-type MRP1 (Fig. 7A) after subtracting basal [3 H]GSH transport by membrane vesicles from the empty vector-transfected control cells. Login to comment
163 Membrane vesicles prepared from transfected cells were incubated at 37 °C with 400 nM [3 H]E217betaG in transport buffer for the times indicated. A, time courses of ATP-dependent [3 H]E217betaG uptake by membrane vesicles prepared from HEK293T cells transfected with wild-type MRP1 (f), TM6 mutants K332D (Œ) and K332L (‚), and the empty pcDNA3.1(-) vector (E). Login to comment
170 A, ATP-dependent [3 H]E217betaG uptake in membrane vesicles from cells expressing wild-type MRP1 (WT-MRP1) (open bars), TM6 mutants K332D and K332L (shaded bars), and the empty pcDNA3.1(-) vector control (solid bars). Login to comment
183 Substitution of Lys332 with a neutral (K332L) or negatively charged (K332D) amino acid had no effect on MTX uptake by MRP1. Login to comment
190 Thus, replacing Lys332 with either Leu or Asp eliminated the ability of MRP1 to transport LTC4 (and markedly reduced GSH transport) without affecting the transport of other organic anions including E217betaG, estrone 3-sulfate, and MTX. Login to comment
204 Membrane vesicles were preincubated with acivicin and then incubated with [3 H]GSH in the presence of 30 ␮M apigenin in transport buffer for 20 min at 37 °C. A, K332D and K332L; B, H335E, H335L, and H335Q; C, D336L and D336R. Login to comment
206 D, WT-MRP1 (f); K332D (Œ); K332L (‚); vector control (E). Login to comment

[hide] Mutations of charged amino acids in or near the tr... Mol Pharmacol. 2004 Jun;65(6):1375-85. Haimeur A, Conseil G, Deeley RG, Cole SP
Mutations of charged amino acids in or near the transmembrane helices of the second membrane spanning domain differentially affect the substrate specificity and transport activity of the multidrug resistance protein MRP1 (ABCC1).
Mol Pharmacol. 2004 Jun;65(6):1375-85., [PMID:15155831]

Abstract [show]
Multidrug resistance protein 1 (MRP1) belongs to the ATP-binding cassette superfamily of transport proteins. In addition to drugs, MRP1 mediates the active transport of many conjugated and unconjugated organic anions. MRP1 consists of two membrane-spanning domains (MSD2 and MSD3) each followed by a nucleotide binding domain plus a third NH2-terminal MSD1. MSD2 contains transmembrane (TM) helices 6 through 11, and previously, we identified two charged residues in TM6 as having important but markedly different roles in MRP1 transport activity and substrate specificity by characterizing mutants containing nonconservative substitutions of Lys332 and Asp336. We have now extended these studies and found that the same-charge TM6 mutant K332R, like the nonconservatively substituted Lys332 mutants, exhibits a selective decrease in leukotriene C4 (LTC4) transport, associated with substantial changes in both Km and Vmax and LTC4 binding. The overall organic anion transport activity of the same-charge mutant of Asp336 (D336E) also remained very low, as observed for D336R. In addition, nonconservative substitutions of TM6-associated Lys319 and Lys347 resulted in a selective decrease in GSH transport. Of eight other charged residues in or proximal to TM7 to TM11 that were investigated, nonconservative substitutions of three of them [Lys396 (TM7), Asp436 (TM8), and Arg593 (TM11)] caused a substantial and global reduction in transport activity. However, unlike TM6 Asp336, wild-type transport activity could be reestablished in these MRP1 mutants by conservative substitutions. We conclude that MSD2-charged residues in or proximal to TM6, TM7, TM8, and TM11 play critical but differential roles in MRP1 transport activity and substrate specificity.

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No. Sentence Comment
100 We showed previously that nonconservative substitutions of Lys332 with either Asp (K332D) or Leu (K332L) led to a selective loss of transport of GSH and the GSH conjugate, LTC4 (Haimeur et al., 2002). Login to comment
121 The same-charge mutant K332R, like the K332D and K332L mutants described previously (Haimeur et al., 2002), exhibited transport levels of the conjugated estrogens E217betaG and E13SO4 and the antifolate MTX that were comparable with wild-type MRP1 (Table 2); however, GSH transport by K332R was very low compared with wild-type MRP1 and similar to that which we reported previously for the K332D/L mutants (Fig. 3B). Login to comment

[hide] Arsenic transport by the human multidrug resistanc... J Biol Chem. 2004 Jul 30;279(31):32700-8. Epub 2004 May 25. Leslie EM, Haimeur A, Waalkes MP
Arsenic transport by the human multidrug resistance protein 1 (MRP1/ABCC1). Evidence that a tri-glutathione conjugate is required.
J Biol Chem. 2004 Jul 30;279(31):32700-8. Epub 2004 May 25., 2004-07-30 [PMID:15161912]

