ABCMdb

PMID: 15755910

Payen L, Gao M, Westlake C, Theis A, Cole SP, Deeley RG
Functional interactions between nucleotide binding domains and leukotriene C4 binding sites of multidrug resistance protein 1 (ABCC1).
Mol Pharmacol. 2005 Jun;67(6):1944-53. Epub 2005 Mar 8., [PubMed]

Sentences

No. Mutations Sentence Comment

44 ABCC1 p.Asp793Glu A D793E mutation in NBD1 enhanced its hydrolytic capacity but caused occlusion of the resultant ADP by the mutant NBD1 in the absence of vanadate. Login to comment 46 ABCC1 p.Glu1455Asp The reciprocal E1455D mutation of NBD2 increased the affinity of NBD2 for both azido-ATP and -ADP, resulting in prolonged binding of both nucleotides. Login to comment 65 ABCC1 p.Lys684Arg ABCC1 p.Lys1333Arg ABCC1 p.Lys1333Glu ABCC1 p.Lys684Glu The forward primers for creating K684R, K684E, K1333R, and K1333E mutations of Walker A motifs were 5Ј-GGCTGCGGAAGGTCGTC- CCTGC-3Ј, 5Ј-GGGCTGCGGAGAGTCGTCCCTGC-3Ј, 5Ј-GGGAGC- TGGGAGGTCGTCCCTGA-3Ј, and 5Ј-GGGAGCTGGGGAGTCGTC- CCTGA-3Ј, respectively. Login to comment 66 ABCC1 p.Gly1433Ala ABCC1 p.Gly771Ala The forward primers for the G771A and G1433A mutations of signature sequences were 5Ј-CCTGTCT- GGGGCCCAGAAGCAGC-3Ј and 5Ј-CCTCAGTGTCGCGCAGCGC- CAG-3Ј, respectively. Login to comment 67 ABCC1 p.Asp1454Asn ABCC1 p.Asp792Asn The forward primers for the D792N and D1454N mutations of the Walker B motifs were 5Ј-CATTTACCTCT- TCAATGATCCCCTC-3Ј and 5Ј-ATCCTTGTGTTGAATGAGGCCA- CG-3Ј, respectively. Login to comment 126 ABCC1 p.Lys1333Met Mutation of the conserved Walker A Lys684 in NBD1 (Fig. 2) to methionine substantially reduces but does not eliminate MRP1 transport activity, whereas the comparable mutation in NBD2, K1333M, essentially inactivates the protein (Gao et al., 2000; Hou et al., 2000). Login to comment 127 ABCC1 p.Lys684Met Despite the retention of ϳ30% of wt LTC4 transport activity by the K684M mutant protein, we were unable to detect photolabeling of either NBD with 8-azido-ATP (Gao et al., 2000). Login to comment 131 ABCC1 p.Lys684Arg ABCC1 p.Lys1333Arg ABCC1 p.Lys1333Glu ABCC1 p.Lys684Glu Densitometry of immunoblots of vesicle proteins indicated that levels of the K684R, K684E, K1333R, and K1333E MRP1 mutants ranged from 30 to 60% those of wt MRP1 (Fig. 3A). Login to comment 133 ABCC1 p.Lys684Arg ABCC1 p.Lys1333Arg It is noteworthy that the K684R substitution in NBD1 decreased ATP-dependent LTC4 uptake by only 40%, whereas the K1333R mutation in NBD2 reduced transport by approximately 80% (Fig. 2B). Login to comment 155 ABCC1 p.Lys684Glu The K684E mutation markedly decreased binding at both the mutant NBD1 and the wt NBD2, as observed previously Fig. 3. Login to comment 158 ABCC1 p.Lys684Arg ABCC1 p.Lys1333Arg ABCC1 p.Lys1333Glu ABCC1 p.Lys684Glu Membrane vesicles (1 ␮g of total protein) prepared from Sf21 cells expressing a combination of a wt and mutant half-molecule containing a K684E, K684R, K1333E, or K1333R mutation were separated by SDS-PAGE on gradient gels and transferred to Immobilon-P membranes. Login to comment 163 ABCC1 p.Lys684Arg ABCC1 p.Lys1333Arg ABCC1 p.Lys1333Glu ABCC1 p.Lys684Glu The relative expression levels of wt and mutant proteins evaluated by densitometry are indicated in the figure. B, effect of K684E, K684R, K1333E, and K1333R mutations on