ABCMdb

PMID: 19398503

Maeno K, Nakajima A, Conseil G, Rothnie A, Deeley RG, Cole SP
Molecular basis for reduced estrone sulfate transport and altered modulator sensitivity of transmembrane helix (TM) 6 and TM17 mutants of multidrug resistance protein 1 (ABCC1).
Drug Metab Dispos. 2009 Jul;37(7):1411-20. Epub 2009 Apr 27., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu Here we have extended our characterization of mutants K332L and W1246C to further define the different roles these two residues play in determining the substrate and inhibitor specificity of MRP1. Login to comment 7 ABCC1 p.Lys332Leu Finally, the differing abilities of the cysteinyl leukotriene derivatives leukotriene C4, D4, and F4 to inhibit estradiol glucuronide transport by wild-type and K332L mutant MRP1 provide further evidence that TM6-Lys332 is involved in the recognition of the ␥-Glu portion of substrates and modulators containing GSH or GSH-like moieties. Login to comment 29 ABCC1 p.Trp1246Phe In contrast, substitution of TM17-Trp1246 with Phe, Tyr, Ala, or Cys selectively eliminates E217betaG and NNAL-O-glucuronide transport and drug resistance but has little or no effect on LTC4 and GSH transport (Ito et al., 2001a; Leslie et al., 2001a). Login to comment 36 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu B-E, ATP-dependent uptake of 3 H-labeled organic anions was measured in membrane vesicles prepared from HEK293T (HEK) cells transfected with wild-type MRP1 (shaded bar) and K332L and W1246C mutant (open bars) cDNAs as described under Materials and Methods. Login to comment 57 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu The generation of wild-type, K332L, and W1246C mutant MRP1 pcDNA3.1 expression constructs has been described previously (Ito et al., 2001a; Haimeur et al., 2002). Login to comment 85 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu Equilibrium binding of [3 H]estrone 3-sulfate to wild-type MRP1 and the K332L and W1246C mutants was determined as described previously (Rothnie et al., 2006, 2008). Login to comment 91 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu To investigate the effect of estrone 3-sulfate concentration on [3 H]estrone 3-sulfate binding, 10 ␮g of membrane protein from wild-type and mutant MRP1-transfected cells were incubated with various concentrations of [3 H]estrone 3-sulfate (50 nM to 10 ␮M) and 3 mM S-MeGSH in the presence or absence of the competitive substrates E217betaG (1 mM) in the case of the K332L mutant and LTC4 (10 ␮M) in the case of the W1246C mutant. Login to comment 95 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu In the first series of experiments, the ability of the K332L and W1246C mutants to transport methotrexate and estrone 3-sulfate (in the presence of GSH and S-MeGSH) was measured. Login to comment 96 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu After transfecting HEK293T cells with the wild-type and mutant constructs, membrane vesicles were prepared, and relative expression levels of the K332L and W1246C mutants were determined by immunoblotting and found to be similar to wild-type MRP1 (data not shown), as reported previously (Ito et al., 2001a; Haimeur et al., 2002). Login to comment 98 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu As shown in Fig. 1B, uptake of [3 H]methotrexate by the K332L mutant was comparable with wild-type MRP1, whereas uptake by W1246C was reduced by Ͼ75%. Login to comment 100 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu As reported previously (Ito et al., 2001a; Haimeur et al., 2002), [3 H]LTC4 transport by the K332L mutant was also comparable with the untransfected HEK293T negative control, whereas that of the W1246C mutant was similar to wild-type MRP1 (Fig. 1D). Login to comment 101 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu On the other hand, the K332L mutant transported [3 H]E217betaG at levels comparable with wild-type MRP1, whereas the W1246C mutant showed no detectable transport of this conjugated estrogen, as expected from previous studies (Ito et al., 2001a) (Fig. 1E). Login to comment 102 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu S-MeGSH Fails to Increase Estrone 3-Sulfate Binding to the K332L and W1246C Mutants. Login to comment 103 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu