ABCMdb

ABCC1 p.Asp436Lys

Predicted by SNAP2:	A: D (75%), C: D (75%), E: D (59%), F: D (80%), G: D (80%), H: D (85%), I: D (85%), K: D (91%), L: D (85%), M: D (75%), N: D (75%), P: D (91%), Q: D (75%), R: D (85%), S: D (71%), T: D (75%), V: D (85%), W: D (91%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: 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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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. 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
162 In contrast, when Asp436 was replaced with Lys to create D436K, the mutant was expressed but showed a significant decrease in overall organic anion transport activity (Fig. 6 and Table 2). Login to comment
163 Thus, vesicular uptake by the D436K mutant was decreased by approximately 60% for LTC4 (Fig. 6A), by 80% for E217betaG (Fig. 6B), by 70% for E13SO4 (Fig. 6C), by 70% for GSH (Fig. 6D), and by 45% for MTX (Fig. 6E). Login to comment
164 Kinetic analyses showed that the apparent Km (LTC4) of the D436K mutant was increased 2-fold and Vmax decreased Ͼ3-fold, resulting in an overall 7-fold decrease in LTC4 transport efficiency for this mutant (Table 1). Login to comment
165 Likewise, the apparent Km (E217betaG) of D436K was increased 3-fold and Vmax decreased 3-fold, resulting in a 7.5-fold decrease in E217betaG transport efficiency (Table 3). Login to comment
166 When the Lys residue in the D436K mutant was replaced with Glu to create the like-charged D(K)436E mutant, wild-type levels of transport activity were observed (Fig. 6, A-E). Login to comment
170 To determine whether the substantially reduced [3 H]LTC4 transport activity of the Asp436 mutant D436K was associated with a decrease in substrate binding, photolabeling experiments were carried out with [3 H]LTC4. Login to comment
172 As shown in Fig. 6F, [3 H]LTC4 photolabeling of the D436K mutant was reduced by approximately 50% compared with wild-type MRP1. 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
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
214 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 D436K () and D(K)436E (Ⅺ), and empty pcDNA3.1(-) vector control (E). Login to comment
215 D, apigenin-stimulated [3 H]GSH uptake and [3 H]MTX uptake (E) at 20 min by wild-type MRP1 (f), mutants D436K and D(K)436E (u), and empty pcDNA3.1(-) vector control (Ⅺ). Login to comment
239 Unlike the oppositely charged mutant of Asp430 discussed earlier, the TM8 D436K mutant was expressed at levels comparable with those of wild-type MRP1. Login to comment
249 In the present study, the reduced transport activity of the D436K mutant was associated with reduced LTC4 binding, and in this way, the D436K mutant is more similar to the Asp336 mutants than to the Asp1084 mutants described previously. 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