ATP-dependent LTC4 transport activity. Login to comment 175 ABCC1 p.Lys684Arg The conservative K684R mutation also decreased photolabeling of both NBDs but to a lesser extent than either the aspartic acid or methionine mutations (Fig. 3C). Login to comment 176 ABCC1 p.Lys1333Arg ABCC1 p.Lys1333Glu In contrast, both the K1333R and the K1333E mutations essentially eliminated binding at NBD2 but had little or no effect on the labeling of NBD1 (Fig. 3C). Login to comment 179 ABCC1 p.Lys1333Arg ABCC1 p.Lys1333Glu Likewise, both the K1333R and K1333E mutations eliminated trapping by the mutant NBD2 (Fig. 3D). Login to comment 189 ABCC1 p.Asp1454Asn ABCC1 p.Asp792Asn The D792N mutation diminished transport activity by approximately 65%, whereas the D1454N mutation essentially inactivated the protein (Fig. 5B). Login to comment 190 ABCC1 p.Asp792Asn As observed with the NBD1 Walker A mutations, the D792N mutation drastically decreased binding by NBD1 and, to a lesser extent, binding by NBD2. Login to comment 191 ABCC1 p.Asp1454Asn In contrast, the D1454N mutation eliminated photolabeling of only NBD2 and had no effect on photolabeling of NBD1 (Fig. 5C). Login to comment 192 ABCC1 p.Asp1454Asn ABCC1 p.Asp792Asn The D792N mutation also strongly decreased ADP trapping by both NBD1 and NBD2, although the D1454N mutation eliminated trapping by NBD2 but only modestly decreased trapping by NBD1 (Fig. 6B). Login to comment 193 ABCC1 p.Asp792Asn Despite the partial retention of vanadate-dependent trapping at NBD2 of the D792N mutant, we were unable to detect an ATP/vanadate-dependent decrease in LTC4 binding with either mutant (Fig. 6C). Login to comment 196 ABCC1 p.Gly1433Ala ABCC1 p.Gly771Ala The G771A and G1433A mutants were expressed at 90 and 50%, respectively, of the level of wt MRP1. Login to comment 198 ABCC1 p.Gly1433Ala ABCC1 p.Gly771Ala In contrast to the results obtained with the Walker A and B mutants, the NBD1 ABC signature mutation G771A eliminated LTC4 transport, whereas the NBD2 G1433A mutant retained approximately 30% of the activity of the wt protein (Fig. 7B). Login to comment 200 ABCC1 p.Gly1433Ala ABCC1 p.Gly771Ala Under vanadate-induced trapping conditions, both of the G771A and G1433A mutations markedly decreased the trapping of ADP at NBD2 but had relatively little effect on the low level of trapping typically observed at NBD1 (Fig. 7D). Login to comment 204 ABCC1 p.Lys684Arg ABCC1 p.Lys1333Arg ABCC1 p.Lys1333Glu ABCC1 p.Lys684Glu Membrane vesicles (50 ␮g of total protein) containing wt and the K684R, K684E, K1333R, and K1333E mutant MRP1 half-molecules were incubated in transport buffer at 23°C for 20 min in the absence or presence of ATP␥S (4 mM) or ATP (1 mM) plus vanadate (1 mM) before the addition of [3 H]LTC4 (200 nM, 0.13 ␮Ci). Login to comment 228 ABCC1 p.Asp1454Asn ABCC1 p.Asp792Asn The relative expression levels of wt and mutant half-molecules were evaluated by densitometry and are indicated on the figure. B, effect of D792N and D1454N mutations on ATP-dependent LTC4 transport activity. Login to comment 229 ABCC1 p.Asp1454Asn ABCC1 p.Asp792Asn Membrane vesicles (2 ␮g) containing wt and the D792N and D1454N mutant MRP1 half-molecules or control beta-gus were assayed for ATP-dependent LTC4 transport activity by incubation