ABCMdb

ABCC1 p.Lys332Arg

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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

4 We have now extended these studies and found that the same-charge TM6 mutant K332R, like the nonconservatively substituted Lys332 mutants, exhibits a selective decrease in leukotriene C4 (LTC4) transport, associated with substantial changes in both Km and Vmax and LTC4 binding. Login to 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
103 To test whether the charge and/or steric properties (side-chain volume) of Lys332 were important for the substrate selective loss of transport activity, Lys332 was also replaced with Arg to create the same-charge mutant K332R. Login to comment
104 Unlike the K332D mutant, the conservatively substituted K332R had a significant level of LTC4 transport activity (approximately 40% of wild-type MRP1 levels). Login to comment
106 Analysis of kinetic parameters showed that the decrease in LTC4 transport by the same-charge K332R mutant was largely caused by a decrease in its apparent uptake affinity for LTC4 (Km ϭ 552 nM versus 115 nM for wild-type MRP1), leading to an overall 6-fold decrease in LTC4 transport efficiency (Vmax/Km ϭ 0.2 versus 1.2 for wild-type MRP1) (Table 1). Login to comment
113 A, time course of [3 H]LTC4 uptake by wild-type MRP1 (f), mutants K332D (), K(D)332K (F), K332R (Œ), and empty pcDNA3.1(-) vector control (E). Login to comment
114 B, [3 H]GSH uptake at 20 min by wild-type MRP1 (f) and mutants K332R, K332D, and K(D)332K (u), and empty pcDNA3.1(-) vector control (Ⅺ). Login to comment
115 C, [3 H]E217betaG uptake at 1 min by wild-type (WT) MRP1 (f) and mutants K332D () and K332R (Œ) was measured in the presence of three different concentrations (300, 600, and 900 nM) of LTC4. Login to comment
118 charge K332R mutant that retained some LTC4 transport activity could still be inhibited by this conjugated leukotriene, the effect of LTC4 on [3 H]E217betaG uptake by K332R was determined. Login to comment
119 The dose-response curves shown in Fig. 3C indicate that LTC4 had a greater inhibitory effect on E217betaG uptake by the K332R mutant than by the K332D mutant, but this effect was significantly less (50-70%) than the effect of LTC4 on E217betaG uptake by wild-type MRP1. Login to comment
121 The same-charge mutant K332R, like the K332D and K332L mutants described previously (Haimeur et al., 2002), exhibited transport levels of the conjugated estrogens E217betaG and E13SO4 and the antifolate MTX that were comparable with wild-type MRP1 (Table 2); however, GSH transport by K332R was very low compared with wild-type MRP1 and similar to that which we reported previously for the K332D/L mutants (Fig. 3B). Login to comment
126 In contrast to the like-charge K332R mutant in which LTC4 transport activity was partially restored, the like-charge D(K)336E mutant remained essentially inactive. Login to comment
145 These data indicate that neither Asp360 nor Arg394 plays an important role in the transport mechanism TABLE 1 Kinetic parameters of LTC4 uptake by MRP1 MSD2 mutants containing substitutions of Lys332 , Lys396 , Asp436 , and Arg593 Transfectants Km Vmax Vmax/Km nM pmol/mg/min ϫ 103 Wild-type MRP1 115 135 1.2 K332R 552 137 0.2 K396E 448 62 0.1 K(E)396R 113 121 1.1 D436K 196 41 0.2 D(K)436E 126 150 1.2 R593E 464 56 0.1 R(E)593K 123 139 1.1 TABLE 2 Summary of organic anion transport activity of MRP1 mutants with substitutions of charged amino acids in and proximal to the TM helices (TM6-11) of MSD2 The values shown are means of triplicate determinations and are derived from this study (Figs. 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
204 The critical importance of TM6 Lys332 as a highly substrate-selective determinant of MRP1 LTC4 and GSH transport activities was first confirmed by demonstrating that, despite maintaining a positive charge at position 332, transport of LTC4 and GSH by the K332R mutant remained significantly lower than wild-type MRP1. Login to comment
205 The reduced LTC4 transport activity of K332R was associated with a substantial reduction (5-fold) in the apparent LTC4 uptake affinity (Km), whereas Vmax was unchanged. Login to comment

PMID: 17295059 [PubMed] Chang XB et al: "A molecular understanding of ATP-dependent solute transport by multidrug resistance-associated protein MRP1."

No. Sentence Comment

154 Mutations in TM6, such as K332D or K332R, significantly reduced ATP-dependent LTC4 or GSH transport, but did not have any effect on ATP-dependent E217βG or E13SO4 transport [94]. Login to comment

PMID: 18775981 [PubMed] Grant CE et al: "Structural determinants of substrate specificity differences between human multidrug resistance protein (MRP) 1 (ABCC1) and MRP3 (ABCC3)."

No. Sentence Comment

303 Most significantly, the conservative substitution of Lys332 by Arg increased the Km for LTC4 ϳ5-fold without affecting the Vmax, as is the case with the Y440F mutation. Login to comment