Abstract [show]
Inorganic arsenic is an established human carcinogen, but its metabolism is incompletely defined. The ATP binding cassette protein, multidrug resistance protein (MRP1/ABCC1), transports conjugated organic anions (e.g. leukotriene C(4)) and also co-transports certain unmodified xenobiotics (e.g. vincristine) with glutathione (GSH). MRP1 also confers resistance to arsenic in association with GSH; however, the mechanism and the species of arsenic transported are unknown. Using membrane vesicles prepared from the MRP1-overexpressing lung cancer cell line, H69AR, we found that MRP1 transports arsenite (As(III)) only in the presence of GSH but does not transport arsenate (As(V)) (with or without GSH). The non-reducing GSH analogs L-gamma-glutamyl-L-alpha-aminobutyryl glycine and S-methyl GSH did not support As(III) transport, indicating that the free thiol group of GSH is required. GSH-dependent transport of As(III) was 2-fold higher at pH 6.5-7 than at a more basic pH, consistent with the formation and transport of the acid-stable arsenic triglutathione (As(GS)(3)). Immunoblot analysis of H69AR vesicles revealed the unexpected membrane association of GSH S-transferase P1-1 (GSTP1-1). Membrane vesicles from an MRP1-transfected HeLa cell line lacking membrane-associated GSTP1-1 did not transport As(III) even in the presence of GSH but did transport synthetic As(GS)(3). The addition of exogenous GSTP1-1 to HeLa-MRP1 vesicles resulted in GSH-dependent As(III) transport. The apparent K(m) of As(GS)(3) for MRP1 was 0.32 microM, suggesting a remarkably high relative affinity. As(GS)(3) transport by MRP1 was osmotically sensitive and was inhibited by several conjugated organic anions (MRP1 substrates) as well as the metalloid antimonite (K(i) 2.8 microM). As(GS)(3) transport experiments using MRP1 mutants with substrate specificities differing from wild-type MRP1 suggested a commonality in the substrate binding pockets of As(GS)(3) and leukotriene C(4). Finally, human MRP2 also transported As(GS)(3). In conclusion, MRP1 transports inorganic arsenic as a tri-GSH conjugate, and GSTP1-1 may have a synergistic role in this process.

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No. Sentence Comment
69 MRP1 and MRP2 Expression Vectors and Transfections in HEK293T Cells-The construction and expression of wild-type MRP1 (WT-MRP1), wild-type MRP2 (WT-MRP2), and the MRP1 mutants K332L, D336K, K319D, and K347D in HEK293T cells have been described previously (31, 35-37). Login to comment
204 To determine whether these amino acid residues are critical for transport of As(GS)3, transport assays were done using membrane vesicles prepared from HEK293T cells transfected with pcDNA3.1(-) (empty vector), WT-MRP1, D336K-MRP1, K332L-MRP1, K319D-MRP1, and K347D-MRP1 cDNAs. Login to comment
206 Consistent with the selective loss of LTC4 and GSH transport by K332L-MRP1 and the general loss of transport function by D336K-MRP1, these mutants also did not transport As(GS)3 (Fig. 8B). Login to comment
222 As(GS)3 transport by wild-type and mutant K332L, D336K, K319D, K347D-MRP1. Login to comment
223 A, membrane vesicles prepared from HEK293T cells transfected with empty vector (pcDNA3.1(-)), wild-type MRP1 (WT-MRP1), or mutant MRP1 (K332L-MRP1, D336K-MRP1, K319D-MRP1, or K347D-MRP1) were immunoblotted with the MRP1-specific mAb QCRL-1 as described under "Experimental Procedures." Login to comment

[hide] A molecular understanding of ATP-dependent solute ... Cancer Metastasis Rev. 2007 Mar;26(1):15-37. Chang XB
A molecular understanding of ATP-dependent solute transport by multidrug resistance-associated protein MRP1.
Cancer Metastasis Rev. 2007 Mar;26(1):15-37., [PMID:17295059]

