ABCMdb

ABCC1 p.Pro600Ala

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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

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
124 [3 H]LTC4 uptake by the P464A and P600A mutants was comparable with wild-type MRP1 (Fig. 2A). Login to comment
127 In contrast, as observed for [3 H]LTC4 uptake, [3 H]E217betaG uptake by the P464A and P600A mutants was comparable with wild-type MRP1. 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