in transport buffer containing [3 H]LTC4 (50 nM, 0.13 ␮Ci) at 23°C for 2 min in the presence and absence of ATP (4 mM) as described under Materials and Methods. Results shown are means Ϯ S.D. of triplicate determinations in a single experiment. Login to comment 230 ABCC1 p.Asp1454Asn ABCC1 p.Asp792Asn Similar results were obtained in three additional independent experiments. C, effect of D792N and D1454N mutations on photolabeling with 8-azido-[␣-32 P]ATP. Login to comment 242 ABCC1 p.Asp1454Asn ABCC1 p.Asp792Asn The effect of the MRP1 Walker B D792N and D1454N mutations on ATP binding and ADP trapping was similar to that of the Walker A lysine mutations; the level of transport activity of the NBD1 aspartic acid-to-asparagine mutation was comparable with that of the Walker A lysine-to-methionine mutation (Gao et al., 2000). Login to comment 258 ABCC1 p.Asp1454Asn Densitometry indicated that the level of the D1454N mutant protein in the vesicle preparation used was approximately 2-fold higher than in control vesicles (A). Login to comment 260 ABCC1 p.Asp1454Asn ABCC1 p.Asp792Asn B, effect of D792N and D1454N mutations on vanadate-dependent nucleotide trapping. Login to comment 265 ABCC1 p.Asp1454Asn ABCC1 p.Asp792Asn C, effect of D792N and D1454N mutations on LTC4 photolabeling in the presence or absence of nucleotide. Login to comment 266 ABCC1 p.Asp1454Asn ABCC1 p.Asp792Asn Membrane vesicles (50 ␮g of total protein) containing wt and the D792N and D1454N mutant MRP1 half-molecules were incubated in transport buffer at 23°C for 20 min in the absence or presence of ATP␥S (4 mM) or ATP (1 mM) plus vanadate (1 mM) before the addition of [3 H]LTC4 (200 nM, 0.13 ␮Ci). Login to comment 276 ABCC1 p.Gly1433Ala ABCC1 p.Gly771Ala A, expression levels of wt and G771A and G1433A mutant MRP1 half-molecules. Login to comment 278 ABCC1 p.Gly1433Ala ABCC1 p.Gly771Ala The relative expression levels of wt and mutant proteins were evaluated by densitometry and are indicated in the figure. B, effect of G771A and G1433A mutations on ATP-dependent LTC4 transport activity. Login to comment 279 ABCC1 p.Gly1433Ala ABCC1 p.Gly771Ala Membrane vesicles (2 ␮g) containing wt and the G771A and G1433A mutant MRP1 half-molecules or control beta-gus were assayed for ATP-dependent LTC4 transport activity by incubation in transport buffer containing [3 H]LTC4 (50 nM, 0.13 ␮Ci) at 23°C for 2 min in the presence and absence of ATP (4 mM) as described under Materials and Methods. Results shown are means Ϯ S.D. of triplicate determinations in a single experiment. Login to comment 280 ABCC1 p.Gly1433Ala ABCC1 p.Gly771Ala Similar results were obtained in three additional independent experiments. C, effect of G771A and G1433A mutations on photolabeling with 8-azido-[␥-32 P]ATP. Login to comment 284 ABCC1 p.Gly1433Ala ABCC1 p.Gly771Ala D, effect of G771A and G1433A mutations on vanadate-dependent nucleotide trapping. Login to comment 291 ABCC1 p.Gly1433Ala ABCC1 p.Gly771Ala Membrane vesicles (50 ␮g of total protein) containing wt and the G771A and G1433A mutant MRP1 half-molecules were incubated in transport buffer at 23°C for 20 min in the absence or presence of ATP␥S (4 mM) or ATP (1 mM) plus vanadate (1 mM) before the addition of [3 H]LTC4 (200 nM, 0.13 ␮Ci). Login to comment