ABCMdb

PMID: 15208328

Situ D, Haimeur A, Conseil G, Sparks KE, Zhang D, Deeley RG, Cole SP
Mutational analysis of ionizable residues proximal to the cytoplasmic interface of membrane spanning domain 3 of the multidrug resistance protein, MRP1 (ABCC1): glutamate 1204 is important for both the expression and catalytic activity of the transporter.
J Biol Chem. 2004 Sep 10;279(37):38871-80. Epub 2004 Jun 18., 2004-09-10 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC1 p.Arg1202Gly ABCC1 p.Arg1202Leu ABCC1 p.Arg1202Lys In addition, organic anion transport levels of the R1202L, R1202G, and R1202K mutants were comparable with wild-type MRP1. Login to comment 5 ABCC1 p.Glu1204Asp ABCC1 p.Glu1204Leu In contrast, organic anion transport by E1204L was substantially reduced, whereas transport by E1204D was comparable with wild-type MRP1, with the notable exception of GSH. Login to comment 8 ABCC1 p.Glu1204Leu ABCC1 p.Glu1204Leu In contrast, substrate binding by the transport-compromised E1204L mutant remained intact. Furthermore, vanadate-induced trapping of azido-ADP by E1204L was dramatically increased, indicating that this mutation may cause a partial uncoupling of the catalytic and transport activities of MRP1. Login to comment 82 ABCC1 p.Arg1131Asp ABCC1 p.Arg1046Asp RESULTS Expression and Transport Properties of MRP1 Mutants Containing Opposite and Same Charge Substitutions of Arg1046 , Asp1084 , and Arg1131 in or Near TM13-15-In the first series of experiments, the importance of ionizable amino acids located in or near predicted TM13-15 for MRP1 protein expression and function were investigated by replacing Arg1046 and Arg1131 with Asp and Glu, respectively. Login to comment 83 ABCC1 p.Arg1046Asp ABCC1 p.Arg1131Glu Levels of expression of the R1046D and R1131E mutant proteins were determined by the immunoblotting of membrane vesicles prepared from the transfected HEK293T cells with mAb QCRL-1. Login to comment 84 ABCC1 p.Asp1084Arg The TM14-associated D1084R mutant described previously (27) was included for comparison. Login to comment 87 ABCC1 p.Asp1084Arg ABCC1 p.Arg1046Asp ABCC1 p.Arg1131Glu To determine the functional consequences of replacing Arg1046 , Asp1084 , and Arg1131 with an oppositely charged amino acid, the ability of the R1046D, D1084R, and R1131E mutants to transport different organic anions was tested. Login to comment 88 ABCC1 p.Asp1084Arg ABCC1 p.Arg1046Asp ABCC1 p.Arg1131Glu Fig. 2, B and C, shows that uptake levels of E217betaG and LTC4, respectively, by the R1046D and R1131E mutants were moderately reduced (by 30-50%) compared with wild-type MRP1, whereas uptake by the D1084R mutant was substantially reduced by Ͼ90%. Login to comment 89 ABCC1 p.Asp1084Arg ABCC1 p.Arg1046Asp ABCC1 p.Arg1131Glu Similarly, levels of E1SO4 and MTX uptake by the R1046D and R1131E mutants were comparable with or moderately different from wild-type MRP1, whereas uptake of these organic anions by D1084R was substantially diminished (Յ25% of wild-type MRP1) (Fig. 2, D and E). Login to comment 93 ABCC1 p.Asp1084Glu ABCC1 p.Asp1084Arg ABCC1 p.Arg1046Asp ABCC1 p.Arg1131Glu A, MRP1 expression in membrane vesicles prepared from HEK293T cells transfected with empty vector [pcDNA3.1] and vector containing wild-type (WT-MRP1) and mutant (R1046D, D1084R, D1084E, and R1131E) cDNAs was determined by immunoblotting with mAb QCRL-1. Shown is a representative immunoblot of membrane vesicles (1 and 2 ␮g of protein) from a single transfection. Login to comment 95 ABCC1 p.Asp1084Glu ABCC1 p.Asp1084Arg ABCC1 p.Arg1046Asp ABCC1 p.Arg1131Glu B-F, levels of 3 H-labeled organic anion uptake by the membrane vesicles shown in A were determined and corrected to take into account any differences in MRP1 protein expression (empty pcDNA3.1 vector control (open bars), wild-type MRP1 (black bars), and mutants R1046D, D1084R, D1084E, and R1131E (gray bars)). Login to comment 98 