ABCMdb

ABCC1 p.Lys1333Leu

Predicted by SNAP2:	A: D (95%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
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: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Allosteric interactions between the two non-equiva... J Biol Chem. 2000 Jul 7;275(27):20280-7. Hou Y, Cui L, Riordan JR, Chang X
Allosteric interactions between the two non-equivalent nucleotide binding domains of multidrug resistance protein MRP1.
J Biol Chem. 2000 Jul 7;275(27):20280-7., 2000-07-07 [PMID:10781583]

Abstract [show]
Membrane transporters of the adenine nucleotide binding cassette (ABC) superfamily utilize two either identical or homologous nucleotide binding domains (NBDs). Although the hydrolysis of ATP by these domains is believed to drive transport of solute, it is unknown why two rather than a single NBD is required. In the well studied P-glycoprotein multidrug transporter, the two appear to be functionally equivalent, and a strongly supported model proposes that ATP hydrolysis occurs alternately at each NBD (Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett 377, 285-289). To assess how applicable this model may be to other ABC transporters, we have examined adenine nucleotide interactions with the multidrug resistance protein, MRP1, a member of a different ABC family that transports conjugated organic anions and in which sequences of the two NBDs are much less similar than in P-glycoprotein. Photoaffinity labeling experiments with 8-azido-ATP, which strongly supports transport revealed ATP binding exclusively at NBD1 and ADP trapping predominantly at NBD2. Despite this apparent asymmetry in the two domains, they are entirely interdependent as substitution of key lysine residues in the Walker A motif of either impaired both ATP binding and ADP trapping. Furthermore, the interaction of ADP at NBD2 appears to allosterically enhance the binding of ATP at NBD1. Glutathione, which supports drug transport by the protein, does not enhance ATP binding but stimulates the trapping of ADP. Thus MRP1 may employ a more complex mechanism of coupling ATP utilization to the export of agents from cells than P-glycoprotein.

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33 Stable cell lines expressing wild-type and mutant MRP1s, K684L, D792L/D793L, K1333L, and D1454L/E1455L, were generated by using procedures described previously (11). Login to comment
205 Fig. 5C shows that although labeling by N3[␣-32 P]ATP was not abolished by the mutations, it was greatly reduced: K684L was ϳ10% of wild-type; K1333L, ϳ5%; D1454L/D1455L, ϳ15%. Login to comment
207 Fig. 5D demonstrates that labeling of K684L by N3[␥-32 P]ATP was almost eliminated and labeling of K1333L and D1454L/E1455L were decreased to ϳ10% and ϳ15% of the wild-type levels, respectively. Login to comment
213 The K1333L mutation in NBD2 nearly abolished ATP-dependent uptake as did the NBD2 Walker B substitution, whereas the K684L substitution reduced it by approximately half. Login to comment
232 Lane 1, 10 ␮g of wild-type MRP1; lane 2, 20 ␮g of K684L; lane 3, 10 ␮g of K1333L; lane 4, 10 ␮g of D1454L/ E1455L. Login to comment
234 Lane 1, 10 ␮g of wild-type MRP1; lane 2, 20 ␮g of K684L; lane 3, 10 ␮g of K1333L; lane 4, 10 ␮g of D1454L/ E1455L. Login to comment
235 E, ATP-dependent LTC4 uptake by membrane vesicles containing wild-type (closed diamonds) and mutant MRPs: NBD1 Walker A lysine mutant K684L (open circles), NBD2 Walker A lysine mutant K1333L (open square), NBD2 Walker B aspartate mutant D1454L/ E1455L (closed circles). Login to comment

[hide] ATP binding to the first nucleotide-binding domain... J Biol Chem. 2002 Feb 15;277(7):5110-9. Epub 2001 Dec 7. Hou YX, Cui L, Riordan JR, Chang XB
ATP binding to the first nucleotide-binding domain of multidrug resistance protein MRP1 increases binding and hydrolysis of ATP and trapping of ADP at the second domain.
J Biol Chem. 2002 Feb 15;277(7):5110-9. Epub 2001 Dec 7., 2002-02-15 [PMID:11741902]

Abstract [show]
Multidrug resistance protein (MRP1) utilizes two non-equivalent nucleotide-binding domains (NBDs) to bind and hydrolyze ATP. ATP hydrolysis by either one or both NBDs is essential to drive transport of solute. Mutations of either NBD1 or NBD2 reduce solute transport, but do not abolish it completely. How events at these two domains are coordinated during the transport cycle have not been fully elucidated. Earlier reports (Gao, M., Cui, H. R., Loe, D. W., Grant, C. E., Almquist, K. C., Cole, S. P., and Deeley, R. G. (2000) J. Biol. Chem. 275, 13098-13108; Hou, Y., Cui, L., Riordan, J. R., and Chang, X. (2000) J. Biol. Chem. 275, 20280-20287) indicate that intact ATP is observed bound at NBD1, whereas trapping of the ATP hydrolysis product, ADP, occurs predominantly at NBD2 and that trapping of ADP at NBD2 enhances ATP binding at NBD1 severalfold. This suggested transmission of a positive allosteric interaction from NBD2 to NBD1. To assess whether ATP binding at NBD1 can enhance the trapping of ADP at NBD2, photoaffinity labeling experiments with [alpha-(32)P]8-N(3)ADP were performed and revealed that when presented with this compound labeling of MRP1 occurred at both NBDs. However, upon addition of ATP, this labeling was enhanced 4-fold mainly at NBD2. Furthermore, the nonhydrolyzable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP), bound preferentially to NBD1, but upon addition of a low concentration of 8-N(3)ATP, the binding at NBD2 increased severalfold. This suggested that the positive allosteric stimulation from NBD1 actually involves an increase in ATP binding at NBD2 and hydrolysis there leading to the trapping of ADP. Mutations of Walker A or B motifs in either NBD greatly reduced their ability to be labeled by [alpha-(32)P]8-N(3)ADP as well as by either [alpha-(32)P]- or [gamma-(32)P]8-N(3)ATP (Hou et al. (2000), see above). These mutations also strongly diminished the enhancement by ATP of [alpha-(32)P]8-N(3)ADP labeling and the transport activity of the protein. Taken together, these results demonstrate directly that events at NBD1 positively influence those at NBD2. The interactions between the two asymmetric NBDs of MRP1 protein may enhance the catalytic efficiency of the MRP1 protein and hence of its ATP-dependent transport of conjugated anions out of cells.

