ABCMdb

PMID: 15155831

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., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC1 p.Lys332Arg 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 5 ABCC1 p.Asp336Arg ABCC1 p.Asp336Glu The overall organic anion transport activity of the same-charge mutant of Asp336 (D336E) also remained very low, as observed for D336R. Login to comment 48 ABCC1 p.Lys332Asp ABCC1 p.Lys332Arg ABCC1 p.Lys396Glu ABCC1 p.Asp336Lys ABCC1 p.Asp336Lys ABCC1 p.Lys347Asp ABCC1 p.Asp430Lys ABCC1 p.Lys396Ile ABCC1 p.Lys347Leu ABCC1 p.Arg394Asp ABCC1 p.Lys319Asp ABCC1 p.Asp360Lys ABCC1 p.Arg394Ile 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 51 ABCC1 p.Arg593Leu ABCC1 p.Glu573Arg ABCC1 p.Arg593Glu ABCC1 p.Asp436Lys ABCC1 p.Asp430Arg ABCC1 p.Asp578Arg ABCC1 p.Asp572Arg 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 96 ABCC1 p.Asp430Lys The exception was the Asp430 mutant D430K which, like endogenous MRP1 in vector control membrane vesicles, was undetectable under the conditions used. Login to comment 100 ABCC1 p.Lys332Asp ABCC1 p.Lys332Leu We showed previously that nonconservative substitutions of Lys332 with either Asp (K332D) or Leu (K332L) led to a selective loss of transport of GSH and the GSH conjugate, LTC4 (Haimeur et al., 2002). Login to comment 102 ABCC1 p.Lys332Asp When the Asp residue of the K332D mutant was mutated back to the wild-type Lys residue to create K(D)332K, LTC4 and GSH transport was restored as expected (Fig. 3, A and B). Login to comment 103 ABCC1 p.Lys332Arg 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 ABCC1 p.Lys332Asp ABCC1 p.Lys332Arg 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 ABCC1 p.Lys332Arg 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 ABCC1 p.Lys332Asp ABCC1 p.Lys332Arg 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 ABCC1 p.Lys332Asp ABCC1 p.Lys332Arg 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 ABCC1 p.Lys332Asp ABCC1 p.Lys332Arg 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 ABCC1 p.Lys332Arg ABCC1 p.Lys332Arg 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 ABCC1 p.Lys332Asp ABCC1 p.Lys332Arg 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 ABCC1 p.Lys332Asp ABCC1 p.Lys332Asp ABCC1 p.Lys332Leu ABCC1 p.Lys332Arg ABCC1 p.Lys332Arg 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 123 ABCC1 p.Asp336Arg ABCC1 p.Asp336Leu In our earlier study, we showed that substitution of the negatively charged TM6 Asp336 residue with Arg or Leu essentially eliminated the transport of both conjugated and unconjugated MRP1 organic anion substrates (Haimeur et al., 2002). Login to comment 124 ABCC1 p.Asp336Lys ABCC1 p.Asp336Lys In the present study, Asp336 was replaced with Lys to create D336K, and a comparable global loss of transport activity was observed (Fig. 4A and Table 2). Login to comment 125 ABCC1 p.Asp336Lys To test whether the charge of Asp336 or the steric bulk of its side chain was responsible for the functional importance of this amino acid, the Lys in the D336K mutant was replaced with Glu to create the like-charge mutant D(K)336E. Login to comment 126 ABCC1 p.Lys332Arg 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 130 ABCC1 p.Lys332Asp ABCC1 p.Asp336Lys To test this idea, the K332D/D336K double mutant in which these two residues were exchanged was created. Login to comment 132 ABCC1 p.Lys332Asp ABCC1 p.Asp336Lys However, as shown in Fig. 4B, LTC4 transport by K332D/ D336K was not detectable. Login to comment 136 ABCC1 p.Lys347Asp ABCC1 p.Lys347Asp ABCC1 p.Lys347Leu ABCC1 p.Lys319Asp ABCC1 p.Lys319Asp Lys319 was replaced with Asp (K319D), and Lys347 was replaced with Asp (K347D) as well as Leu (K347L). Login to comment 138 ABCC1 p.Lys347Asp ABCC1 p.Lys347Leu ABCC1 p.Lys319Asp Thus the ability of the K319D, K347D, and K347L mutants to transport GSH was reduced by approximately 50% (Fig. 4C), whereas the transport of four other substrates (LTC4, E217betaG, E13SO4 and MTX) by these mutants remained comparable with wild-type MRP1 (Table 2). Login to comment 142 ABCC1 p.Lys396Glu ABCC1 p.Lys396Ile ABCC1 p.Arg394Asp ABCC1 p.Asp360Lys ABCC1 p.Arg394Ile 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 ABCC1 p.Lys332Arg ABCC1 p.Lys396Glu ABCC1 p.Arg593Glu ABCC1 p.Asp436Lys 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 ABCC1 p.Lys332Asp ABCC1 p.Lys332Asp ABCC1 p.Lys332Arg ABCC1 p.Lys396Glu ABCC1 p.Asp336Lys ABCC1 p.Asp336Lys ABCC1 p.Lys347Asp ABCC1 p.Arg394Asp ABCC1 p.Lys319Asp ABCC1 p.Asp360Lys ABCC1 p.Glu573Arg ABCC1 p.Arg593Glu ABCC1 p.Asp436Lys ABCC1 p.Asp578Arg ABCC1 p.Asp572Arg 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 ABCC1 p.Lys396Glu ABCC1 p.Lys396Ile 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 