ABCMdb

ABCC1 p.Lys396Glu

Predicted by SNAP2:	A: D (71%), C: D (71%), D: D (85%), E: D (85%), F: D (80%), G: D (80%), H: D (66%), I: D (71%), L: D (75%), M: D (66%), N: D (71%), P: D (85%), Q: D (59%), R: N (93%), S: D (63%), T: D (71%), V: D (71%), W: D (75%), Y: D (80%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: N, F: D, G: D, H: D, I: D, L: D, M: D, N: D, P: D, Q: N, R: N, S: D, T: D, V: D, W: D, Y: D,

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[hide] Mutations of charged amino acids in or near the tr... Mol Pharmacol. 2004 Jun;65(6):1375-85. Haimeur A, Conseil G, Deeley RG, Cole SP
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).
Mol Pharmacol. 2004 Jun;65(6):1375-85., [PMID:15155831]

Abstract [show]
Multidrug resistance protein 1 (MRP1) belongs to the ATP-binding cassette superfamily of transport proteins. In addition to drugs, MRP1 mediates the active transport of many conjugated and unconjugated organic anions. MRP1 consists of two membrane-spanning domains (MSD2 and MSD3) each followed by a nucleotide binding domain plus a third NH2-terminal MSD1. MSD2 contains transmembrane (TM) helices 6 through 11, and previously, we identified two charged residues in TM6 as having important but markedly different roles in MRP1 transport activity and substrate specificity by characterizing mutants containing nonconservative substitutions of Lys332 and Asp336. 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. The overall organic anion transport activity of the same-charge mutant of Asp336 (D336E) also remained very low, as observed for D336R. In addition, nonconservative substitutions of TM6-associated Lys319 and Lys347 resulted in a selective decrease in GSH transport. Of eight other charged residues in or proximal to TM7 to TM11 that were investigated, nonconservative substitutions of three of them [Lys396 (TM7), Asp436 (TM8), and Arg593 (TM11)] caused a substantial and global reduction in transport activity. However, unlike TM6 Asp336, wild-type transport activity could be reestablished in these MRP1 mutants by conservative substitutions. We conclude that MSD2-charged residues in or proximal to TM6, TM7, TM8, and TM11 play critical but differential roles in MRP1 transport activity and substrate specificity.

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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
142 Asp360 was replaced with an oppositely charged Lys residue (D360K), whereas Arg394 and Lys396 were substituted with Asp and Glu, respectively, to introduce the opposite charge (R394D and K396E) and with a nonpolar neutral amino acid (Ile) to eliminate charge (R394I and K396I). 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
149 In contrast, substitution of the nearby Lys396 residue with either an oppositely charged (K396E) or neutral (K396I) residue resulted in a substantial decrease in overall MRP1 transport activity (Fig. 5). Login to comment
150 Thus, LTC4, E217betaG, and E13SO4 transport by the K396E and K396I mutants was reduced by 60 to 75% (Fig. 5, A-C) whereas GSH and MTX transport by these two mutants was reduced by approximately 50% (Fig. 5, D and E). Login to comment
151 Kinetic analyses indicated that both the apparent uptake affinity and the transport capacity of the K396E mutant for LTC4 and E217betaG were altered. Login to comment
154 To test whether the positive charge of the Lys396 side chain was the physical property critical for MRP1 transport function, the Glu in the K396E mutant was replaced with Arg to create the same-charge mutant K(E)396R. 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
197 Time courses of [3 H]LTC4 uptake (A), [3 H]E217betaG uptake (B), and GSH-stimulated [3 H]E13SO4 uptake(C) by wild-type (WT) MRP1 (f), mutants K396E (F), K396I (‚), and K(E)396R (ƒ), and empty pcDNA3.1(-) vector control (E). Login to comment
198 D, apigenin-stimulated [3 H]GSH uptake and [3 H]MTX uptake (E) at 20 min by wild-type MRP1 (f), mutants K396E, K396I, and K(E)396R (u), and empty pcDNA3.1(-) vector control (Ⅺ). Login to comment
213 Nevertheless, in a study of rat Mrp2, TABLE 3 Kinetic parameters of E217betaG uptake by MRP1 MSD2 mutants containing substitutions of Lys396 and Asp436 Transfectants Km Vmax Vmax/Km ␮M pmol/mg/min ϫ 103 Wild-type MRP1 2.6 219 0.9 K396E 4.7 84 0.2 K396(E)R 2.3 195 0.9 D436K 7.7 87 0.1 D(K)436E 2.9 250 0.9 Fig. 6. ATP-dependent uptake of 3 H-labeled organic anions into membrane vesicles enriched for MRP1 mutants containing substitutions of Asp436 . Login to comment
250 Opposite-charge substitutions of either Lys396 or Asp436 substantially reduced transport activity, although the global structure of MRP1 was not severely disrupted because the K396E and D436K mutants retained some low level of activity. 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