ABCMdb

PMID: 10777615

Gao M, Cui HR, Loe DW, Grant CE, Almquist KC, Cole SP, Deeley RG
Comparison of the functional characteristics of the nucleotide binding domains of multidrug resistance protein 1.
J Biol Chem. 2000 Apr 28;275(17):13098-108., 2000-04-28 [PubMed]

Sentences

No. Mutations Sentence Comment

80 ABCC1 p.Lys1333Met ABCC1 p.Lys684Met The primers with the mismatched bases (boldface type) for K684M and K1333M were 5Ј-GGCTGCGGAATGTCGTC- CCTG-3Ј and 5Ј-GGAGCTGGGATGTCGTCCCTG-3Ј, respectively. Login to comment 83 ABCC1 p.Lys684Met ABCC1 p.Lys684Met ABCC1 p.Lys684Met The Bsu36I-SphI fragment bearing the K684M mutation was isolated from pGEM-NBD1 and used to replace the same region in pFB-MRP1 (35) and pFBDual-halves to create pFB-MRP1/K684M and pFBDual-halves/ K684M, respectively. Login to comment 84 ABCC1 p.Lys1333Met ABCC1 p.Lys1333Met The EcoRI-KpnI fragment with the K1333M mutation was isolated from pGEM-NBD2 and used to replace the equivalent region in pBSMRP1-fc-ATG to generate pBSMRP1-fc-ATG/ K1333M. Login to comment 85 ABCC1 p.Lys1333Met ABCC1 p.Lys684Met The SphI-KpnI fragment was then isolated from the resulting plasmid and used to replace the same region in pFB-MRP1 and pFB-MRP1/K684M to give pFB-MRP1/K1333M and pFB-MRP1/Double Km, respectively. Login to comment 86 ABCC1 p.Lys1333Met To generate pFBDual-halves/Double KM, the NcoI-KpnI fragment of pBSMRP1-fc-ATG/K1333M was isolated and used to replace the equivalent region of pFBDual-MRP1932-1531. Login to comment 87 ABCC1 p.Lys684Met Then the SalI-XbaI fragment of pFBDual-halves/K684M was isolated and cloned into the resulting vector as described for pFBDual-halves. Login to comment 204 ABCC1 p.Lys684Met The rate of ATP/ MRP1-dependent LTC4 uptake by vesicles from cells expressing the NBD1 mutant, MRP1/K684M, was approximately 25% (at 1 min and in the presence of 50 nM LTC4) of that obtained with vesicles containing the wild-type protein (Fig. 6B). Login to comment 205 ABCC1 p.Lys1333Met In contrast, the rates of ATP-dependent LTC4 uptake by membranes containing either MRP1/K1333M or MRP1/Double KM were less than 5% of that of the membranes expressing the wild-type protein. Login to comment 217 ABCC1 p.Lys1333Met ABCC1 p.Lys684Met Time and ATP-dependence of uptake is shown for MRP1 (f), NBD1 mutant MRP1/K684M (Œ), NBD2 mutant MRP1/K1333M (), and the double mutant MRP1/Double KM (ࡗ). Login to comment 226 ABCC1 p.Lys684Met Thus, despite the transport activity observed with the K684M mutant, no trapping of 8-azido-ADP was detectable. Login to comment 227 ABCC1 p.Lys1333Met ABCC1 p.Lys684Met LTC4 Transport by Co-expressed Half-molecules of MRP1 Containing Walker A Lysine Mutations-To further characterize the effect of the Walker A K684M and K1333M substitutions on the ability to photolabel each NBD with 8-azido-␣- [32 P]ATP, these mutations were introduced into each of the half-molecules, which were then expressed either together, or with the appropriate wild-type half-molecule, using dual expression vectors. Login to comment 230 ABCC1 p.Lys684Met The rate of LTC4 uptake by vesicles containing the K684M mutation was approximately 35% (at 1 min and in the presence of 50 nM LTC4) of that obtained with vesicles containing both halves of the wild-type protein (Fig. 7B). Login to comment 231 ABCC1 p.Lys1333Met The rates of LTC4 uptake by both halves of MRP1/K1333M and MRP1/Double KM were 8 and 5% that of the wild-type protein, respectively. Login to comment 234 ABCC1 p.Lys684Met ABCC1 p.Lys684Met Despite similar levels of the NH2-proximal half-molecules in the membrane vesicles used (Fig. 7A), labeling of the NH2-proximal half-molecule containing the K684M mutation (for both Dual-halves/K684M and Dual-halves/Double KM) was not detectable with either 8-azido-␣- (Fig. 8B) or 8-azido-␥-[32 P]ATP (Fig. 8A), regardless of whether it was expressed with a wild-type or mutant COOH-proximal half-molecule. Login to comment 235 ABCC1 p.Lys684Met In addition, the K684M mutation eliminated any LTC4 enhancement of the photolabeling of NBD1 and all labeling of a co-expressed wild-type COOH-proximal half-molecule. Login to comment 237 ABCC1 p.Lys1333Met As expected, the K1333M mutation also eliminated all labeling of NBD2. Login to comment 242 ABCC1 p.Lys1333Met ABCC1 p.Lys684Met Consistent with the results obtained with the full-length protein, no labeling of either NBD was observed when either the K1333M or K684M mutant half-molecules were co-expressed with the appropriate wild-type half of