ABCC1 p.Lys396Glu ABCC1 p.Lys396Ile 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 ABCC1 p.Lys396Glu 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 ABCC1 p.Lys396Glu 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 161 ABCC1 p.Asp430Lys ABCC1 p.Asp430Lys When Asp430 was replaced with Lys, the resulting D430K mutant was not expressed (Fig. 2). Login to comment 162 ABCC1 p.Asp436Lys ABCC1 p.Asp436Lys 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 ABCC1 p.Asp436Lys 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 ABCC1 p.Asp436Lys 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 ABCC1 p.Asp436Lys 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 ABCC1 p.Asp436Lys 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 ABCC1 p.Asp436Lys 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 ABCC1 p.Asp436Lys 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 175 ABCC1 p.Asp336Lys A, time course of [3 H]LTC4 uptake by wild-type MRP1 (f), mutants D336K (‚), D(K)336E (ƒ), and empty pcDNA3.1(-) control (E). Login to comment 176 ABCC1 p.Lys332Asp ABCC1 p.Asp336Lys B, time course of [3 H]LTC4 uptake by wild-type MRP1 (f), TM6 double mutant K332D/D336K (ࡗ), and empty pcDNA3.1(-) vector control (E). Login to comment 177 ABCC1 p.Lys347Asp ABCC1 p.Lys347Leu ABCC1 p.Lys319Asp C, apigenin-stimulated [3 H]GSH uptake at 20 min by wild-type (WT) MRP1 (f), mutants K319D, K347D, and K347L (u), and empty pcDNA3.1(-) vector control (Ⅺ). Login to comment 182 ABCC1 p.Arg593Glu 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 183 ABCC1 p.Glu573Arg ABCC1 p.Asp578Arg ABCC1 p.Asp572Arg The D572R, E573R, and D578R mutants exhibited transport activity profiles similar to wild-type MRP1, indicating that none of these negatively charged amino acids are critical for MRP1 transport activity (Table 2). Login to comment 184 ABCC1 p.Arg593Leu ABCC1 p.Arg593Glu In marked contrast, substitutions of TM11 Arg593 with Glu or Leu substantially reduced MRP1 transport activity. Login to comment 185 ABCC1 p.Arg593Leu ABCC1 p.Arg593Glu 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 ABCC1 p.Arg593Glu 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 ABCC1 p.Arg593Glu 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 ABCC1 p.Lys332Asp ABCC1 p.Asp336Glu ABCC1 p.Lys396Glu ABCC1 p.Asp336Lys ABCC1 p.Arg593Glu ABCC1 p.Asp436Lys 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 ABCC1 p.Lys396Glu ABCC1 p.Lys396Ile 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 ABCC1 p.Lys396Glu ABCC1 p.Lys396Ile 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 204 ABCC1 p.Lys332Arg 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 ABCC1 p.Lys332Arg 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 208 ABCC1 p.Asp336Glu In a similar way, a like-charge substitution of Glu for Asp at position 336 in the same TM helix of MRP1 resulted in a loss of overall transport activity comparable with that observed with Arg, Lys, or Leu substitutions (Haimeur et al., 2002). Login to comment 211 ABCC1 p.Lys332Asp ABCC1 p.Asp336Lys However, the inactivity of the double-exchange mutant K332D/D336K suggests that this is unlikely to be the case (Fig. 2). Login to comment 213 ABCC1 p.Lys396Glu ABCC1 p.Asp436Lys 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 ABCC1 p.Asp436Lys 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 ABCC1 p.Asp436Lys 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 232 ABCC1 p.Asp430Lys The exception was the Asp430 mutant D430K. Login to comment 233 ABCC1 p.Asp430Lys Northern blot analyses showed that the D430K-expressing cells contained levels of the mutant MRP1 mRNA that were comparable with those of wild-type MRP1 transfected cells, indicating a post-transcriptional event is probably involved (results not shown). Login to comment 239 ABCC1 p.Asp436Lys 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 242 ABCC1 p.Arg593Leu ABCC1 p.Arg593Glu 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 ABCC1 p.Arg593Leu ABCC1 p.Arg593Glu 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 249 ABCC1 p.Asp436Lys ABCC1 p.Asp436Lys 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 ABCC1 p.Lys396Glu ABCC1 p.Asp436Lys 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 255 ABCC1 p.Arg593Leu ABCC1 p.Arg593Glu 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 256 ABCC1 p.Arg593Lys On the other hand, the like-charge substitution of Arg593 with Lys resulted in a fully functional MRP1 R(E)593K. Login to comment 260 ABCC1 p.Arg593Glu 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 ABCC1 p.Lys332Asp ABCC1 p.Asp336Glu ABCC1 p.Lys396Glu ABCC1 p.Asp336Lys ABCC1 p.Arg593Glu ABCC1 p.Asp436Lys 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