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50 Stable cell lines expressing wild-type and mutant MRP1s, K684L, D792A, K1333L, and D1454L/E1455L were established previously (2, 31). Login to comment
117 Essentially the stimulation was much reduced in the NBD1 mutants, K684L and D792A (Fig. 4, C and D), and in the NBD2 mutants, K1333L and D1454L/E1455L (Fig. 4, E and F), and the stimulation effects were shifted to higher ATP concentrations (Fig. 4, C-F). Login to comment
118 Trypsin digestion of either [␣-32 P]8-N3ATP or [␣-32 P]8N3ADP-labeled K1333L and D1454L/E1455L proved that the mutated NBD2 fragment can still be labeled (data not shown). Login to comment
173 Lane 1, 10 ␮g of wild-type MRP1; lane 2, 15 ␮g of K684L; lane 3, 20 ␮g of D792A; lane 4, 10 ␮g of K1333L; lane 5, 10 ␮g of D1454L/E1455L. Login to comment
175 The results for K684L and D792A are the average of three independent experiments and for K1333L and D1454L/E1455L are the average of two independent experiments. C, influence of ATP on the [␣-32 P]8-N3ADP labeling of K684L. Login to comment
178 E, influence of ATP on the [␣-32 P]8-N3ADP labeling of K1333L. Login to comment
179 10 ␮g of K1333L was labeled in the presence of varying amounts of ATP indicated above each lane. Login to comment

[hide] Verapamil and its derivative trigger apoptosis thr... Cancer Res. 2004 Jul 15;64(14):4950-6. Trompier D, Chang XB, Barattin R, du Moulinet D'Hardemare A, Di Pietro A, Baubichon-Cortay H
Verapamil and its derivative trigger apoptosis through glutathione extrusion by multidrug resistance protein MRP1.
Cancer Res. 2004 Jul 15;64(14):4950-6., 2004-07-15 [PMID:15256468]

Abstract [show]
This study demonstrates that verapamil and a newly synthesized verapamil derivative, NMeOHI(2), behave as apoptogens in multidrug resistance protein 1 (MRP1)-expressing cells. When treated with either verapamil or NMeOHI(2), surprisingly, baby hamster kidney-21 (BHK) cells transfected with human MRP1 were killed. Because parental BHK cells were not, as well as cells expressing an inactive (K1333L) MRP1 mutant, this indicated that cell death involved functional MRP1 transporter. Cell death was identified as apoptosis by using annexin V-fluorescein labeling and was no longer observed in the presence of the caspase inhibitor Z-Val-Ala-Asp(OMe)-CH(2)F (Z-VAD-FMK). In vitro, both verapamil and its derivative inhibited leukotriene C4 transport by MRP1-enriched membrane vesicles in a competitive manner, with a K(i) of 48.6 microm for verapamil and 5.5 microm for NMeOHI(2,) and stimulated reduced glutathione (GSH) transport 3-fold and 9-fold, respectively. Treatment of MRP1-expressing cells with either verapamil or the derivative quickly depleted intracellular GSH content with a strong decrease occurring in the first hour of treatment, which preceded cell death beginning at 8-16 h. Furthermore, addition of GSH to the media efficiently prevented cell death. Therefore, verapamil and its derivative trigger apoptosis through stimulation of GSH extrusion mediated by MRP1. This new information on the mechanism of induced apoptosis of MDR cells may represent a novel approach in the selective treatment of MRP1-positive tumors.

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5 Because parental BHK cells were not, as well as cells expressing an inactive (K1333L) MRP1 mutant, this indicated that cell death involved functional MRP1 transporter. Login to comment
33 BHK-21 cells stably transfected with either wild-type or (K1333L) mutant MRP1 have been described previously (20, 21). Login to comment
108 To check whether the MRP1 transporter was directly involved in hypersensitivity, BHK-21 cells transfected with an inactive MRP1 (K1333L) mutant (20) were analyzed for their sensitivity toward verapamil and its derivative. Login to comment
127 The lactate dehydrogenase release induced by verapamil (A) or NMeOHI2 (B) on BHK-21 control cells (᭛), or BHK-21 cells transfected with either wild-type (᭜) or K1333L mutant MRP1 (E), was determined in the incubation medium after 24-h treatment with the indicated concentrations. Login to comment
130 Both verapamil (Fig. 7A) and NMeOHI2 (Fig. 7B) induced a strong and fast (in Ͻ1 h) decrease in total cellular gluthatione content, whereas no significant decrease was observed with untransfected control cells (Fig. 7) or BHK-21 cells expressing the inactive MRP1 (K1333L) mutant (data not shown). Login to comment
184 Taking into account that no cytotoxicity and no GSH decrease were observed on either control cells or cells expressing an inactive (K1333L) MRP1 mutant, the involvement of active MRP1 clearly emerged as being responsible for direct efflux of GSH leading to intracellular depletion. Login to comment

[hide] Replacement of the positively charged Walker A lys... Biochem J. 2006 Jul 1;397(1):121-30. Buyse F, Hou YX, Vigano C, Zhao Q, Ruysschaert JM, Chang XB
Replacement of the positively charged Walker A lysine residue with a hydrophobic leucine residue and conformational alterations caused by this mutation in MRP1 impair ATP binding and hydrolysis.
Biochem J. 2006 Jul 1;397(1):121-30., 2006-07-01 [PMID:16551273]

