ABCMdb

ABCC1 p.Pro478Ala

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Publications

PMID: 14722114 [PubMed] Koike K et al: "Identification of proline residues in the core cytoplasmic and transmembrane regions of multidrug resistance protein 1 (MRP1/ABCC1) important for transport function, substrate specificity, and nucleotide interactions."

No. Sentence Comment

47 In addition, five mutants containing substitutions of Pro residues predicted to be in or proximal to the TM helices of MSD2 (TM6-Pro343 , TM8-Pro448 , TM10-Pro557 , and TM11-Pro595 ) and MSD3 (TM14-Pro1088 ) exhibited significantly reduced transport of five structurally diverse organic anion substrates of MRP1, whereas TM9 mutant P478A showed a selective reduction in LTC4 and GSH transport and an increase in E217betaG, E13SO4, and MTX transport. Login to comment
57 Proline substitutions were generated in the pBluescriptSK(ϩ) and pGEM-3Z plasmids above according to the manufacturer`s instructions with the following mutagenic primers (substituted nucleotides are underlined): MSD2 Pro mutants, P323A, 5Ј-GTG TTA TAC AAG ACC TTT GGC GCC TAC TTC CTC ATG AGC-3Ј; P343A, 5Ј-G ATG ATG TTT TCC GGG GCG CAG ATC TTA AAG TTG C-3Ј; P359A, 5Ј-G AAT GAC ACG AAG GCC GCA GAC TGG CAG GG-3Ј; P448A, 5Ј-G ATC TGG TCA GCC GCC CTG CAA GTC ATC CTT GC-3Ј; P464A, 5Ј-G CTG AAT CTG GGC GCT TCC GTC CTG GCT GG-3Ј; P478A, 5Ј-G GTC CTC ATG GTG GCC GTC AAT GCT GTG-3Ј; P557A, 5Ј-CC TGG GTC TGC ACG GCC TTT CTG GTG GCC-3Ј; P595A, 5Ј-C AAC ATC CTC CGG TTT GCC CTG AAC ATT CTC C-3Ј; P600A, 5Ј-CCC CTG AAC ATT CTC GCG ATG GTC ATC TABLE I Conservation of MRP1 MSD2 and MSD3 Pro residues in human ABCC family members Sequences are from Swiss-Prot/TrEMBL entries P33527 (MRP1/ ABCC1), O15438 (MRP3,/ABCC3), Q92887 (MRP2,/ABCC2), O95255 (MRP6/ ABCC6), Q09428 (SUR1/ABCC8), O60706 (SUR2/ABCC9), Q8NHX7 (MRP7/ABCC10), O15439 (MRP4/ABCC4), O15440 (MRP5/ABCC5), and P13569 (CFTR/ABCC7). Login to comment
115 The locations of the Pro residues mutated in the present study are highlighted, and the approximate boundaries of the TM helices are indicated by dashed lines. B, shown is a representative immunoblot of membrane vesicles prepared from HEK293T cells transfected with empty vector (pcDNA3.1(-)), wild-type (WT-MRP1), and mutant (P323A, P343A, P359A, P448A, P464A, P478A, P557A, P595A, and P600A) MRP1 cDNAs. MRP1 proteins were detected with mAb QCRL-1, and relative levels of expression estimated by densitometry are indicated; equal protein loading was confirmed by Amido Black staining of the polyvinylidene difluoride membrane and is shown below the blot. Login to comment
123 [3 H]LTC4 uptake by the MSD2 mutants P323A and P359A was moderately reduced (by ϳ25%), whereas uptake by the P343A, P448A, P478A, P557A, and P595A TM mutants was substantially reduced (by 55-70%). Login to comment
126 As shown in Fig. 2B, ATP-dependent uptake levels of this substrate by mutants P323A and P359A relative to wild-type MRP1 were moderately reduced (by ϳ30%), whereas uptake by mutants P343A, P448A, P557A, and P595A was substantially reduced (by 75-90%), and uptake by the P478A mutant was increased 1.5-fold. Login to comment
130 In contrast, [3 H]MTX uptake by the P464A and P478A mutants was moderately increased by ϳ30% relative to wild-type MRP1, whereas uptake by the P600A mutant was comparable with wild-type MRP1 (Fig. 2C). Login to comment
132 Relative to wild-type MRP1, GSH uptake by six of the nine MSD2 Pro mutants (P323A, P343A, P448A, P478A, P557A, and P595A) was substantially reduced (60-93%), whereas GSH uptake levels by the P359A, P464A, and P600A mutants were comparable with wild-type MRP1 (Table II). Login to comment
