ABCMdb

ABCC1 p.Arg593Glu

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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

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
182 Asp572 , Glu573, and Asp578 were substituted with Arg, whereas Arg593 was substituted with Glu as well as with the neutral, nonpolar Leu. Login to comment
184 In marked contrast, substitutions of TM11 Arg593 with Glu or Leu substantially reduced MRP1 transport activity. Login to comment
185 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. Login to comment
187 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). Login to comment
188 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. 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
242 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). Login to comment
243 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). Login to comment
255 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. Login to comment
260 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. 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