Abstract [show]
Over a million new cases of cancers are diagnosed each year in the United States and over half of these patients die from these devastating diseases. Thus, cancers cause a major public health problem in the United States and worldwide. Chemotherapy remains the principal mode to treat many metastatic cancers. However, occurrence of cellular multidrug resistance (MDR) prevents efficient killing of cancer cells, leading to chemotherapeutic treatment failure. Numerous mechanisms of MDR exist in cancer cells, such as intrinsic or acquired MDR. Overexpression of ATP-binding cassette (ABC) drug transporters, such as P-glycoprotein (P-gp or ABCB1), breast cancer resistance protein (BCRP or ABCG2) and/or multidrug resistance-associated protein (MRP1 or ABCC1), confers an acquired MDR due to their capabilities of transporting a broad range of chemically diverse anticancer drugs. In addition to their roles in MDR, there is substantial evidence suggesting that these drug transporters have functions in tissue defense. Basically, these drug transporters are expressed in tissues important for absorption, such as in lung and gut, and for metabolism and elimination, such as in liver and kidney. In addition, these drug transporters play an important role in maintaining the barrier function of many tissues including blood-brain barrier, blood-cerebral spinal fluid barrier, blood-testis barrier and the maternal-fetal barrier. Thus, these ATP-dependent drug transporters play an important role in the absorption, disposition and elimination of the structurally diverse array of the endobiotics and xenobiotics. In this review, the molecular mechanism of ATP-dependent solute transport by MRP1 will be addressed.

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109 P343A, K332L and K332D mutations in TM6 resulted in significantly reduced transport of some organic anion substrates [75, 76]. Login to comment

[hide] Structural determinants of substrate specificity d... Drug Metab Dispos. 2008 Dec;36(12):2571-81. Epub 2008 Sep 5. Grant CE, Gao M, DeGorter MK, Cole SP, Deeley RG
Structural determinants of substrate specificity differences between human multidrug resistance protein (MRP) 1 (ABCC1) and MRP3 (ABCC3).
Drug Metab Dispos. 2008 Dec;36(12):2571-81. Epub 2008 Sep 5., [PMID:18775981]

Abstract [show]
Multidrug resistance proteins (MRPs) are members of the "C" branch of the ATP-binding cassette transporter superfamily. Human MRP1 transports a wide range of natural product drugs and structurally diverse conjugated and unconjugated organic anions. Its closest relative is MRP3. Despite their structural similarity, the homologs differ substantially in their substrate specificity. It is noteworthy that MRP1 transports glutathione (GSH) and GSH conjugates and displays GSH-stimulated transport of a number of unconjugated and conjugated compounds. In contrast, MRP3 does not transport GSH and is a poor transporter of GSH conjugates. However, both proteins transport glucuronide conjugates, such as 17beta-estradiol 17-(beta-D-glucuronide). We have constructed a series of MRP1/MRP3 hybrids and used them to identify a region of MRP1 that is critical for binding and transport of GSH conjugates such as leukotriene C(4) (LTC(4)). Substitution of this region encompassing transmembrane helices 8 and 9 and portions of cytoplasmic loops 4 and 5 of MRP1 with the equivalent region of MRP3 eliminated LTC(4) transport. Transport of other substrates was either unaffected or enhanced. We identified three residues in this region: Tyr(440), Ile(441), and Met(443), mutation of which differentially affected transport. It is noteworthy that substitution of Tyr(440) with Phe, as found in MRP3, reduced LTC(4) and GSH-stimulated estrone-3-sulfate transport without affecting transport of other substrates tested. The mutation increased the K(m) for LTC(4) 5-fold and substantially reduced photolabeling of MRP1 by both [3H]LTC(4) and the GSH derivative, azidophenacyl-[35S]GSH. These results suggest that Tyr(440) makes a major contribution to recognition of GSH and the GSH moiety of conjugates such as LTC(4).

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305 In addition, photolabeling with [3 H]LTC4 of K332D and K332L mutant proteins was severely reduced compared with wild-type MRP1. Login to comment

[hide] Molecular basis for reduced estrone sulfate transp... Drug Metab Dispos. 2009 Jul;37(7):1411-20. Epub 2009 Apr 27. Maeno K, Nakajima A, Conseil G, Rothnie A, Deeley RG, Cole SP
Molecular basis for reduced estrone sulfate transport and altered modulator sensitivity of transmembrane helix (TM) 6 and TM17 mutants of multidrug resistance protein 1 (ABCC1).
Drug Metab Dispos. 2009 Jul;37(7):1411-20. Epub 2009 Apr 27., [PMID:19398503]