134 In contrast, levels of [3 H]E13SO4 uptake by the P478A mutant were increased 1.5-fold, and those of the P323A, P359A, P464A, and P600A mutants were comparable with wild-type MRP1. Login to comment
140 TABLE II Summary of effects of MSD2 Pro substitutions on MRP1 vesicular transport activities Substrate % wild-type MRP1 activitya TM6 ECL3 P359A TM8 P448A ECL4 P464A TM9 P478A TM10 P557A TM11 P323A P343A P595A P600A LTC4 75 35 75 40 100 45 35 30 100 E217betaG 70 10 70 25 100 155 10 Ͻ5 100 MTX 60 20 70 20 135 125 25 10 100 GSH 40 35 100 15 100 20 Ͻ10 10 100 E13SO4 100 70 100 60 100 155 25 15 100 a The values shown are means of triplicate determinations in a single experiment and are representative of results obtained in 2-3 independent experiments (for details, see legend to Fig. 2 and text). Login to comment
185 The horizontal white scale bar in the image represents 20 ␮m. Kinetic Parameters of [3 H]LTC4 and [3 H]E217betaG Uptake by MRP1 Pro Mutants-MSD2 TM mutants P343A, P448A, P478A, P557A, and P595A, MSD3 CL7 mutant P1150A, and TM14 mutant P1088A whose [3 H]LTC4 or [3 H]E217betaG transport properties were substantially altered relative to wild-type MRP1 were further characterized by kinetic analyses (Table IV). Login to comment
189 The apparent Km(LTC4) values for MSD2 TM mutants P343A, P448A, P478A, and P557A (range 39-63) were all somewhat lower than wild-type MRP1 (72-115 nM) except for P595A (TM6), which was increased by nearly 5-fold (485 versus 115 nM). Login to comment
191 Consequently, the overall LTC4 transport efficiency (Vmax/Km) of the P343A, P448A, P478A, and P557A mutants was moderately reduced (range 3.03 to 4.08) compared with wild-type MRP1 (5.83, 5.84). Login to comment
198 Photolabeling of Wild-type and Pro Mutant MRP1 Proteins with [3 H]LTC4-To investigate further whether the reduced [3 H]LTC4 transport activity of the TM mutants P343A, P448A, P478A, P557A, P595A, and P1088A and the CL7 mutant P1150A was associated with a decrease in substrate binding, photolabeling experiments were carried out with this intrinsically photoactivable arachidonic acid derivative. Login to comment
204 Radiolabeled vesicles enriched for wild-type MRP1 (WT-MRP1) and MRP1 mutants P343A, P448A, P1088A, P1150A, and empty vector control (pcDNA3.1(-)) are shown in the upper panel, and radiolabeled vesicles enriched for WT-MRP1 and mutants P478A, P557A, P595A, P1150A, and pcDNA3.1(-) are shown in the lower panel. Login to comment
205 TABLE IV Kinetic parameters of vesicular LTC4 and E217beta G uptake by selected Pro mutants of MRP1 Km Vmax Vmax/ Km Mutant:WT nM pmol mg-1 min-1 ϫ10-3 Vmax/Km LTC4 WT-MRP1 72 422 5.86 1.0 P343A 50 203 4.06 0.7 P448A 39 155 3.97 0.7 P1088A 55 229 4.15 0.7 P1150A 40 182 4.55 0.8 WT-MRP1 115 674 5.85 1.0 P478A 49 160 3.24 0.6 P557A 63 191 3.02 0.5 P595A 485 239 0.49 0.1 E217betaG WT-MRP1 1017 176 0.17 1.0 P1088A 1390 165 0.12 0.7 P1150A 243 160 0.66 3.9 take of this conjugated organic anion at different ATP concentrations (Fig. 8A). Login to comment
245 Between these two extremes of transport activity were mutants P323A and P478A. Login to comment
246 Replacement of Pro323 at the TM6 membrane-cytosol interface with Ala caused a significant and relatively specific loss of GSH transport, whereas Ala substitution of Pro478 (TM9) was associated with a substantial loss of LTC4 and GSH transport on the one hand, and a moderate increase in E217betaG, MTX, and E13SO4 transport on the other. Login to comment
247 The partial and substrate-selective alterations in transport activity observed for the P323A and P478A mutants provide evidence that although these Pro residues are not essential for function, they may be involved in some substrate-specific interactions with the transporter. Login to comment