ABCMdb

PMID: 16551273

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 [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC1 p.Trp653Cys 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. Login to comment 3 ABCC1 p.Lys684Leu ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 4 ABCC1 p.Lys684Leu 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. Login to comment 5 ABCC1 p.Lys684Leu Although K684L-mutated NBD1 can bind ATP at higher concentrations, the bound nucleotide at that site is not efficiently hydrolysed. Login to comment 8 ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys1333Leu ABCC7 p.Lys684Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 107 ABCC1 p.Lys684Leu 0.564 (K684L) or approx. Login to comment 108 ABCC1 p.Lys1333Leu 1.608 (K1333L) compared with wild-type MRP1 (Figure 1A). Login to comment 109 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu (B) Relative LTC4 transport activity by membrane vesicles containing wild-type, K684L- and K1333L-mutated MRP1. Login to comment 120 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu ABCC1 p.Lys1333Leu ABCC7 p.Lys684Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys1333Leu ABCC1 p.Lys1333Leu 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 130 ABCC1 p.Lys684Leu Using the same rationale, the labelling of K684L-mutated MRP1 should mainly occur at the unmutated NBD2. Login to comment 132 ABCC1 p.Lys684Leu ABCC1 p.Lys684Leu However, the Kd value of K684L is increased to 56 µM, which is much higher than that of wild-type MRP1 (Table 1) or the Kd value of wild-type NBD2 determined by digesting the labelled wild-type MRP1 with trypsin [20], implying that K684L mutation impairs the regulatory effect of ATP binding to NBD1 [19]. Login to comment 134 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu For example, the labelling intensity of K684L is much weaker than either wild-type or K1333L-mutated MRP1. Login to comment 136 ABCC1 p.Lys684Leu Due to very low levels of labelling, the Kd (AMP-PNP) of K684L-mutated MRP1 cannot be accurately determined. Login to comment 137 ABCC1 p.Lys1333Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys1333Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 142 ABCC1 p.Lys684Leu 83.9% (K684L) and approx. Login to comment 143 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu ABCC1 p.Lys1333Leu 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 152 ABCC1 p.Lys684Leu ABCC1 p.Lys684Leu NBD2 of K684L, whereas [γ -32 P]8-N3ATP barely labelled NBD1 or NBD2 of K684L; (iii) [α-32 P]8-N3ATP labelled both NBD1 and NBD2 of wild-type MRP1, whereas [γ -32 P]8-N3ATP mainly labelled NBD1 of wild-type MRP1. Login to comment 153 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 154 ABCC1 p.Lys684Leu ABCC1 p.Lys684Leu ABCC1 p.Lys684Leu Interestingly, the [γ -32 P]8-N3ATP labelling intensity of the unmutated NBD2 of K684L-mutated MRP1 is much weaker than the [α-32 P]8-N3ATP labelling, whereas the [γ -32 P]8-N3ATP labelling intensity of the K684L-mutated NBD1 is similar to that of [α-32 P]8-N3ATP labelling, implying that ATP bound to the K684L-mutated NBD1 is not efficiently hydrolysed. Login to comment 155 ABCC1 p.Lys1333Leu ABCC1 p.Lys1333Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys684Leu ABCC1 p.Lys684Leu ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu ABCC1 p.Lys1333Leu ABCC1 p.Lys1333Leu ABCC1 p.Lys1333Leu 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 181 ABCC1 p.Lys684Leu For the K684L mutant, non-hydrolysable ATP analogue AMP-PNP binding increased the amount of unexchanged amide hydrogen from 21 to 25% (Figure 5C), whereas binding of hydrolysable ATP in the presence of Vi increased the amount of unexchanged amide hydrogen from 21 to 31% (Figure 5C). Login to comment 182 ABCC1 p.Lys684Leu These results indicate that although nucleotide binding to the K684L-mutated NBD1 is significantly reduced (Figure 2), AMP-PNP binding made the protein more 'compact` than the original state, and ATP binding, hydrolysis and trapping of ADP (probably at the unmutated NBD2) led the protein to an even more 'compact` structure (Figure 5C). Login to comment 183 ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys1333Leu ABCC1 p.Lys1333Leu ABCC7 p.Lys684Leu ABCC7 p.Lys684Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys1333Leu Indeed, the cells expressing K1333L-mutated MRP1 are not resistant to daunomycin (Figure 6A), colchicine (Figure 6B) or vincristine (Figure 6C). Login to comment 189 ABCC1 p.Lys684Leu However, the cells expressing K684L-mutated MRP1 are also not resistant to daunomycin (Figure 6A), colchicine (Figure 6B) or vincristine (Figure 6C). Login to comment 190 ABCC1 p.Lys684Leu ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu ABCC1 p.Lys1333Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu ABCC1 p.Lys1333Leu 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 192 ABCC1 p.Lys684Leu This lower transport activity resulted in the cells expressing K684L-mutated MRP1 not being resistant to vincristine (Figure 6C). Login to comment 193 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu Since K684L- or K1333L-mutated MRP1 has approx. Login to comment 195 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys1333Leu Indeed, the cells expressing K1333L-mutated MRP1 have similar sensitivity to verapamil as the parental BHK cells (Figure 6D). Login to comment 197 ABCC1 p.Lys684Leu ABCC1 p.Lys684Leu However, the cells expressing K684L-mutated MRP1s are not more sensitive to verapamil than its parental BHK cells (Figure 6D), implying that the intracellular glutathione is not efficiently co-transported out with verapamil by the K684L-mutated MRP1. Login to comment 199 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 212 ABCC1 p.Lys684Leu In contrast, replacement of the positively charged Walker A Lys684 in NBD1 with a hydrophobic leucine greatly decreased the ATP binding at the mutated NBD1 (Figure 2) and significantly diminished the enhancing effect of ATP on the [α-32 P]8-N3ADP trapping [19] and the ATP-dependent LTC4 transport [15,16], probably due to the elimination of a positively charged group and the conformational alterations caused by the K684L mutation (Figure 5A). Login to comment 214 ABCC1 p.Lys1333Leu However, ATP can still bind to the K1333L-mutated NBD2 at a higher concentration (Figure 3C). Login to comment 217 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys1333Leu ABCC1 p.Glu1455Gln ABCC1 p.Tyr1302Cys ABCC1 p.His1486Leu ABCC1 p.His1486Phe 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 ABCC1 p.Lys684Leu ABCC1 p.Lys1333Leu 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 231 ABCC1 p.His1486Leu ABCC1 p.His1486Phe ABCC1 p.His1486Phe Interestingly, although replacement of His1486 (H-loop in NBD2), which forms a hydrogen bond with the γ -phosphate of the bound ATP in NBD2, with an aromatic phenylalanine residue or a hydrophobic leucine residue has different effects on ATP binding, i.e. the H1486F mutation had no effect on ATP binding at the unmutated NBD1 and the H1486F-mutated NBD2, whereas the H1486L mutation increased the Kd value by approx. Login to comment 232 ABCC1 p.His1486Leu ABCC1 p.His1486Phe 2-fold, the mutation of either H1486F or H1486L abolished the ATP-dependent LTC4 transport activity (R. Yang and X.-b. Chang, unpublished work). Login to comment