To determine whether changes in the ability of S-MeGSH to stimulate binding might contribute to the low levels of estrone 3-sulfate transport displayed by the K332L and W1246C mutants, equilibrium binding assays with MRP1 in the nucleotide-free state were carried out. Login to comment 107 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu In contrast, S-MeGSH had little or no effect on estrone 3-sulfate binding to the K332L (Fig. 2A) or W1246C (Fig. 2B) mutants. Login to comment 108 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu The EC50 for S-MeGSH of wild-type MRP1 was 1.24 Ϯ 0.07 mM, whereas the binding curves for the K332L and W1246C mutants were almost flat, precluding reliable estimations of their EC50s for this tripeptide. Login to comment 109 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu Estrone 3-sulfate binding values for both the K332L and W1246C mutants at apparent saturation were just 20% that of wild-type MRP1 (after subtraction of nonspecific binding). Login to comment 110 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu To exclude the possibility that S-MeGSH-stimulated estrone 3-sulfate binding to the K332L and W1246C mutants might be underestimated in the above experiments because the initial concentration of estrone 3-sulfate used (50 nM) was too low, the binding assays were repeated at higher concentrations of this conjugated estrogen. Login to comment 112 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu K332L Decreases Ability of S-MeGSH to Stimulate Estrone 3-Sulfate Uptake, Whereas W1246C Decreases Affinity for Estrone 3-Sulfate. Login to comment 114 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu S-MeGSH stimulated binding of [3 H]estrone 3-sulfate by MRP1 mutants K332L and W1246C. Login to comment 115 ABCC1 p.Lys332Leu A, binding of [3 H]estrone 3-sulfate (50 nM) to wild-type MRP1 (f), K332L mutant (F), and control HEK (E) membranes (10 ␮g of protein) was measured at 23°C for 60 min in the presence of increasing concentrations (0.01-18 mM) of S-MeGSH. Login to comment 117 ABCC1 p.Trp1246Cys B, binding of [3 H]estrone 3-sulfate to wild-type MRP1 (f), W1246C mutant (Œ), and control HEK (E) membranes (10 ␮g of protein) measured in the presence of S-MeGSH (0.01-18 mM) as described in A. Login to comment 119 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu C, specific binding of [3 H]estrone 3-sulfate to wild-type MRP1 (f), W1246C (Œ), K332L (F), and control HEK (E) membranes (10 ␮g of protein) was measured at estrone 3-sulfate concentrations ranging from 0.01 to 10 mM in the presence of 3 mM S-MeGSH. Login to comment 123 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu To help distinguish between these possibilities, ATP-dependent estrone 3-sulfate transport by the K332L and W1246C mutants was measured in the absence of S-MeGSH (or GSH) and compared with transport by wild-type MRP1. Login to comment 124 ABCC1 p.Lys332Leu As shown in Fig. 3A, [3 H]estrone 3-sulfate uptake in the absence of S-MeGSH (or GSH) by K332L was similar to that of wild-type MRP1. Login to comment 125 ABCC1 p.Trp1246Cys In contrast, ATP-dependent uptake of estrone 3-sulfate by W1246C was very low and similar to that of negative control HEK293T membrane vesicles, and this did not change when S-MeGSH was present (Figs. Login to comment 127 ABCC1 p.Lys332Leu ABCC1 p.Lys332Leu These observations indicate that like wild-type MRP1, the K332L mutant can still bind and transport estrone 3-sulfate in the absence of S-MeGSH (or GSH), but unlike wild-type MRP1, estrone 3-sulfate uptake by the K332L mutant is no longer stimulated by S-MeGSH. Login to comment 128 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu Conversely, unlike wild-type and K332L mutant MRP1, W1246C apparently no longer binds estrone 3-sulfate; thus, no transport is observed in either the presence or absence of S-MeGSH (or GSH). Login to comment 129 ABCC1 p.Lys332Leu Loss of Synergistic Inhibition of E217betaG Uptake by GSH and Apigenin for the K332L Mutant. Login to comment 131 ABCC1 p.Lys332Leu Therefore, we reasoned that if the K332L mutant could no longer bind GSH (or S-MeGSH), then GSH (or S-MeGSH) would no longer be expected to act synergistically with apigenin to inhibit uptake of LTC4 or other organic