Abstract [show]
MRP1 (multidrug resistance protein 1) couples ATP binding/hydrolysis at its two non-equivalent NBDs (nucleotide-binding domains) with solute transport. Some of the NBD1 mutants, such as W653C, decreased affinity for ATP at the mutated site, but increased the rate of ATP-dependent solute transport. In contrast, other NBD1 mutants, such as K684L, had decreased ATP binding and rate of solute transport. We now report that mutations of the Walker A lysine residue, K684L and K1333L, significantly alter the tertiary structure of the protein. Due to elimination of the positively charged group and conformational alterations, the K684L mutation greatly decreases the affinity for ATP at the mutated NBD1 and affects ATP binding at the unmutated NBD2. Although K684L-mutated NBD1 can bind ATP at higher concentrations, the bound nucleotide at that site is not efficiently hydrolysed. All these alterations result in decreased ATP-dependent solute transport to approx. 40% of the wild-type. In contrast, the K1333L mutation affects ATP binding and hydrolysis at the mutated NBD2 only, leading to decreased ATP-dependent solute transport to approx. 11% of the wild-type. Consistent with their relative transport activities, the amount of vincristine accumulated in cells is in the order of K1333L> or =CFTR (cystic fibrosis transmembrane conductance regulator)>K684L>>>wild-type MRP1. Although these mutants retain partial solute transport activities, the cells expressing them are not multidrug-resistant owing to inefficient export of the anticancer drugs by these mutants. This indicates that even partial inhibition of transport activity of MRP1 can reverse the multidrug resistance caused by this drug transporter.