Abstract [show]
Multidrug resistance protein 1 (MRP1) confers drug resistance and also mediates cellular efflux of many organic anions. MRP1 also transports glutathione (GSH); furthermore, this tripeptide stimulates transport of several substrates, including estrone 3-sulfate. We have previously shown that mutations of Lys(332) in transmembrane helix (TM) 6 and Trp(1246) in TM17 cause different substrate-selective losses in MRP1 transport activity. Here we have extended our characterization of mutants K332L and W1246C to further define the different roles these two residues play in determining the substrate and inhibitor specificity of MRP1. Thus, we have shown that TM17-Trp(1246) is crucial for conferring drug resistance and for binding and transport of methotrexate, estradiol glucuronide, and estrone 3-sulfate, as well as for binding of the tricyclic isoxazole inhibitor N-[3-(9-chloro-3-methyl-4-oxo-4H-isoxazolo-[4,3-c]quinolin-5-yl)-cyclohexy lmethyl]-benzamide (LY465803). In contrast, TM6-Lys(332) is important for enabling GSH and GSH-containing compounds to serve as substrates (e.g., leukotriene C(4)) or modulators (e.g., S-decyl-GSH, GSH disulfide) of MRP1 and, further, for enabling GSH (or S-methyl-GSH) to enhance the transport of estrone 3-sulfate and increase the inhibitory potency of LY465803. On the other hand, both mutants are as sensitive as wild-type MRP1 to the non-GSH-containing inhibitors (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3- oxopropyl]thio]methyl]thio]-propanoic acid (MK571), 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazol-5-yl)butoxy]phenyl]-ethanone (LY171883), and highly potent 6-[4'-carboxyphenylthio]-5[S]-hydroxy-7[E], 11[Z]14[Z]-eicosatetrenoic acid (BAY u9773). Finally, the differing abilities of the cysteinyl leukotriene derivatives leukotriene C(4), D(4), and F(4) to inhibit estradiol glucuronide transport by wild-type and K332L mutant MRP1 provide further evidence that TM6-Lys(332) is involved in the recognition of the gamma-Glu portion of substrates and modulators containing GSH or GSH-like moieties.