anion substrates. Login to comment 132 ABCC1 p.Lys332Leu Accordingly, ATP-dependent E217betaG uptake by K332L-enriched membrane vesicles was measured in the absence or presence of GSH (3 mM) and apigenin (10 ␮M). Login to comment 135 ABCC1 p.Lys332Leu In contrast, neither GSH or apigenin (10 ␮M) alone nor the combination of GSH and apigenin had any effect on E217betaG uptake by the K332L mutant. Login to comment 137 ABCC1 p.Lys332Leu ABCC1 p.Lys332Leu Because higher concentrations of apigenin (30 ␮M) inhibited E217betaG transport by wild-type and K332L MRP1 similarly (data not shown), these observations support the conclusion that GSH (and S-MeGSH) binding to the K332L mutant is severely impaired. Login to comment 138 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu Effect of MRP1 Modulators on the Activity of MRP1 Mutants K332L and W1246C. Login to comment 139 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu Because of the marked differences in the substrate specificity of the K332L and W1246C mutants, we next investigated whether the two mutants differed in their sensitivity to compounds reported previously to be modulators of MRP1 transport activity. Login to comment 140 ABCC1 p.Lys332Leu Thus, the effect of four small molecule inhibitors (MK571, LY465803, LY171883, and BAY u9773) (Fig. 4A) and two GSH derivatives (S-decyl-GSH and GSSG) (Fig. 4B) were tested for their ability to inhibit E217betaG transport by the K332L mutant. Login to comment 141 ABCC1 p.Trp1246Cys Three of the small molecules (MK571, BAY u9773, and LY465803) and S-decyl-GSH were also examined for their ability to inhibit LTC4 transport by the W1246C mutant. Login to comment 145 ABCC1 p.Lys332Leu As summarized in Table 1 and shown in Fig. 5, MK571 and LY171883 were moderately (2-fold) less potent inhibitors of E217betaG uptake by wild-type MRP1 than by the K332L mutant, whereas the inhibitory potency of BAYu9773 was not significantly different for the mutant and wild-type transporters. Login to comment 146 ABCC1 p.Lys332Leu In contrast, LY465803 (ϩGSH) showed a far greater potency (16-fold) to inhibit wild-type compared with K332L mutant MRP1. Login to comment 147 ABCC1 p.Lys332Leu In a similar manner, the GSH derivative S-decyl-GSH and GSSG inhibited wild-type MRP1 activity with IC50s Ͼ100-fold and Ͼ30-fold lower than for K332L, respectively. Login to comment 148 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu In contrast to its more potent inhibitory effect on E217betaG transport by the K332L mutant, MK571 was a less potent inhibitor (ϳ3-fold) of LTC4 transport by the W1246C mutant than by wild-type MRP1 (Table 2; Fig. 6A). Login to comment 149 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu However, similar to K332L, the inhibitory potency of BAY u9773 for LTC4 transport by wild-type and W1246C was comparable (Table 2; Fig. 6B). Login to comment 150 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu Also similar to K332L, LY465803 (ϩGSH) showed a far greater (Ͼ250-fold) potency to inhibit wild-type MRP1 than the W1246C mutant (Table 2; Fig. 6C). Login to comment 151 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu Finally, in contrast to K332L, the sensitivity of W1246C and wild-type MRP1 to FIG. 3. Login to comment 152 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu [3 H]Estrone 3-sulfate and [3 H]E217betaG vesicular uptake by K332L and W1246C mutant and wild-type MRP1 proteins. Login to comment 153 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu A, ATP-dependent [3 H]estrone 3-sulfate uptake by membrane vesicles prepared from untransfected control cells (HEK) (solid bar) and cells transfected with cDNA vectors containing wild-type MRP1 (shaded bar), and K332L and W1246C mutant MRP1 (open bars) was measured in the absence of S-MeGSH. Login to comment 154 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu Inset, [3 H]estrone 3-sulfate uptake by membrane vesicles prepared from untransfected HEK cells (solid bars) and HEK cells expressing comparable levels of wild-type MRP1 (shaded bars), and MRP1 mutants K332L and W1246C (open bars) was measured in the absence and presence of S-MeGSH as described under Materials and Methods. Login to comment 157 ABCC1 p.Lys332Leu B, ATP-dependent [3 H]E217betaG uptake was measured