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3 In contrast, other NBD1 mutants, such as K684L, had decreased ATP binding and rate of solute transport. We now report that mutations of the Walker A lysine residue, K684L and K1333L, significantly alter the tertiary structure of the protein. Login to comment
8 In contrast, the K1333L mutation affects ATP binding and hydrolysis at the mutated NBD2 only, leading to decreased ATP-dependent solute transport to approx. Login to comment
10 Consistent with their relative transport activities, the amount of vincristine accumulated in cells is in the order of K1333L CFTR (cystic fibrosis transmembrane conductance regulator) > K684L wild-type MRP1. Login to comment
40 However, this speculation cannot explain why the K684L mutation decreases affinity for ATP at the mutated NBD1, in other words, increases the release rate from the mutated NBD1, but does not increase the rate of ATP-dependent solute transport. We have now found that replacement of the Walker A lysine residue with a leucine residue in either NBD1 (K684L) or NBD2 (K1333L) significantly alters the tertiary structure of the protein and affects ATP binding/hydrolysis and ATP-dependent solute transport. Login to comment
47 Cell culture and cell lines expressing MRP1 Cell lines expressing wild-type, K684L- and K1333L-mutated MRP1s and CFTR (cystic fibrosis transmembrane conductance regulator) were established previously [16,24,25] (but see [25a]). Login to comment
66 Reconstitution of wild-type, K684L- and K1333L-mutated MRP1 was achieved by employing SM-2 Bio-Beads to remove detergent from the protein/detergent/ lipid mixture as described previously [28]. Login to comment
70 Sample preparation Reconstituted wild-type, K684L- or K1333L-mutated MRP1s (20 µg) was mixed with either ATP + Vi or AMP-PNP (the molar ratio of protein to ATP is 1:6). Login to comment
105 RESULTS Mutation of NBD2 has a greater effect on the ATP-dependent LTC4 transport than the corresponding mutation of NBD1 We have found that the K1333L-mutated MRP1 almost completely abolished ATP-dependent solute uptake, whereas the corresponding mutation in NBD1, K684L, reduced it by approximately half [16]. Login to comment
108 1.608 (K1333L) compared with wild-type MRP1 (Figure 1A). Login to comment
109 The ATP-dependent LTC4 transport activities of K684L- and K1333L-mutated MRP1, after adjusting the amount of MRP1 in the membrane vesicles to a similar amount with membrane vesicles containing CFTR, are approx. Login to comment
110 40% (K684L) and 11% (K1333L) of the wild-type MRP1 (Figure 1B), indicating that mutation of NBD2 has a larger effect on the ATP-dependent LTC4 transport than the corresponding mutation of NBD1. Login to comment
112 8% of the wild-type MRP1, implying that Figure 1 Expression and functional analysis of wild-type and Walker A lysine mutants (A) Expression of wild-type (WT), K684L- and K1333L-mutated MRP1 in BHK cells. Login to comment
113 Membrane vesicles were prepared from BHK cells expressing wild-type, K684L- or K1333L-mutated MRP1 and used in Western-blot analyses. Login to comment
117 The ratios of the band intensities are: 1.000 (wild-type MRP1), 0.564 + - 0.093 (K684L, n = 3) and 1.608 + - 0.175 (K1333L, n = 3). Login to comment
118 (B) Relative LTC4 transport activity by membrane vesicles containing wild-type, K684L- and K1333L-mutated MRP1. Login to comment
120 Since the amounts of MRP1 proteins in the membrane vesicles containing wild-type, K684L- and K1333L-mutated MRP1 were different, they were adjusted to a similar amount of MRP1 with membrane vesicles containing CFTR (1.692 µg of wild-type MRP1 + 1.308 µg of CFTR; 3 µg of K684L; 1.05 µg of K1333L + 1.95 µg of CFTR; 3 µg of CFTR) to determine the ATP-dependent LTC4 transport activity. Login to comment
123 Thus the ATP-dependent LTC4 transport activity of K684L- or K1333L-mutated MRP1 should be less than 40% or 11% of the wild-type MRP1. Login to comment
125 To test this hypothesis, membrane vesicles containing wild-type, K684L- and K1333L-mutated MRP1s were labelled with various concentrations of [α-32 P]8-N3ATP on ice (Figures 2A-2C). Login to comment
127 Figures 2(A) and 2(C) show that wild-type and K1333L-mutated MRP1 are heavily labelled by 16 µM [α-32 P]8-N3ATP, whereas K684L is not heavily labelled even at 256 µM (Figure 2B), implying that ATP binding to K684L-mutated NBD1 and unmutated NBD2 is significantly decreased. Login to comment
128 Unexpectedly, the Kd value of K1333L is only approximately one-third of the wild-type MRP1 (Table 1), similar to the Kd value of wild-type NBD1 determined by digesting the labelled wild-type MRP1 with trypsin [20], implying that the labelling of K1333L mainly occurs at the unmutated NBD1 and mutation of the NBD2 does not have a significant effect on the unmutated NBD1. Login to comment
134 We then decided to use the non-hydrolysable ATP analogue, [α-32 P]8-N3-AMP-PNP, to label the MRP1 protein at 37◦ C. Figures 2(D-F) (wild-type MRP1, K684L and K1333L respectively) show that the labelling patterns of these MRP1 proteins with [α-32 P]8-N3-AMP-PNP at 37◦ C are similar to those performed on ice with [α-32 P]8-N3ATP (Figures 2A-2C). Login to comment
135 For example, the labelling intensity of K684L is much weaker than either wild-type or K1333L-mutated MRP1. Login to comment
137 In addition, the labelling of K1333L-mutated MRP1 at a higher AMP-PNP concentration is greatly increased (Figure 2F), implying that the K1333L-mutated NBD2 could also be labelled. Login to comment
138 ATP bound to the mutated NBDs is not efficiently hydrolysed Since the ATP-dependent LTC4 transport activity of K1333L is extremely low (Figure 1B), we expected that even if nucleotide can bind to the K1333L-mutated NBD2 at higher concentrations, the bound nucleotide would not be efficiently hydrolysed by this mutated NBD2. Login to comment
139 To test this hypothesis, wild-type, K684L- and K1333L-mutated MRP1s were labelled with 8 µM of either [α-32 P]8-N3ATP or [γ -32 P]8-N3ATP at 37◦ C in the presence of Vi (Figure 3A). Login to comment
143 109.3% (K1333L), implying that the ATP bound to K684L is more efficiently hydrolysed than the ATP bound to K1333L and less efficiently hydrolysed than the ATP bound to wild-type MRP1. Login to comment
144 These conclusions are supported by the results in Figure 3(B), i.e. (i) both [α-32 P]8-N3ATP and [γ -32 P]8-N3ATP labelled the unmutated NBD1 of K1333L-mutated MRP1 with similar intensity; (ii) [α-32 P]8-N3ATP mainly labelled the unmutated Figure 2 Nucleotide binding to wild-type and mutant MRP1s Samples were mixed in 10 µl of a solution containing 10 µg of wild-type MRP1 (A), K684L (B) or K1333L (C) membrane proteins and various concentrations of [α-32 P]8-N3ATP, incubated on ice for 1 min and UV-irradiated on ice for 2 min. Login to comment
145 Due to high background at a higher concentration of [α-32 P]8-N3ATP, the labelled K684L and K1333L proteins were immunoprecipitated with MRP1-specific antibodies 42.4 and 897.2 [16]. Login to comment
149 Samples were mixed in 10 µl of a solution containing 10 µg of wild-type MRP1 (D), K684L (E) or K1333L (F) and various concentrations of [α-32 P]8-N3-AMP-PNP, incubated at 37◦C for 10 min and UV-irradiated after washing with 500 µl of ice-cold Tris/EGTA buffer (0.1 mM EGTA and 40 mM Tris/HCl, pH 7.5) [16]. Login to comment
150 In order to prove that the labelling at K684L- or K1333L-mutated MRP1 mainly occurs at the unmutated NBD, the labelled wild-type (WT), K684L- and K1333L-mutated MRP1 were digested with trypsin (G). Login to comment
153 At a higher concentration of ATP (128 µM in Figure 3C), K684L-mutated NBD1 and K1333L-mutated NBD2 are clearly labelled by either [α-32 P]8-N3ATP or [γ -32 P]8-N3ATP. Login to comment
155 In contrast, the [γ -32 P]8-N3ATP labelling intensity of the unmutated NBD1 of K1333L-mutated MRP1 is much weaker than the [α-32 P]8-N3ATP labelling, whereas the [γ -32 P]8-N3ATP labelling intensity of the K1333L-mutated NBD2 is similar to that of the [α-32 P]8-N3ATP labelling, implying that ATP bound to the K1333L-mutated NBD2 is not efficiently hydrolysed. Login to comment
