ABCMdb

ABCC1 p.Asp336Lys

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Publications

PMID: 15155831 [PubMed] Haimeur A et al: "Mutations of charged amino acids in or near the transmembrane helices of the second membrane spanning domain differentially affect the substrate specificity and transport activity of the multidrug resistance protein MRP1 (ABCC1)."

No. Sentence Comment

48 The sequences of the individual sense strands (with the corresponding amino acid changes indicated in parentheses, the altered codons underlined, and silent mutations introducing new restriction sites italicized) were as follows: K(D)332K, 5Ј-G AGC TTC TTC TTC AAG GCC ATC CAC GAC CTG-3Ј; K332R, 5Ј-G AGC TTC TTC TTC AGG GCC ATC CAC GAC CTG-3Ј; D(K)336E, 5Ј-C AAG GCC ATC CAC GAG CTC ATG ATG TTT TCC-3Ј; D336K, 5Ј-C AAG GCC ATC CAC AAG CTT ATG ATG TTT TCC-3Ј; K332D/D336K, 5Ј-GC TTC TTC TTC GAC GCC ATC CAC AAA CTG ATG ATG-3Ј; K319D, 5Ј-G TTT AAG GTG TTA TAC GAC ACG TTT GGG CCC-3Ј; K347D, 5Ј-GGG CCG CAG ATA TTA GAC TTG CTC ATC AAG-3Ј; K347L, 5Ј-GGG CCG CAA ATC TTA CTT TTG CTC ATC AAG-3Ј; D360K, 5Ј-GAC ACG AAG GCG CCA AAG TGG CAG GGC TAC-3Ј; R394D, 5Ј-C GTC AGT GGC ATG GAG ATC AAG ACC GCT GTC-3Ј; R394I, 5Ј-C GTC AGT GGC ATG ATC ATC AAG ACC GCT GTC-3Ј; K396E, 5Ј-GT GGC ATG AGG ATC GAG ACC GCT GTC ATT GGG-3Ј; K396I, 5Ј-GT GGC ATG AGG ATC ATC ACC GCT GTC ATT GGG-3Ј; K(E)396R, 5Ј-GGC ATG AGG ATC AGG ACC GCT GTC ATT GGG GC-3Ј; D430K, 5Ј-C AAC CTC ATG TCT GTG AAG GCT CAG AGG Fig. 1. Login to comment
124 In the present study, Asp336 was replaced with Lys to create D336K, and a comparable global loss of transport activity was observed (Fig. 4A and Table 2). Login to comment
125 To test whether the charge of Asp336 or the steric bulk of its side chain was responsible for the functional importance of this amino acid, the Lys in the D336K mutant was replaced with Glu to create the like-charge mutant D(K)336E. Login to comment
130 To test this idea, the K332D/D336K double mutant in which these two residues were exchanged was created. Login to comment
132 However, as shown in Fig. 4B, LTC4 transport by K332D/ D336K was not detectable. Login to comment
148 MRP1 Mutant % Wild-Type MRP1 Transport Activity LTC4 GSH E217betaG E13SO4 MTX TM6 K332D Ͻ10 Ͻ10 100 100 100 K332R 40 20 100 100 100 D336K Ͻ10 Ͻ10 Ͻ10 15 45 D(K)336E 20 Ͻ10 25 25 45 K332D/D336K Ͻ10 Ͻ10 30 25 50 K319D 100 50 100 100 100 K347D 100 45 100 100 100 TM7 D360K 100 100 100 100 100 R394D 100 100 100 100 100 K396E 25 50 25 25 35 K(E)396R 100 100 100 100 100 TM8 D436K 40 30 20 30 55 D(K)436E 100 100 100 100 100 ECL5/TM11 D572R 100 100 100 100 100 E573R 100 100 100 100 100 D578R 100 100 100 100 100 R593E 35 30 Ͻ10 Ͻ10 40 R(E)593K 100 100 100 100 100 or substrate specificity of MRP1. Login to comment
175 A, time course of [3 H]LTC4 uptake by wild-type MRP1 (f), mutants D336K (‚), D(K)336E (ƒ), and empty pcDNA3.1(-) control (E). Login to comment
176 B, time course of [3 H]LTC4 uptake by wild-type MRP1 (f), TM6 double mutant K332D/D336K (ࡗ), and empty pcDNA3.1(-) vector control (E). Login to comment
194 For these experiments, GFP-tagged constructs encoding wild-type MRP1 and the mutant MRP1 proteins (D336E, K332D/D336K, K396E, D436K, and R593E) were generated and transfected into HEK 293T cells (Koike et al., 2002). Login to comment
211 However, the inactivity of the double-exchange mutant K332D/D336K suggests that this is unlikely to be the case (Fig. 2). Login to comment
272 HEK 293T cells were transfected with the wild-type pcDNA3.1(-)-MRP1-GFP and mutant pcDNA3.1(-)-MRP1- D336E-GFP, pcDNA3.1(-)-MRP1-K332D/D336K-GFP, pcDNA3.1(-)- MRP1-K396E-GFP, pcDNA3.1(-)-MRP1-D436K-GFP, and pcDNA3.1(-)- MRP1-R593E-GFP expression vectors as indicated, and cells were viewed 48 h later under the confocal microscope. Login to comment

PMID: 15161912 [PubMed] Leslie EM et al: "Arsenic transport by the human multidrug resistance protein 1 (MRP1/ABCC1). Evidence that a tri-glutathione conjugate is required."

No. Sentence Comment

69 MRP1 and MRP2 Expression Vectors and Transfections in HEK293T Cells-The construction and expression of wild-type MRP1 (WT-MRP1), wild-type MRP2 (WT-MRP2), and the MRP1 mutants K332L, D336K, K319D, and K347D in HEK293T cells have been described previously (31, 35-37). Login to comment
204 To determine whether these amino acid residues are critical for transport of As(GS)3, transport assays were done using membrane vesicles prepared from HEK293T cells transfected with pcDNA3.1(-) (empty vector), WT-MRP1, D336K-MRP1, K332L-MRP1, K319D-MRP1, and K347D-MRP1 cDNAs. Login to comment
206 Consistent with the selective loss of LTC4 and GSH transport by K332L-MRP1 and the general loss of transport function by D336K-MRP1, these mutants also did not transport As(GS)3 (Fig. 8B). Login to comment
222 As(GS)3 transport by wild-type and mutant K332L, D336K, K319D, K347D-MRP1. Login to comment
223 A, membrane vesicles prepared from HEK293T cells transfected with empty vector (pcDNA3.1(-)), wild-type MRP1 (WT-MRP1), or mutant MRP1 (K332L-MRP1, D336K-MRP1, K319D-MRP1, or K347D-MRP1) were immunoblotted with the MRP1-specific mAb QCRL-1 as described under "Experimental Procedures." Login to comment