in membrane vesicles prepared from untransfected cells (HEK) (solid bars), and wild-type MRP1 (shaded bars), and K332L-MRP1 (open bars) transfected cells with and without GSH (3 mM) and with and without apigenin (10 ␮M) as indicated. Login to comment 160 ABCC1 p.Lys332Leu Effect of Leukotriene Derivatives on E217betaG Transport Activity by K332L. Login to comment 162 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu ABCC1 p.Lys332Leu Thus, the equal sensitivity of the transport activities of the K332L and W1246C mutants to inhibition by BAY u9773, despite their profoundly different abilities to transport LTC4, suggests that the K332L mutant has lost its ability to bind to the GSH moiety of LTC4. Login to comment 163 ABCC1 p.Lys332Leu To determine whether this loss of binding involves one or more of the three amino acids that comprise the GSH moiety of LTC4, E217betaG transport assays with wild-type MRP1 and the K332L mutant were carried out in the presence of various concentrations of the cysteinyl leukotriene derivatives LTC4, LTD4, and LTF4. Login to comment 164 ABCC1 p.Lys332Leu As expected from previous studies (Haimeur et al., 2002), the IC50 of LTC4 (containing ␥-Glu, Cys, and Gly) of the K332L mutant was significantly increased (ϳ25-fold) compared with that of wild-type MRP1 (Table 3; Fig. 7A). Login to comment 165 ABCC1 p.Lys332Leu ABCC1 p.Lys332Leu When E217betaG transport was carried out in the presence of LTD4 (lacking a ␥-Glu moiety but containing Gly and Cys), the IC50s of wild-type MRP1 and the K332L mutant were the same, although the inhibitor potency of LTD4 compared with LTC4 was ϳ40-fold less for wild-type MRP1 but just ϳ2-fold less for K332L (Table 3; Fig. 7B). Login to comment 166 ABCC1 p.Lys332Leu The IC50s of LTF4 (containing a ␥-Glu moiety but lacking Gly) for K332L and wild-type MRP1 differed by 4-fold, with the inhibitor potency of LTF4 comparable with LTD4 (Table 3; Fig. 7C). Login to comment 167 ABCC1 p.Lys332Leu Thus, the K332L mutant protein is considerably less sensitive than wild-type MRP1 with respect to inhibition by the ␥-Glu-containing LTC4 and LTF4 but not the ␥-Glu-less LTD4, suggesting that Lys332 is critical for recognizing the ␥-Glu moiety of GSH. Login to comment 168 ABCC1 p.Lys332Leu TABLE 1 Effect of modulators on E217betaG uptake by wild-type and K332L mutant MRP1 The values shown represent the means (ϮS.D.) of IC50 values obtained in three independent experiments. Login to comment 170 ABCC1 p.Lys332Leu E217betaG Uptake IC50 Modulator Wild-type K332L ␮M ␮M MK571 0.4 Ϯ 0.1 0.20 Ϯ 0.07a LY171883 19.8, 14.7 8.8, 8.1 BAY u9773 0.20 Ϯ 0.03 0.3 Ϯ 0.1 LY465803 (ϩGSH) 0.2 Ϯ 0.1 3.3 Ϯ 2.5b S-Decyl-GSH 0.07 Ϯ 0.01 Ͼ10, Ͼ10c GSSG 16.3, 13.0 503.0, 505.8 a Significantly different from IC50 obtained with wild-type MRP1 (p Ͻ 0.05). Login to comment 180 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu In the present study, we have further characterized the substrate specificity of two of these, the TM6 mutant K332L and the TM17 mutant W1246C, and have also determined their inhibitor profiles. Login to comment 189 ABCC1 p.Lys332Leu ABCC1 p.Lys332Leu Effect of MRP1 modulators and GSH derivatives on [3 H]E217betaG transport by wild-type and K332L mutant MRP1. ATP-dependent vesicular uptake of [3 H]E217betaG by wild-type MRP1 (open symbols, dotted lines) and K332L mutant MRP1 (closed symbols, solid lines) was measured in the presence of the indicated concentrations of modulators and GSH derivatives. Login to comment 194 ABCC1 p.Trp1246Cys TABLE 2 Effect of modulators on LTC4 uptake by wild-type and W1246C mutant MRP1 The values shown represent the means (ϮS.D.) of IC50 values obtained in three independent experiments. Login to comment 195 ABCC1 p.Trp1246Cys Modulator LTC4 Uptake IC50 Wild-type W1246C ␮M ␮M MK571 0.50 Ϯ 0.16 1.53 Ϯ 0.67a BAY u9773 0.4 Ϯ 0.1 0.57 Ϯ 0.35 LY465803 (ϩGSH) 0.046 Ϯ 0.007 13.2 Ϯ 9.1a S-Decyl-GSH 0.09 Ϯ 0.02 0.09 Ϯ 0.06 a Significantly different from IC50 in wild-type MRP1 (p Ͻ 0.01). Login to comment 198 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu However, this was not the case for membranes prepared from transfected HEK cells expressing either the K332L or W1246C mutants. Login to comment 200 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu We found that