156 Table 1 Mean Kd (ATP) of wild-type and mutant MRP1 Protein Kd (µM ATP)* Wild-type MRP1 32.3 + - 10.9 K684L 55.9 + - 16.5 K1333L 10.8 + - 5.3 * The Kd values of wild-type MRP1 (n = 6), K684L (n = 6) and K1333L (n = 6) were derived from Figures 2(A)-2(C). Login to comment
167 The kinetics of H/2 H exchange of wild-type, K684L- and K1333L-mutated MRP1s in the absence of nucleotide were different from each other, with 37, 21 and 43% of amide hydrogen remaining unexchanged after a 2 h exposure to 2 H2O (Figure 5A), implying that replacement of the lysine residue with leucine altered the tertiary structure of the protein. Login to comment
169 Thus the protein with the K684L mutation becomes more 'relaxed` (greater extent of water accessibility) than that of the wild-type MRP1, whereas the protein with the K1333L mutation becomes slightly more 'compact` (lesser extent of water accessibility) than that of the wild-type MRP1. Login to comment
170 Conformational changes induced by nucleotide binding/hydrolysis at NBD1 are different from that at NBD2 Since binding of ATP to NBD1 induced conformational changes of the protein and enhanced ATP binding to NBD2 [19], we expected that conformational changes induced by ATP binding/ Figure 3 Walker A lysine mutations affect ATP binding and hydrolysis Samples were mixed in 10 µl of a solution containing wild-type (10 µg), K684L- (15 µg) or K1333L-mutated MRP1 (10 µg), 800 µM Vi and 8 µM of either [α-32 P]8-N3ATP or [γ -32 P]8-N3ATP,incubatedat37◦Cfor4 minandUV-irradiatedonicefor2 min(A).Thelabelled samples were subjected to SDS/PAGE (7% gel) and electroblotted on to a nitrocellulose membrane. Login to comment
171 The amounts of radioactivity incorporated into MRP1 were determined by using a Packard instant imager, yielding a ratio ([γ -32 P]8-N3ATP labelling versus [α-32 P]8-N3ATP labelling) of 60.4 + - 16.6% (wild-type, n = 7); 83.9 + - 17.5% (K684L, n = 4); and 109.3 + - 20.6% (K1333L, n = 5). Login to comment
172 Samples were mixed in 10 µl of a solution containing wild-type (10 µg), K684L- (15 µg) or K1333L-mutated MRP1 (10 µg), 800 µM Vi and 8 µM (B) or 128 µM (C) of either [α-32 P]8-N3ATP or [γ -32 P]8-N3ATP, incubated at 37◦C for 10 min, UV-irradiated on ice for 2 min, washed with 500 µl of ice-cold Tris-EGTA buffer and digested with trypsin (trypsin/protein ratio = 1:16). Login to comment
180 Table 2 Secondary structure analysis of wild-type, K684L- and K1333L-mutated MRP1 in the presence or in the absence of nucleotide Proportion (%) Protein and substrate α-Helix β-Sheet β-Turn Random coil MRP1 42 + - 3 29 + - 3 12 + - 4 17 + - 6 MRP1 + AMP-PNP 40 + - 2 28 + - 2 11 + - 3 21 + - 4 MRP1 + ATP + Vi 43 + - 2 27 + - 3 12 + - 3 18 + - 3 K684L 41 + - 2 31 + - 4 9 + - 4 19 + - 3 K684L + AMP-PNP 39 + - 3 30 + - 4 10 + - 3 21 + - 5 K684L + ATP + Vi 40 + - 2 32 + - 3 11 + - 3 17 + - 4 K1333L 42 + - 2 28 + - 4 11 + - 3 19 + - 3 K1333L + AMP-PNP 41 + - 3 29 + - 2 12 + - 2 18 + - 4 K1333L + ATP + Vi 39 + - 3 30 + - 3 13 + - 4 18 + - 4 (Figure 5B), indicating that ATP binding, hydrolysis and trapping of the hydrolysis product ADP by Vi induced conformational changes slightly different from those observed after AMP-PNP binding. Login to comment
183 Interestingly, non-hydrolysable ATP analogue AMP-PNP binding to the K1333L-mutated MRP1 induced the same (from 43 to 26% with 17% extra amide hydrogen exchanged) extent of conformational changes as did ATP + Vi (Figure 5D). Login to comment
184 The fact that the H/2 H exchange patterns in the presence of either AMP-PNP or ATP + Vi are almost identical indicates that the bound ATP at either unmutated NBD1 or K1333L-mutated NBD2 is not efficiently hydrolysed. Login to comment
186 Cells expressing K684L- or K1333L-mutated MRP1s are not multidrug-resistant Considering that the transport activities of K684L, K1333L and CFTR (or parental BHK) are approx. Login to comment
187 40, 11 and 8% of the wild-type MRP1, we expected that cells expressing K684L-mutated MRP1 should be partially resistant to anticancer drugs, whereas the cells expressing K1333L-mutated MRP1 should not. Login to comment
188 Indeed, the cells expressing K1333L-mutated MRP1 are not resistant to daunomycin (Figure 6A), colchicine (Figure 6B) or vincristine (Figure 6C). Login to comment
190 The amount of vincristine that accumulated in cells expressing K1333L-mutated MRP1 is similar to that in cells expressing CFTR (Figure 7), whereas the accumulation of vincristine in cells expressing either wild-type or K684L-mutated MRP1 is slightly less than in cells expressing K1333L-mutated MRP1 or CFTR within 15 min incubation at 37◦ C (Figure 7), implying that either wild-type or K684L-mutated MRP1 has greater ability to transport vincristine out of the cells than the K1333L-mutated MRP1. Login to comment
191 The amount of vincristine that accumulated in cells expressing K684L-mutated MRP1 is more than in cells expressing wild-type MRP1, but less than in cells expressing either K1333L-mutated MRP1 or CFTR after 30, 60 or 120 min incubation at 37◦ C (Figure 7), implying that K684L-mutated MRP1 is more active than K1333L-mutated MRP1, but less active than wild-type MRP1. Login to comment
193 Cells expressing either K684L- or K1333L-mutated MRP1s are not hypersensitive to verapamil We have found that cells expressing wild-type MRP1 are hypersensitive to verapamil [38]. Login to comment
194 Since K684L- or K1333L-mutated MRP1 has approx. Login to comment
195 40 or 11% of the wild-type MRP1 transport activity (Figure 1B), we expected that cells expressing K1333L-mutated MRP1 should have similar sensitivity to verapamil as the cells without MRP1 expression, whereas the cells expressing K684L-mutated MRP1s might be more sensitive to verapamil than the cells without MRP1 expression. Login to comment
196 Indeed, the cells expressing K1333L-mutated MRP1 have similar sensitivity to verapamil as the parental BHK cells (Figure 6D). Login to comment
199 Both NBDs of MRP1 can bind nucleotide and contribute to solute transport Figure 5 Evolution of the proportion of unexchanged amide hydrogen in wild-type and mutant MRP1 as a function of the deuteration time The data were derived from Figure 4 by using wild-type (A, B), K684L- (A, C) or K1333L-mutated (A, D) MRP1s in the absence or presence of AMP-PNP or ATP + Vi. Login to comment
206 This asymmetry is visualized by IR spectroscopy that shows distinct conformational alterations for the Walker A lysine mutant K684L in NBD1, and the corresponding NBD2 mutant K1333L (Figure 5A), suggesting that the original tertiary structures of the two NBDs are different. Login to comment
214 However, ATP can still bind to the K1333L-mutated NBD2 at a higher concentration (Figure 3C). Login to comment
217 The results in Figure 3(C) indicate that the ATP bound to either K684L-mutated NBD1 or K1333L-mutated NBD2 is not efficiently hydrolysed. Login to comment
220 Considering the data accumulated from other NBD2 mutants, such as Y1302C, E1455Q, H1486F and H1486L, the conformational alterations caused by the K1333L mutation may not be the only reason preventing ATP hydrolysis at the mutated site. Login to comment
222 In contrast, replacement of the putative catalytic base Glu1455 , Figure 6 BHK cells expressing K684L- or K1333L-mutated MRP1 are neither multidrug-resistant nor hypersensitive to verapamil Cell survival experiments were performed according to the chemosensitivity assay as described in the Materials and methods section. Login to comment
223 Various concentrations of daunomycin (A), colchicine (B), vincristine (C) and verapamil (D) were applied to 96-well plates containing parental, wild-type MRP1-, K684L- or K1333L-transfected BHK cells. Login to comment
226 Figure 7 Vincristine accumulation in cells expressing wild-type, K684L- and K1333L-mutated MRP1 Intracellular accumulation of 3 H-labelled vincristine was carried out according to the vincristine accumulation method as described in the Materials and methods section. Login to comment
227 3 H-labelled vincristine (1 µM) was applied to the 24-well plate containing CFTR-, MRP1-, K684L- or K1333L-transfected BHK cells. Login to comment