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3 Here we have extended our characterization of mutants K332L and W1246C to further define the different roles these two residues play in determining the substrate and inhibitor specificity of MRP1. Login to comment
7 Finally, the differing abilities of the cysteinyl leukotriene derivatives leukotriene C4, D4, and F4 to inhibit estradiol glucuronide transport by wild-type and K332L mutant MRP1 provide further evidence that TM6-Lys332 is involved in the recognition of the ␥-Glu portion of substrates and modulators containing GSH or GSH-like moieties. Login to comment
36 B-E, ATP-dependent uptake of 3 H-labeled organic anions was measured in membrane vesicles prepared from HEK293T (HEK) cells transfected with wild-type MRP1 (shaded bar) and K332L and W1246C mutant (open bars) cDNAs as described under Materials and Methods. Login to comment
57 The generation of wild-type, K332L, and W1246C mutant MRP1 pcDNA3.1 expression constructs has been described previously (Ito et al., 2001a; Haimeur et al., 2002). Login to comment
85 Equilibrium binding of [3 H]estrone 3-sulfate to wild-type MRP1 and the K332L and W1246C mutants was determined as described previously (Rothnie et al., 2006, 2008). Login to comment
91 To investigate the effect of estrone 3-sulfate concentration on [3 H]estrone 3-sulfate binding, 10 ␮g of membrane protein from wild-type and mutant MRP1-transfected cells were incubated with various concentrations of [3 H]estrone 3-sulfate (50 nM to 10 ␮M) and 3 mM S-MeGSH in the presence or absence of the competitive substrates E217betaG (1 mM) in the case of the K332L mutant and LTC4 (10 ␮M) in the case of the W1246C mutant. Login to comment
95 In the first series of experiments, the ability of the K332L and W1246C mutants to transport methotrexate and estrone 3-sulfate (in the presence of GSH and S-MeGSH) was measured. Login to comment
96 After transfecting HEK293T cells with the wild-type and mutant constructs, membrane vesicles were prepared, and relative expression levels of the K332L and W1246C mutants were determined by immunoblotting and found to be similar to wild-type MRP1 (data not shown), as reported previously (Ito et al., 2001a; Haimeur et al., 2002). Login to comment
98 As shown in Fig. 1B, uptake of [3 H]methotrexate by the K332L mutant was comparable with wild-type MRP1, whereas uptake by W1246C was reduced by Ͼ75%. Login to comment
100 As reported previously (Ito et al., 2001a; Haimeur et al., 2002), [3 H]LTC4 transport by the K332L mutant was also comparable with the untransfected HEK293T negative control, whereas that of the W1246C mutant was similar to wild-type MRP1 (Fig. 1D). Login to comment
101 On the other hand, the K332L mutant transported [3 H]E217betaG at levels comparable with wild-type MRP1, whereas the W1246C mutant showed no detectable transport of this conjugated estrogen, as expected from previous studies (Ito et al., 2001a) (Fig. 1E). Login to comment
102 S-MeGSH Fails to Increase Estrone 3-Sulfate Binding to the K332L and W1246C Mutants. Login to comment
103 To determine whether changes in the ability of S-MeGSH to stimulate binding might contribute to the low levels of estrone 3-sulfate transport displayed by the K332L and W1246C mutants, equilibrium binding assays with MRP1 in the nucleotide-free state were carried out. Login to comment
107 In contrast, S-MeGSH had little or no effect on estrone 3-sulfate binding to the K332L (Fig. 2A) or W1246C (Fig. 2B) mutants. Login to comment
108 The EC50 for S-MeGSH of wild-type MRP1 was 1.24 Ϯ 0.07 mM, whereas the binding curves for the K332L and W1246C mutants were almost flat, precluding reliable estimations of their EC50s for this tripeptide. Login to comment
109 Estrone 3-sulfate binding values for both the K332L and W1246C mutants at apparent saturation were just 20% that of wild-type MRP1 (after subtraction of nonspecific binding). Login to comment
110 To exclude the possibility that S-MeGSH-stimulated estrone 3-sulfate binding to the K332L and W1246C mutants might be underestimated in the above experiments because the initial concentration of estrone 3-sulfate used (50 nM) was too low, the binding assays were repeated at higher concentrations of this conjugated estrogen. Login to comment
112 K332L Decreases Ability of S-MeGSH to Stimulate Estrone 3-Sulfate Uptake, Whereas W1246C Decreases Affinity for Estrone 3-Sulfate. Login to comment
114 S-MeGSH stimulated binding of [3 H]estrone 3-sulfate by MRP1 mutants K332L and W1246C. Login to comment
115 A, binding of [3 H]estrone 3-sulfate (50 nM) to wild-type MRP1 (f), K332L mutant (F), and control HEK (E) membranes (10 ␮g of protein) was measured at 23°C for 60 min in the presence of increasing concentrations (0.01-18 mM) of S-MeGSH. Login to comment
119 C, specific binding of [3 H]estrone 3-sulfate to wild-type MRP1 (f), W1246C (Œ), K332L (F), and control HEK (E) membranes (10 ␮g of protein) was measured at estrone 3-sulfate concentrations ranging from 0.01 to 10 mM in the presence of 3 mM S-MeGSH. Login to comment