GSH-independent estrone 3-sulfate transport by W1246C was similar to negative controls, whereas transport by K332L was comparable with wild-type MRP1. Login to comment 201 ABCC1 p.Lys332Leu Thus, in the case of the K332L mutant, impaired GSH-stimulated estrone 3-sulfate transport seems to be caused by a loss of GSH binding. Login to comment 202 ABCC1 p.Trp1246Cys In contrast, impaired transport by the W1246C mutant is caused by a loss of binding of the sulfated estrogen. Login to comment 203 ABCC1 p.Lys332Leu Furthermore, unlike wild-type MRP1, GSH no longer acted synergistically with apigenin to inhibit E217betaG uptake by the K332L mutant. Login to comment 205 ABCC1 p.Lys332Leu The latter conclusion is also consistent with the inhibitor profile displayed by the K332L mutant (Table 1; Fig. 5). Login to comment 206 ABCC1 p.Lys332Leu The inhibitory potencies of modulators such as MK571, LY171883, and BAY u9773, which do not contain a GSH moiety and are not GSH-dependent, on the transport activities of wild-type and K332L mutant MRP1 were much more similar (1.5-2-fold difference in IC50s) than those of modulators that contain a GSH or GSH-like moiety (S-decyl-GSH, GSSG) or are dependent on GSH (LY465803) for their activity (15-30-fold difference in IC50s). Login to comment 207 ABCC1 p.Trp1246Cys On the other hand, as might be expected because mutation of Trp1246 has little or no effect on transport of either GSH or the GSH-containing LTC4 (Table 2; Fig. 6), the inhibitory potency of S-decyl-GSH was the same for the W1246C mutant and wild-type MRP1. Login to comment 208 ABCC1 p.Trp1246Cys The inhibitory potencies of the non-GSH-containing modulators MK571 and BAY u9773 on LTC4 transport by wild-type and W1246C mutant MRP1 were also comparable. Login to comment 211 ABCC1 p.Trp1246Cys However, TM17-Trp1246 seems to be important for recognition of the tricyclic isoxazole derivative LY465803 because the IC50 for this modulator (in the presence of GSH) was approximately 280-fold greater for the W1246C mutant than for wild-type MRP1 (13.2 ␮M versus 46 nM). Login to comment 212 ABCC1 p.Trp1246Cys This observation is consistent with our earlier finding that radiolabeling of the COOH-proximal half of the W1246C mutant by [125 I]LY475776, a photoactivateable derivative of LY465803, was substantially reduced relative to the same region of wild-type MRP1 (Mao et al., 2002). Login to comment 213 ABCC1 p.Lys332Leu When transport assays were carried out in the presence of three cysteinyl leukotriene derivatives to determine whether reduced binding of compounds that contain GSH-like moieties by the K332L mutant involves one or more of the three amino acids that comprise FIG. 6. Login to comment 214 ABCC1 p.Trp1246Cys ABCC1 p.Trp1246Cys ABCC1 p.Trp1246Cys ABCC1 p.Trp1246Cys Effect of small molecule modulators and S-decyl-GSH on [3 H]LTC4 transport by wild-type and W1246C mutant MRP1. ATP-dependent vesicular uptake of [3 H]LTC4 by wild-type MRP1 (open symbols, dotted lines) and W1246C mutant MRP1 (closed symbols, solid lines) was measured in the presence of the indicated concentrations of modulators and S-decyl-GSH. Login to comment 219 ABCC1 p.Lys332Leu TABLE 3 Effect of leukotriene derivatives on E217betaG uptake by wild-type and K332L mutant MRP1 The values shown represent the means (ϮS.D.) of IC50 values obtained in three or four independent experiments (number shown in parentheses). Login to comment 220 ABCC1 p.Lys332Leu E217betaG Uptake IC50 Leukotriene Derivative Wild-type K332L ␮M ␮M LTC4 0.1 Ϯ 0.03 (3) 2.5 Ϯ 0.5 (3)a LTD4 4.3 Ϯ 0.4 (3) 4.2 Ϯ 2.3 (3) LTF4 3.7 Ϯ 0.1 (4) 15.3 Ϯ 7.3 (4)a a Significantly different from IC50 in wild-type MRP1 (p Ͻ 0.01). Login to comment 222 ABCC1 p.Lys332Leu Thus, E217betaG transport by the K332L mutant was considerably less sensitive than wild-type MRP1 to inhibition by the ␥-Glu- containing LTC4 and LTF4 (25- and 4-fold differences in IC50s, respectively) than for the ␥-Glu-less LTD4, for which the IC50s were the same (Fig. 7; Table 3). Login to comment 229 ABCC1 p.Trp1246Cys ABCC1 p.Lys332Leu In conclusion, we have extended our characterization of the MRP1 mutants K332L and W1246C and further defined the different roles of these two amino acids in determining the substrate and inhibitor specificity of MRP1. Login to comment