the protein despite the demonstrable transport activity of the latter combination. Login to comment 247 ABCC1 p.Lys1333Met ABCC1 p.Lys684Met A, membrane proteins from Sf21 cells expressing both halves of wild-type MRP1 (Dual-halves), NBD1 mutant (Dual-halves/ K684M), NBD2 mutant (Dual-halves/K1333M) and double mutant (Dual-halves/Double KM) were separated by SDS-PAGE on a 5-15% gradient gel and transferred to Immobilon-P membranes. Login to comment 250 ABCC1 p.Lys1333Met ABCC1 p.Lys684Met Time and ATP dependence of uptake is shown for Dual-halves (f), Dual-halves/K684M (Œ), Dual-halves/K1333M (), and Dual-halves/ Double KM (ࡗ). Login to comment 255 ABCC1 p.Lys684Met Thus, the results of LTC4 transport studies were very similar to those obtained with K684M mutation. Login to comment 264 ABCC1 p.Lys684Met Thus, the effect of the Ins708 mutation was similar to that of the K684M mutation both with respect to LTC4 transport activity and labeling by 8-azido-ATP and -ADP. Login to comment 317 ABCC1 p.Lys684Met The K684M mutation had a similar effect on the activity of the reconstituted transporter, and we confirmed that binding of 8-azido-ATP by the mutated NBD1 had been abolished. Login to comment 318 ABCC1 p.Lys684Met However, despite the retention of 30% of the wild-type level of ATP-dependent transport activity, the K684M mutation also eliminated detectable trapping of 8-azido-ADP by NBD2. Login to comment 319 ABCC1 p.Lys684Met Consequently, to ensure that the transport activity detected in the K684M mutation was indeed dependent on ATP hydrolysis, assays were carried out with the nonhydrolyzable ATP analogue, ATP␥S, and no transport activity could be detected (data not shown). Login to comment 324 ABCC1 p.Lys684Met Furthermore, like the K684M mutation, it reduced LTC4 transport by approximately 70%. Login to comment 325 ABCC1 p.Lys684Met Thus, the Ins708 mutation behaved in a manner indistinguishable from the K684M mutation with respect to nucleotide binding, vanadate-induced trapping, and transport activity. Login to comment 326 ABCC1 p.Lys684Met No major differences could be detected between the ATP dependence of the initial rates of transport of the K684M and Ins708 mutations when compared with the wild-type protein. Login to comment 327 ABCC1 p.Lys684Met As observed with the wild-type protein, initial rates of LTC4 transport of both the K684M and Ins708 mutations reached a maximum between 0.5 and 1 mM ATP (data not shown). Login to comment 329 ABCC1 p.Lys684Met The inability to trap nucleotide at NBD2 in the K684M and the Ins708 mutant proteins suggests that in the absence of ATP binding and possibly hydrolysis at NBD1, either 8-azidoADP is released rapidly from NBD2 even in the presence of vanadate or that the conformation in which NBD2 binds the 8-azido-ADP vanadate complex cannot be efficiently photoaffinity-labeled. Login to comment 330 ABCC1 p.Lys1333Met In contrast, the K1333M mutation essentially eliminated transport and abolished 8-azido-ADP trapping by NBD2 and the low level of trapping detectable at NBD1. Login to comment 332 ABCC1 p.Lys1333Met ABCC1 p.Lys684Met The combined results of LTC4 transport and photoaffinity labeling studies with the K684M, Ins708, and K1333M mutants are consistent with a model in which ATP hydrolysis at NBD1 is obligatorily coupled to hydrolysis at NBD2 but not vice versa. Login to comment 334 ABCC1 p.Lys1333Met ABCC1 p.Lys684Met The lack of reciprocal coupling between the two NBDs of MRP1 and the different consequences of the K684M and K1333M mutations raise an important question with respect to the role played by NBD1 in substrate transport. Login to comment 336 ABCC1 p.Lys684Met However, it is not possible to determine with the data presently available for MRP1 whether the decrease in LTC4 transport efficiency seen with the K684M mutation is a direct consequence of the inactivation of NBD1 or the result of a decrease in the efficiency of ATP hydrolysis at NBD2. Login to comment 337 ABCC1 p.Lys684Met ABCC1 p.Lys684Met Several observations, including (i) the LTC4-dependent stimulation of 8-azido-ATP binding by NBD1, (ii) the retention of partial transport activity following inactivation of NBD1 but not NBD2 and, (iii) loss of the ability to trap and photolabel NBD2 in the K684M and Ins708 mutants with 8-azido-ADP, are equally compatible with a mechanism in which the role of NBD1 is to regulate, in a substrate-responsive manner, the efficiency of ATP binding and hydrolysis at NBD2. Login to comment