123 To help distinguish between these possibilities, ATP-dependent estrone 3-sulfate transport by the K332L and W1246C mutants was measured in the absence of S-MeGSH (or GSH) and compared with transport by wild-type MRP1. Login to comment
124 As shown in Fig. 3A, [3 H]estrone 3-sulfate uptake in the absence of S-MeGSH (or GSH) by K332L was similar to that of wild-type MRP1. Login to comment
127 These observations indicate that like wild-type MRP1, the K332L mutant can still bind and transport estrone 3-sulfate in the absence of S-MeGSH (or GSH), but unlike wild-type MRP1, estrone 3-sulfate uptake by the K332L mutant is no longer stimulated by S-MeGSH. Login to comment
128 Conversely, unlike wild-type and K332L mutant MRP1, W1246C apparently no longer binds estrone 3-sulfate; thus, no transport is observed in either the presence or absence of S-MeGSH (or GSH). Login to comment
129 Loss of Synergistic Inhibition of E217betaG Uptake by GSH and Apigenin for the K332L Mutant. Login to comment
131 Therefore, we reasoned that if the K332L mutant could no longer bind GSH (or S-MeGSH), then GSH (or S-MeGSH) would no longer be expected to act synergistically with apigenin to inhibit uptake of LTC4 or other organic anion substrates. Login to comment
132 Accordingly, ATP-dependent E217betaG uptake by K332L-enriched membrane vesicles was measured in the absence or presence of GSH (3 mM) and apigenin (10 ␮M). Login to comment
135 In contrast, neither GSH or apigenin (10 ␮M) alone nor the combination of GSH and apigenin had any effect on E217betaG uptake by the K332L mutant. Login to comment
137 Because higher concentrations of apigenin (30 ␮M) inhibited E217betaG transport by wild-type and K332L MRP1 similarly (data not shown), these observations support the conclusion that GSH (and S-MeGSH) binding to the K332L mutant is severely impaired. Login to comment
138 Effect of MRP1 Modulators on the Activity of MRP1 Mutants K332L and W1246C. Login to comment
139 Because of the marked differences in the substrate specificity of the K332L and W1246C mutants, we next investigated whether the two mutants differed in their sensitivity to compounds reported previously to be modulators of MRP1 transport activity. Login to comment
140 Thus, the effect of four small molecule inhibitors (MK571, LY465803, LY171883, and BAY u9773) (Fig. 4A) and two GSH derivatives (S-decyl-GSH and GSSG) (Fig. 4B) were tested for their ability to inhibit E217betaG transport by the K332L mutant. Login to comment
145 As summarized in Table 1 and shown in Fig. 5, MK571 and LY171883 were moderately (2-fold) less potent inhibitors of E217betaG uptake by wild-type MRP1 than by the K332L mutant, whereas the inhibitory potency of BAYu9773 was not significantly different for the mutant and wild-type transporters. Login to comment
146 In contrast, LY465803 (ϩGSH) showed a far greater potency (16-fold) to inhibit wild-type compared with K332L mutant MRP1. Login to comment
147 In a similar manner, the GSH derivative S-decyl-GSH and GSSG inhibited wild-type MRP1 activity with IC50s Ͼ100-fold and Ͼ30-fold lower than for K332L, respectively. Login to comment
148 In contrast to its more potent inhibitory effect on E217betaG transport by the K332L mutant, MK571 was a less potent inhibitor (ϳ3-fold) of LTC4 transport by the W1246C mutant than by wild-type MRP1 (Table 2; Fig. 6A). Login to comment
149 However, similar to K332L, the inhibitory potency of BAY u9773 for LTC4 transport by wild-type and W1246C was comparable (Table 2; Fig. 6B). Login to comment
150 Also similar to K332L, LY465803 (ϩGSH) showed a far greater (Ͼ250-fold) potency to inhibit wild-type MRP1 than the W1246C mutant (Table 2; Fig. 6C). Login to comment
151 Finally, in contrast to K332L, the sensitivity of W1246C and wild-type MRP1 to FIG. 3. Login to comment
152 [3 H]Estrone 3-sulfate and [3 H]E217betaG vesicular uptake by K332L and W1246C mutant and wild-type MRP1 proteins. Login to comment
153 A, ATP-dependent [3 H]estrone 3-sulfate uptake by membrane vesicles prepared from untransfected control cells (HEK) (solid bar) and cells transfected with cDNA vectors containing wild-type MRP1 (shaded bar), and K332L and W1246C mutant MRP1 (open bars) was measured in the absence of S-MeGSH. Login to comment
154 Inset, [3 H]estrone 3-sulfate uptake by membrane vesicles prepared from untransfected HEK cells (solid bars) and HEK cells expressing comparable levels of wild-type MRP1 (shaded bars), and MRP1 mutants K332L and W1246C (open bars) was measured in the absence and presence of S-MeGSH as described under Materials and Methods. Login to comment
157 B, ATP-dependent [3 H]E217betaG uptake was measured in membrane vesicles prepared from untransfected cells (HEK) (solid bars), and wild-type MRP1 (shaded bars), and K332L-MRP1 (open bars) transfected cells with and without GSH (3 mM) and with and without apigenin (10 ␮M) as indicated. Login to comment
160 Effect of Leukotriene Derivatives on E217betaG Transport Activity by K332L. Login to comment
162 Thus, the equal sensitivity of the transport activities of the K332L and W1246C mutants to inhibition by BAY u9773, despite their profoundly different abilities to transport LTC4, suggests that the K332L mutant has lost its ability to bind to the GSH moiety of LTC4. Login to comment
