ABCMdb

ABCC1 p.Pro196Ala

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Publications

PMID: 11278867 [PubMed] Ito K et al: "Mutation of a single conserved tryptophan in multidrug resistance protein 1 (MRP1/ABCC1) results in loss of drug resistance and selective loss of organic anion transport."

No. Sentence Comment

103 RESULTS Identification of Trp1246 as a Functionally Important Amino Acid in MRP1-During the course of a mutational analysis of proline residues in MRP1, we generated an MRP1 mutant in which proline at position 196 was replaced with alanine (P196A). Login to comment
105 In contrast, a second independently derived P196A mutant HeLa cell line displayed a phenotype similar to that of cells expressing wild-type MRP1.2 Using three sets of overlapping primers, the integrated MRP1-transfected cDNAs of the wild-type MRP1 and the first P196A mutant cell lines were amplified by polymerase chain reaction. Login to comment
106 The DNA fragments obtained were of the size expected, and when sequenced, it was discovered that, in addition to the P196A mutation, the transfected copies of MRP1 cDNA in the first cell mutant line with an altered phenotype contained a G 3 C nucleotide mutation, resulting in substitution of cysteine for tryptophan at position 1246. Login to comment
191 In the course of a mutational analysis of this region of MRP1, we derived a mutant in which Pro196 was replaced with Ala by site-directed mutagenesis (34). Login to comment
205 In contrast, a second independently derived transfected cell line also expressing an MRP1 protein with the same P196A mutation displayed a phenotype similar to that of cells expressing wild-type MRP1.2 It was subsequently discovered that the transfected MRP1 in the first P196A mutant cell line had acquired a second non-engineered mutation causing the substitution of Cys for Trp at position 1246. Login to comment
206 Consequently, we generated a single W1246C mutant of MRP1 so that its phenotype could be compared with that of the double P196A/W1246C mutant. Login to comment
209 However, as observed for the double P196A/ W1246C mutant, transport of E217betaG by W1246C-MRP1 membrane vesicles was not detectably different from that obtained with vesicles from vector control-transfected cells, and although LTC4 transport appeared unchanged in the W1246C mutants, it could no longer be inhibited by E217betaG. Login to comment
210 Taken together, these results allowed us to conclude that the altered phenotype of the P196A/W1246C-transfected cells was attributable to the single tryptophan substitution at position 1246 in MSD3 and did not require the additional proline substitution at position 196 in the cytoplasmic loop between MSD1 and MSD2. Login to comment

PMID: 12948592 [PubMed] Ito K et al: "Mutation of proline residues in the NH(2)-terminal region of the multidrug resistance protein, MRP1 (ABCC1): effects on protein expression, membrane localization, and transport function."

No. Sentence Comment

47 For substitution of Pro residues in the CL3 loop connecting MSD1 to MSD2, the mutagenic primers were: MRP1-P196A (5V-CA GAT CGC TCA GCC CTG TTC TC-3V), MRP1-P205A (5V-C ATC CAC GAC GCT AAT CCC TG-3V), MRP1-P207A (5V-C GAC CCT AAT GCC TGC CCA GAG-3V), MRP1-P209A (5V-CTA ATC CCT GCG CAG AGT CCA G-3V), MRP1-P235A (5V-C TAC CGC CAG GCC CTG GAG GG-3V), MRP1-P255A (5V-CG GAA CAA GTC GTG GCT GTT TTG G-3V), MRP1-P272A (5V-CT AGG AAG CAG GCG GTG AAG G-3V). Login to comment
49 The quadruple MRP1-P196/205/207/209A construct was generated by introducing the P205/207/209A mutations into the MRP1-P196A construct. Login to comment
107 The seven single (P196A, P205A, P207A, P209A, P235A, P255A, and P272A) and two multiply substituted CL3 mutants (P196/205/207/209A and P235/255A) could also be stably expressed in HeLa cells. Login to comment
188 Results were expressed relative to wild-type Table 1 Kinetic parameters of [3 H]LTC4 uptake by membrane vesicles prepared from transfected HeLa cells expressing MRP1-Pro mutants Transfected cell line Km (nM) Vmax (pmol minÀ 1 mgÀ 1 ) Normalized Vmax MSD1 mutants WT-MRP1 128 80 80 P42A 139 35 41 P51A 109 55 50 P42/51A 103 15 48 P69A 142 46 42 P110A 150 73 124 CL3 mutants WT-MRP1 88 57 57 P196A 143 25 52 P205A 63 14 88 P207A 81 75 110 P209A 112 121 85 P196/205/207/209A 90 87 94 P235A 91 45 30 P255A 99 110 67 P235/255A 97 81 35 P272A 80 59 109 The kinetic parameters of [3 H]LTC4 uptake were determined by regression analysis of the Eadie-Hofstee transformed data. Login to comment
187 Results were expressed relative to wild-type Table 1 Kinetic parameters of [3 H]LTC4 uptake by membrane vesicles prepared from transfected HeLa cells expressing MRP1-Pro mutants Transfected cell line Km (nM) Vmax (pmol min 1 mg 1 ) Normalized Vmax MSD1 mutants WT-MRP1 128 80 80 P42A 139 35 41 P51A 109 55 50 P42/51A 103 15 48 P69A 142 46 42 P110A 150 73 124 CL3 mutants WT-MRP1 88 57 57 P196A 143 25 52 P205A 63 14 88 P207A 81 75 110 P209A 112 121 85 P196/205/207/209A 90 87 94 P235A 91 45 30 P255A 99 110 67 P235/255A 97 81 35 P272A 80 59 109 The kinetic parameters of [3 H]LTC4 uptake were determined by regression analysis of the Eadie-Hofstee transformed data. Login to comment