ABCC1 p.Arg593Glu
Predicted by SNAP2: | A: D (75%), C: D (80%), D: D (91%), E: D (91%), F: D (85%), G: D (80%), H: D (75%), I: D (85%), K: D (63%), L: D (85%), M: D (75%), N: D (75%), P: D (91%), Q: D (71%), S: D (71%), T: D (71%), V: D (85%), W: D (91%), Y: D (85%), |
Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: N, L: D, M: D, N: D, P: D, Q: D, 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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No. Sentence Comment
51 TTC-3Ј; D430R, 5Ј-C AAC CTC ATG TCT GTG CGC GCT CAG AGG TTC-3Ј; D436K, 5Ј-C GCT CAG AGG TTC ATG AAG TTG GCC ACG TAC-3Ј; D(K)436E, 5Ј-C GCT CAG AGG TTC ATG GAA TTA GCC ACG TAC-3Ј; D572R, 5Ј-C TAC GTG ACC ATT CGC GAG AAC AAC ATC CTG G-3Ј; E573R, 5Ј-C GTG ACC ATT GAC CGG AAC AAC ATC CTG GAT G-3Ј; D578R, 5Ј-G AAC AAC ATC CTG CGG GCC CAG ACA GCC TTC G-3Ј; R593E, 5Ј-G GCC TTG TTC AAC ATC CTC GAG TTT CCC CTG AAC-3Ј; R593L, 5Ј-G GCC TTG TTC AAC ATC CTC CTG TTT CCC CTG AAC-3Ј; R(E)593K, 5Ј-GCC TTG TTC AAC ATC CTT AAG TTT CCC CTG AAC-3Ј.
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ABCC1 p.Arg593Glu 15155831:51:431
status: NEW145 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.
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ABCC1 p.Arg593Glu 15155831:145:409
status: NEW148 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.
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ABCC1 p.Arg593Glu 15155831:148:556
status: NEW182 Asp572 , Glu573, and Asp578 were substituted with Arg, whereas Arg593 was substituted with Glu as well as with the neutral, nonpolar Leu.
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ABCC1 p.Arg593Glu 15155831:182:63
status: NEW184 In marked contrast, substitutions of TM11 Arg593 with Glu or Leu substantially reduced MRP1 transport activity.
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ABCC1 p.Arg593Glu 15155831:184:42
status: NEW185 As shown in Fig. 7, the LTC4 (Fig. 7A), GSH (Fig. 7D), and MTX (Fig. 7E) transport activities of the R593E and the R593L mutants were reduced by 70 to 80% relative to wild-type MRP1.
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ABCC1 p.Arg593Glu 15155831:185:101
status: NEW187 Kinetic analysis of LTC4 transport by the R593E mutant showed that its decreased transport activity was caused by a 4-fold decrease in apparent uptake affinity (Km ϭ 464 versus 115 nM for wild-type MRP1) and in transport capacity (2-fold decrease in Vmax), resulting in an overall 10-fold decrease in LTC4 transport efficiency (Table 1).
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ABCC1 p.Arg593Glu 15155831:187:42
status: NEW188 To investigate the physical properties of Arg593 important for MRP1 transport function, the Glu residue of the R593E mutant was replaced with Lys to create the same-charge mutant R(E)593K.
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ABCC1 p.Arg593Glu 15155831:188:111
status: NEW194 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).
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ABCC1 p.Arg593Glu 15155831:194:137
status: NEW242 Time courses of [3 H]LTC4 uptake (A), [3 H]E217betaG uptake (B), and GSH-stimulated [3 H]E13SO4 uptake (C) by wild-type MRP1 (f), mutants R593E (‚), R593L (ƒ), and R(E)593K (F), and empty pcDNA3.1(-) vector control (E).
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ABCC1 p.Arg593Glu 15155831:242:138
status: NEW243 D, apigenin-stimulated [3 H]GSH uptake and [3 H]MTX uptake (E) at 20 min by wild-type (WT) MRP1 (f), mutants R593E, R593L, and R(E)593K (u), and empty pcDNA3.1(-) vector control (Ⅺ).
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ABCC1 p.Arg593Glu 15155831:243:109
status: NEW255 Thus, the nonconservatively substituted R593E and R593L mutants displayed a complete loss of E217betaG and E13SO4 transport and a substantial loss of LTC4, GSH, and MTX transport.
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ABCC1 p.Arg593Glu 15155831:255:40
status: NEW260 However, the lack of LTC4 binding by R593E and the global reduction in its transport activity indicates that Arg593 is important for high-affinity binding of LTC4 and presumably other substrates as well.
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ABCC1 p.Arg593Glu 15155831:260:37
status: NEW272 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.
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ABCC1 p.Arg593Glu 15155831:272:225
status: NEW