163 To determine whether this loss of binding involves one or more of the three amino acids that comprise the GSH moiety of LTC4, E217betaG transport assays with wild-type MRP1 and the K332L mutant were carried out in the presence of various concentrations of the cysteinyl leukotriene derivatives LTC4, LTD4, and LTF4. Login to comment
164 As expected from previous studies (Haimeur et al., 2002), the IC50 of LTC4 (containing ␥-Glu, Cys, and Gly) of the K332L mutant was significantly increased (ϳ25-fold) compared with that of wild-type MRP1 (Table 3; Fig. 7A). Login to comment
165 When E217betaG transport was carried out in the presence of LTD4 (lacking a ␥-Glu moiety but containing Gly and Cys), the IC50s of wild-type MRP1 and the K332L mutant were the same, although the inhibitor potency of LTD4 compared with LTC4 was ϳ40-fold less for wild-type MRP1 but just ϳ2-fold less for K332L (Table 3; Fig. 7B). Login to comment
166 The IC50s of LTF4 (containing a ␥-Glu moiety but lacking Gly) for K332L and wild-type MRP1 differed by 4-fold, with the inhibitor potency of LTF4 comparable with LTD4 (Table 3; Fig. 7C). Login to comment
167 Thus, the K332L mutant protein is considerably less sensitive than wild-type MRP1 with respect to inhibition by the ␥-Glu-containing LTC4 and LTF4 but not the ␥-Glu-less LTD4, suggesting that Lys332 is critical for recognizing the ␥-Glu moiety of GSH. Login to comment
168 TABLE 1 Effect of modulators on E217betaG uptake by wild-type and K332L mutant MRP1 The values shown represent the means (ϮS.D.) of IC50 values obtained in three independent experiments. Login to comment
170 E217betaG Uptake IC50 Modulator Wild-type K332L ␮M ␮M MK571 0.4 Ϯ 0.1 0.20 Ϯ 0.07a LY171883 19.8, 14.7 8.8, 8.1 BAY u9773 0.20 Ϯ 0.03 0.3 Ϯ 0.1 LY465803 (ϩGSH) 0.2 Ϯ 0.1 3.3 Ϯ 2.5b S-Decyl-GSH 0.07 Ϯ 0.01 Ͼ10, Ͼ10c GSSG 16.3, 13.0 503.0, 505.8 a Significantly different from IC50 obtained with wild-type MRP1 (p Ͻ 0.05). Login to comment
180 In the present study, we have further characterized the substrate specificity of two of these, the TM6 mutant K332L and the TM17 mutant W1246C, and have also determined their inhibitor profiles. Login to comment
189 Effect of MRP1 modulators and GSH derivatives on [3 H]E217betaG transport by wild-type and K332L mutant MRP1. ATP-dependent vesicular uptake of [3 H]E217betaG by wild-type MRP1 (open symbols, dotted lines) and K332L mutant MRP1 (closed symbols, solid lines) was measured in the presence of the indicated concentrations of modulators and GSH derivatives. Login to comment
198 However, this was not the case for membranes prepared from transfected HEK cells expressing either the K332L or W1246C mutants. Login to comment
200 We found that GSH-independent estrone 3-sulfate transport by W1246C was similar to negative controls, whereas transport by K332L was comparable with wild-type MRP1. Login to comment
201 Thus, in the case of the K332L mutant, impaired GSH-stimulated estrone 3-sulfate transport seems to be caused by a loss of GSH binding. Login to comment
203 Furthermore, unlike wild-type MRP1, GSH no longer acted synergistically with apigenin to inhibit E217betaG uptake by the K332L mutant. Login to comment
205 The latter conclusion is also consistent with the inhibitor profile displayed by the K332L mutant (Table 1; Fig. 5). Login to comment
206 The inhibitory potencies of modulators such as MK571, LY171883, and BAY u9773, which do not contain a GSH moiety and are not GSH-dependent, on the transport activities of wild-type and K332L mutant MRP1 were much more similar (1.5-2-fold difference in IC50s) than those of modulators that contain a GSH or GSH-like moiety (S-decyl-GSH, GSSG) or are dependent on GSH (LY465803) for their activity (15-30-fold difference in IC50s). Login to comment
213 When transport assays were carried out in the presence of three cysteinyl leukotriene derivatives to determine whether reduced binding of compounds that contain GSH-like moieties by the K332L mutant involves one or more of the three amino acids that comprise FIG. 6. Login to comment
219 TABLE 3 Effect of leukotriene derivatives on E217betaG uptake by wild-type and K332L mutant MRP1 The values shown represent the means (ϮS.D.) of IC50 values obtained in three or four independent experiments (number shown in parentheses). Login to comment
220 E217betaG Uptake IC50 Leukotriene Derivative Wild-type K332L ␮M ␮M LTC4 0.1 Ϯ 0.03 (3) 2.5 Ϯ 0.5 (3)a LTD4 4.3 Ϯ 0.4 (3) 4.2 Ϯ 2.3 (3) LTF4 3.7 Ϯ 0.1 (4) 15.3 Ϯ 7.3 (4)a a Significantly different from IC50 in wild-type MRP1 (p Ͻ 0.01). Login to comment
222 Thus, E217betaG transport by the K332L mutant was considerably less sensitive than wild-type MRP1 to inhibition by the ␥-Glu- containing LTC4 and LTF4 (25- and 4-fold differences in IC50s, respectively) than for the ␥-Glu-less LTD4, for which the IC50s were the same (Fig. 7; Table 3). Login to comment
229 In conclusion, we have extended our characterization of the MRP1 mutants K332L and W1246C and further defined the different roles of these two amino acids in determining the substrate and inhibitor specificity of MRP1. Login to comment