[hide] Hydrogen-bond formation of the residue in H-loop o... Biochim Biophys Acta. 2007 Feb;1768(2):324-35. Epub 2006 Nov 18. Yang R, Chang XB
Hydrogen-bond formation of the residue in H-loop of the nucleotide binding domain 2 with the ATP in this site and/or other residues of multidrug resistance protein MRP1 plays a crucial role during ATP-dependent solute transport.
Biochim Biophys Acta. 2007 Feb;1768(2):324-35. Epub 2006 Nov 18., [PMID:17187755]

Abstract [show]
MRP1 couples ATP binding/hydrolysis to solute transport. We have shown that ATP binding to nucleotide-binding-domain 1 (NBD1) plays a regulatory role whereas ATP hydrolysis at NBD2 plays a crucial role in ATP-dependent solute transport. However, how ATP is hydrolyzed at NBD2 is not well elucidated. To partially address this question, we have mutated the histidine residue in H-loop of MRP1 to either a residue that prevents the formation of hydrogen-bonds with ATP and other residues in MRP1 or a residue that may potentially form these hydrogen-bonds. Interestingly, substitution of H827 in NBD1 with residues that prevented formation of these hydrogen-bonds had no effect on the ATP-dependent solute transport whereas corresponding mutations in NBD2 almost abolished the ATP-dependent solute transport completely. In contrast, substitutions of H1486 in H-loop of NBD2 with residues that might potentially form these hydrogen-bonds exerted either full function or partial function, implying that hydrogen-bond formation between the residue at 1486 and the gamma-phosphate of the bound ATP and/or other residues, such as putative catalytic base E1455, together with S769, G771, T1329 and K1333, etc., holds all the components necessary for ATP binding/hydrolysis firmly so that the activated water molecule can efficiently hydrolyze the bound ATP at NBD2.

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27 In contrast, mutations of the residue that should interact with the γ-phosphate of the bound ATP [28,29], such as K1333L [20], significantly decreased the ATP binding at the mutated NBD2 [23] and almost abolished the ATP-dependent solute transport activity completely. 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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259 In contrast, mutation of the Walker A motif K1333 residue in NBD2, such as K1333L [40, 141, 148], K1333M [16, 63, 118], K1333R [61] or K1333E [61], mainly affected ATP binding (at 4°C) at the mutated NBD2 [61, 148] and significantly decreased the ATP hydrolysis at the mutated NBD2 [61, 148]. Login to comment

[hide] Interaction between the bound Mg.ATP and the Walke... Biochemistry. 2008 Aug 12;47(32):8456-64. Epub 2008 Jul 18. Yang R, Scavetta R, Chang XB
Interaction between the bound Mg.ATP and the Walker A serine residue in NBD2 of multidrug resistance-associated protein MRP1 plays a crucial role for the ATP-dependent leukotriene C4 transport.
Biochemistry. 2008 Aug 12;47(32):8456-64. Epub 2008 Jul 18., 2008-08-12 [PMID:18636743]

Abstract [show]
Structural analysis of human MRP1-NBD1 revealed that the Walker A S685 forms a hydrogen bond with the Walker B D792 and interacts with the Mg (2+) cofactor and the beta-phosphate of the bound Mg.ATP. We have found that substitution of the S685 with an amino acid that potentially prevents the formation of the hydrogen bond resulted in misfolding of the protein and significantly affect the ATP-dependent leukotriene C4 (LTC4) transport. In this report we tested whether the corresponding substitution in NBD2 would also result in misfolding of the protein. In contrast to the NBD1 mutations, none of the mutations in NBD2, including S1334A, S1334C, S1334D, S1334H, S1334N, and S1334T, caused misfolding of the protein. However, elimination of the hydroxyl group at S1334 in mutations including S1334A, S1334C, S1334D, S1334H, and S1334N drastically reduced the ATP binding and the ATP-enhanced ADP trapping at the mutated NBD2. Due to this low efficient ATP binding at the mutated NBD2, the inhibitory effect of ATP on the LTC4 binding is significantly decreased. Furthermore, ATP bound to the mutated NBD2 cannot be efficiently hydrolyzed, leading to almost completely abolishing the ATP-dependent LTC4 transport. In contrast, S1334T mutation, which retained the hydroxyl group at this position, exerts higher LTC4 transport activity than the wild-type MRP1, indicating that the hydroxyl group at this position plays a crucial role for ATP binding/hydrolysis and ATP-dependent solute transport.