[hide] Molecular mechanism of ATP-dependent solute transp... Methods Mol Biol. 2010;596:223-49. Chang XB
Molecular mechanism of ATP-dependent solute transport by multidrug resistance-associated protein 1.
Methods Mol Biol. 2010;596:223-49., [PMID:19949927]

Abstract [show]
Millions of new cancer patients are diagnosed each year and over half of these patients die from this devastating disease. Thus, cancer causes a major public health problem worldwide. Chemotherapy remains the principal mode to treat many metastatic cancers. However, occurrence of cellular multidrug resistance (MDR) prevents efficient killing of cancer cells, leading to chemotherapeutic treatment failure. Over-expression of ATP-binding cassette transporters, such as P-glycoprotein, breast cancer resistance protein and/or multidrug resistance-associated protein 1 (MRP1), confers an acquired MDR due to their capabilities of transporting a broad range of chemically diverse anticancer drugs across the cell membrane barrier. In this review, the molecular mechanism of ATP-dependent solute transport by MRP1 will be addressed.

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No. Sentence Comment
104 Mutations of C43S in TM1 (112); P343A, K332L and K332D in TM6 (113, 114); W445A and P448A in TM8 (113, 115); T550A, T556A and P557A in TM10 (113, 116); N590A, F594A, P595A, N597A, S604A and S605A in TM11 (113, 117, 118); E1089Q, E1089A, E1089L, E1089N, K1092, S1097 and N1100 in TM14 (119, 120); R1197K in TM16 (121); Y1236F, T1241A, T1242A, T1242C, T1242S, T1242L, Y1243F, N1245A, W1246C, W1246A, W1246F, W1246Y or R1249K in TM17 (121-124) significantly affect MRP1 function. Login to comment

[hide] Structural and functional properties of human mult... Curr Med Chem. 2011;18(3):439-81. He SM, Li R, Kanwar JR, Zhou SF
Structural and functional properties of human multidrug resistance protein 1 (MRP1/ABCC1).
Curr Med Chem. 2011;18(3):439-81., [PMID:21143116]

Abstract [show]
Multidrug ABC transporters such as P-glycoprotein (P-gp/MDR1/ABCB1) and multidrug resistance protein 1 (MRP1/ABCC1) play an important role in the extrusion of drugs from the cell and their overexpression can be a cause of failure of anticancer and antimicrobial chemotherapy. Recently, the mouse P-gp/Abcb1a structure has been determined and this has significantly enhanced our understanding of the structure-activity relationship (SAR) of mammalian ABC transporters. This paper highlights our current knowledge on the structural and functional properties and the SAR of human MRP1/ABCC1. Although the crystal structure of MRP1/ABCC1 has yet to be resolved, the current topological model of MRP1/ABCC1 contains two transmembrane domains (TMD1 and TMD2) each followed by a nucleotide binding domain (NBD) plus a third NH2-terminal TMD0. MRP1/ABCC1 is expressed in the liver, kidney, intestine, brain and other tissues. MRP1/ABCC1 transports a structurally diverse array of important endogenous substances (e.g. leukotrienes and estrogen conjugates) and xenobiotics and their metabolites, including various conjugates, anticancer drugs, heavy metals, organic anions and lipids. Cells that highly express MRP1/ABCC1 confer resistance to a variety of natural product anticancer drugs such as vinca alkaloids (e.g. vincristine), anthracyclines (e.g. etoposide) and epipodophyllotoxins (e.g. doxorubicin and mitoxantrone). MRP1/ABCC1 is associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. However, most compounds that efficiently reverse P-gp/ABCB1-mediated multidrug resistance have only low affinity for MRP1/ABCC1 and there are only a few effective and relatively specific MRP1/ABCC1 inhibitors available. A number of site-directed mutagenesis studies, biophysical and photolabeling studies, SAR and QSAR, molecular docking and homology modeling studies have documented the role of multiple residues in determining the substrate specificity and inhibitor selectivity of MRP1/ABCC1. Most of these residues are located in the TMs of TMD1 and TMD2, in particular TMs 4, 6, 7, 8, 10, 11, 14, 16, and 17, or in close proximity to the membrane/cytosol interface of MRP1/ABCC1. The exact transporting mechanism of MRP1/ABCC1 is unclear. MRP1/ABCC1 and other multidrug transporters are front-line mediators of drug resistance in cancers and represent important therapeutic targets in future chemotherapy. The crystal structure of human MRP1/ABCC1 is expected to be resolved in the near future and this will provide an insight into the SAR of MRP1/ABCC1 and allow for rational design of anticancer drugs and potent and selective MRP1/ABCC1 inhibitors.

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No. Sentence Comment
765 Both Lys332Leu and Trp1246Cys mutants were as sensitive as wild-type MRP1/ABCC1 to MK-571, LY171883, and the potent MRP1/ABCC1 inhibitor 6-[4 -carboxyphenylthio]-5[S]- hydroxy-7[E], 11[Z]14[Z]-eicosatetrenoic acid (BAY u9773, a leukotriene-like dual antagonist that acts on both CysLT1 and CysLT2 receptors) [341]. Login to comment
766 MK-571 and LY171883 were moderately (2-fold) less potent inhibitors of E217 G uptake by wild-type MRP1/ABCC1 than by the Lys332Leu mutant, but LY465803 showed 16-fold greater potency to inhibit wild-type compared with Lys332Leu mutant. Login to comment
767 In a similar manner, the GSH derivative S-decyl-GSH and GSSG inhibited wild-type MRP1/ABCC1 activity with IC50 >100-fold and >30-fold lower than for Lys332Leu, respectively [341]. Login to comment
768 In contrast to its more potent inhibitory effect on E217 G transport by the Lys332Leu mutant, MK-571 was a less potent inhibitor ( 3-fold) of LTC4 transport by the Trp1246Cys mutant than by wild-type MRP1. Login to comment
769 However, similar to Lys332Leu, the inhibitory potency of BAY u9773 for LTC4 transport by wild-type and Trp1246Cys was comparable. Login to comment