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153 However, if a mutant significantly affected the ATP binding, such a mutant, such as the Walker A mutation of K1333L, would abrogate the ATP-enhanced nucleotide binding at NBD2 (16). Login to comment

[hide] Characterization of the ATPase activity of a novel... Arch Biochem Biophys. 2009 May 15;485(2):102-8. Epub 2009 Mar 11. Wan L, Liang X, Huang Y
Characterization of the ATPase activity of a novel chimeric fusion protein consisting of the two nucleotide binding domains of MRP1.
Arch Biochem Biophys. 2009 May 15;485(2):102-8. Epub 2009 Mar 11., 2009-05-15 [PMID:19285030]

Abstract [show]
Nucleotide Binding Domains (NBDs) are responsible for the ATPase activity of the multidrug resistance protein 1 (MRP1). A series of NBD1-linker-NBD2 chimeric fusion proteins were constructed, expressed and purified, and their ATPase activities were analyzed. We report here that a GST linked NBD1(642-890)-GST-NBD2(1286-1531) was able to hydrolyze ATP at a rate of about 4.6 nmol/mg/min (K(m)=2.17 mM, V(max)=12.36 nmol/mg/min), which was comparable to the purified and reconstituted MRP1. In contrast, neither a mixture of NBD1 and GST-NBD2 nor the NBD1-GST-NBD1 fusion protein showed detectable ATPase activity. Additionally, the E1455Q mutant was found to be nonfunctional. Measurements by both MIANS labeling and circular dichroism spectroscopy revealed significant conformational differences in the NBD1-GST-NBD2 chimeric fusion protein compared to the mixture of NBD1 and GST-NBD2. The results suggest a direct interaction mediated by GST between the two NBDs of MRP1 leading to conformational changes which would enhance its ATPase activity.

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163 Among the NBD1-GST-NBD2 mutants, K684L in Walker A of NBD1, K1333L in Walker A of NBD2, and D1454L/E1455L in Walker B of NBD2 were expressed mainly as inclusion bodies in E. coli, and only the E1455Q mutant was expressed in a sufficient quantity of soluble protein to allow activity analysis. Login to comment

[hide] Infection of H69AR cells with retroviral particles... Int J Biochem Mol Biol. 2011;2(2):155-167. Epub 2011 Apr 22. Palaniyandi K, Zhao Q, Chang XB
Infection of H69AR cells with retroviral particles harboring interfering RNAi significantly reduced the multidrug resistance of these small cell lung cancer cells.
Int J Biochem Mol Biol. 2011;2(2):155-167. Epub 2011 Apr 22., [PMID:21968975]

Abstract [show]
Incubation of the drug-sensitive H69, a small cell lung cancer cell line, with increased concentrations of adriamycin yielded multidrug resistant (MDR) H69AR cells that over-express multidrug resistance-associated protein (MRP1). MRP1 co-transports its substrate with glutathione (GSH), leading to lower intracellular GSH. In this report we tested whether depleting intracellular GSH in MRP1-expressing cells could hyper-sensitize them to anticancer drugs or not. We have found that the GSH contents in MRP1-expressing cells are significantly lower than their corresponding control cells. The treatment with MRP1 substrate verapamil or the GSH synthetase inhibitor buthionine sulfoxi-mine significantly reduced the intracellular GSH contents in MRP1-expressing cells. Interestingly, depleting intracellular GSH contents can hyper-sensitize the MRP1-cDNA transfected BHK cells to daunomycin, but not the adriamycin-selected H69AR cells. Further analyses indicated that anti-apoptotic factor Bcl2 might be a factor responsible for the fact that depleting intracellular GSH could not hyper-sensitize H69AR cells to daunomycin. We hypothesized that knocking down the expression of Bcl2 could hyper-sensitize H69AR cells to daunomycin. Interestingly, infection of H69AR cells with retroviral particles harboring Bcl2 interfering RNAi not only reduced the expression of Bcl2, but also many factors that contribute to MDR, such as Bcl-xl, MRP1 and ABCC3, etc., leading to the MDR H69AR cells more sensitive to daunomycin than the parental H69 cell. Thus, although the mechanisms of the down-regulation of the genes contributing to MDR remain to be elucidated, retroviral particles harboring Bcl2 interfering RNAi could be used as an alternative way to sensitize the MDR cancer cells to anticancer drugs.

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52 In order to test whether this is the consequence of MRP1-mediated substrate-GSH co-transport, the GSH contents in BHK cells expressing human CFTR, human MRP1 and K1333L-mutated MRP1 were determined. Login to comment
53 The results in Figure 2A clearly indicate that the GSH content in BHK/K1333L cell is similar to that of BHK/ CFTR, but significantly higher than that in BHK/ MRP1 cell, suggesting that wt MRP1 constantly pumps the GSH out of the cell whereas the functionally inactive K1333L mutant [14] cannot. Login to comment
80 Total intracellular GSH was determined in triplicate [n = 55 (CFTR), 4 (K1333L), 65 (MRP1), 50 (H69) and 50 (H69AR)] according to the method described in Materials and Methods. Login to comment

[hide] Mutations of the Walker B motif in the first nucle... Arch Biochem Biophys. 2001 Aug 1;392(1):153-61. Cui L, Hou YX, Riordan JR, Chang XB
Mutations of the Walker B motif in the first nucleotide binding domain of multidrug resistance protein MRP1 prevent conformational maturation.
Arch Biochem Biophys. 2001 Aug 1;392(1):153-61., [PMID:11469806]

Abstract [show]
ATP-binding cassette (ABC) transporters couple the binding and hydrolysis of ATP to the translocation of solutes across biological membranes. The so-called "Walker motifs" in each of the nucleotide binding domains (NBDs) of these proteins contribute directly to the binding and the catalytic site for the MgATP substrate. Hence mutagenesis of residues in these motifs may interfere with function. This is the case with the MRP1 multidrug transporter. However, interpretation of the effect of mutation in the Walker B motif of NBD1 (D792L/D793L) was confused by the fact that it prevented biosynthetic maturation of the protein. We have determined now that this latter effect is entirely due to the D792L substitution. This variant is unable to mature conformationally as evidenced by its remaining more sensitive to trypsin digestion in vitro than the mature wild-type protein. In vivo, the core-glycosylated form of that mutant is retained in the endoplasmic reticulum and degraded by the proteasome. A different substitution of the same residue (D792A) had a less severe effect enabling accumulation of approximately equal amounts of mature and immature MRP1 proteins in the membrane vesicles but still resulted in defective nucleotide interaction and organic anion transport, indicating that nucleotide hydrolysis at NBD1 is essential to MRP1 function.

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42 Stable cell lines expressing wild-type and mutant MRP1s, K684L, D792L/D793L, K1333L, and D1454L/E1455L were established previously (8). Login to comment
147 This was also true in the case of D793L (Fig. 5E) and mutations of the Walker A lysine residues in both NBDs (Fig. 5B, K684L, and Fig. 5G, K1333L), where the protein matured normally as did a variant in which the Walker B aspartate in NBD2 was mutated (Fig. 5H, D1454L/ E1455L). Login to comment
174 Since hydrolysis is believed to drive MRP1 transport it would be expected that the mature D792A protein would not be capable of active transport. Login to comment
175 The data in Fig. 7 confirm this expectation, i.e., there is not significantly more ATP-dependent LTC4 uptake by vesicles containing D792A protein that does mature than by the other variants that do not mature (Fig. 7, D792L and D792L/D793L), nor by the NBD2 mutants (Fig. 7, K1333L and D1454L/E1455L) that do mature but have difficulties to hydrolyze ATP and to trap the hydrolysis product, ADP (8). Login to comment
207 (F) D792L/D793L, 1.1 ␮g protein in each lane. Login to comment
208 (G) K1333L, 0.3 ␮g protein in each lane. Login to comment
146 This was also true in the case of D793L (Fig. 5E) and mutations of the Walker A lysine residues in both NBDs (Fig. 5B, K684L, and Fig. 5G, K1333L), where the protein matured normally as did a variant in which the Walker B aspartate in NBD2 was mutated (Fig. 5H, D1454L/ E1455L). Login to comment

[hide] Collateral sensitivity of resistant MRP1-overexpre... Biochem Pharmacol. 2014 Aug 1;90(3):235-45. doi: 10.1016/j.bcp.2014.05.017. Epub 2014 May 27. Lorendeau D, Dury L, Genoux-Bastide E, Lecerf-Schmidt F, Simoes-Pires C, Carrupt PA, Terreux R, Magnard S, Di Pietro A, Boumendjel A, Baubichon-Cortay H
Collateral sensitivity of resistant MRP1-overexpressing cells to flavonoids and derivatives through GSH efflux.
Biochem Pharmacol. 2014 Aug 1;90(3):235-45. doi: 10.1016/j.bcp.2014.05.017. Epub 2014 May 27., [PMID:24875445]

Abstract [show]
The multidrug resistance protein 1 (MRP1) is involved in multidrug resistance of cancer cells by mediating drug efflux out of cells, often in co-transport with glutathione (GSH). GSH efflux mediated by MRP1 can be stimulated by verapamil. In cells overexpressing MRP1, we have previously shown that verapamil induced a huge intracellular GSH depletion which triggered apoptosis of the cells. That phenomenon takes place in the more global anticancer strategy called "collateral sensitivity" and could be exploited to eradicate some chemoresistant cancer cells. Seeking alternative compounds to verapamil, we screened a library of natural flavonoids and synthetic derivatives. A large number of these compounds stimulate MRP1-mediated GSH efflux and the most active ones have been evaluated for their cytotoxic effect on MRP1-overexpressing cells versus parental cells. Interestingly, some are highly and selectively cytotoxic for MRP1-cells, leading them to apoptosis. However, some others do not exhibit any cytotoxicity while promoting a strong GSH efflux, indicating that GSH efflux is necessary but not sufficient for MRP1-cells apoptosis. In support to this hypothesis, structure activity relationships show that the absence of a hydroxyl group at position 3 of the flavonoid C ring is an absolute requirement for induction of MRP1-cells death, but is not for GSH efflux stimulation. Chrysin (compound 8) and its derivatives, compounds 11 and 22, exhibit a high selectivity toward MRP1-cells with a IC(5)(0) value of 4.1 muM for compound 11 and 4.9 muM for chrysin and compound 22, making them among the best described selective killer compounds of multidrug ABC transporter-overexpressing cells.

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84 BHK-21 (Baby Hamster Kidney-21) cells stably transfected with wild-type mrp1 or K1333L mrp1 mutant have been previously described [25,26]. Login to comment
175 Flavonoids (flavonoid 8, 11 and 22 for the most active ones), like verapamil, are only effective on BHK-21 cells overexpressing wild-type MRP1 and not on those overexpressing MRP1 mutated in its hydrolytic site (K1333L) (Fig. 5) suggesting that the functional transporter is required to induce selective cell death. Login to comment