ABCC1 p.Asp1084Asn sitely charged amino acid caused a global and almost complete loss of transport activity. We reported previously (27) that substitution of Asp1084 with Asn, Ala, or Val also eliminated LTC4 and E217betaG transport by MRP1. Login to comment 100 ABCC1 p.Asp1084Glu Because the overall transport activity of the oppositely charged Asp1084 mutant was greatly diminished (Fig. 2, B-E), a like-charge substituted D1084E mutant was created to clarify whether it was the loss of the acidic character or a change in size of the amino acid side chain that was responsible for the diminished activity. Login to comment 101 ABCC1 p.Asp1084Glu ABCC1 p.Asp1084Arg Like the D1084R mutant, the D1084E mutant showed substantially reduced LTC4 uptake levels (Ͻ20% of wild-type MRP1) (27) (Fig. 2C). Login to comment 102 ABCC1 p.Asp1084Glu ABCC1 p.Asp1084Arg In contrast to D1084R, however, the D1084E mutant exhibited E217betaG (Fig. 2B), E1SO4 (Fig. 2D), and MTX (Fig. 2E) uptake levels that were similar or only moderately reduced compared with those of wild-type MRP1. Login to comment 103 ABCC1 p.Asp1084Glu ABCC1 p.Asp1084Arg Because of the apparent selectively greater loss of LTC4 transport activity by the D1084E mutant, GSH uptake by this mutant was also examined and compared with that of D1084R. Login to comment 104 ABCC1 p.Asp1084Glu ABCC1 p.Asp1084Arg As shown in Fig. 2F, apigenin-stimulated GSH uptake by the D1084R and D1084E mutants was reduced by Ͼ90 and 80%, respectively. Login to comment 107 ABCC1 p.Arg1249Asp ABCC1 p.Arg1197Glu The relative mean expression levels of the R1197E and R1249D mutants from 2 to 3 independent transfections were comparable with wild-type MRP1 (110 and 70%, respectively). Login to comment 108 ABCC1 p.Arg1202Asp ABCC1 p.Glu1204Lys In contrast, expression levels of the R1202D and E1204K mutants were substantially reduced (by Ͼ75%). Login to comment 109 ABCC1 p.Arg1202Asp ABCC1 p.Arg1202Asp ABCC1 p.Glu1204Lys ABCC1 p.Glu1204Lys A Northern blot analysis performed on total RNA isolated from the transfected cells indicated that the R1202D and E1204K mRNA levels were comparable with wild-type MRP1 mRNA levels, thus excluding the possibility that the low R1202D and E1204K protein levels could be caused by differences in transfection efficiency (results not shown). Login to comment 113 ABCC1 p.Arg1249Asp ABCC1 p.Arg1197Glu ABCC1 p.Arg1202Asp ABCC1 p.Glu1204Lys Membrane vesicles prepared from HEK293T cells transfected with empty vector [pcDNA3.1] and vector containing the wild-type (WT-MRP1) and mutant (R1197E, R1202D, E1204K, and R1249D) cDNAs were immunoblotted with mAb QCRL-1. Shown is a representative immunoblot of membrane vesicles (1 and 2 ␮g of protein) from a single transfection. Login to comment 116 ABCC1 p.Arg1202Gly ABCC1 p.Arg1202Leu MRP1 expression levels in membrane vesicle proteins R1202G and R1202L prepared from cells expressing the neutrally substituted Arg1202 mutants were determined as described in A. Login to comment 118 ABCC1 p.Arg1202Gly ABCC1 p.Asp1084Glu ABCC1 p.Asp1084Arg ABCC1 p.Arg1202Leu ABCC1 p.Glu1204Asp ABCC1 p.Glu1204Asp ABCC1 p.Glu1204Leu ABCC1 p.Glu1204Leu ABCC1 p.Arg1046Asp ABCC1 p.Arg1131Glu ABCC1 p.Arg1249Asp ABCC1 p.Arg1197Glu ABCC1 p.Arg1249Lys ABCC1 p.Arg1197Lys Immunoblots of membrane vesicle proteins prepared from cells expressing the Glu1204 mutants E1204L and E1204D were carried out as described in A. TABLE I Summary of organic anion transport activity of MRP1 mutants with substitutions of ionizable amino acids in and proximal to TM13 to TM17 of MSD3 Mutation % Wild-type MRP1 transport activitya E217betaG LTC4 E1SO4 MTX GSH TM13 R1046D 115 70 80 120 NDb TM14 D1084R Ͻ10 Ͻ10 15 25 Ͻ10 D1084E 80 20 65 90 20 TM15 R1131E 70 50 80 60 ND TM16 R1197E Ͻ10 Ͻ10 Ͻ15 Ͻ10 ND R1197K 20 Ͻ25 Ͻ20 Ͻ10 ND R1202G 115 115 75 70 ND R1202L 115 120 50 110 ND E1204L Ͻ10 50 10 110 Ͻ25 E1204D 100 115 100 115 Ͻ25 TM17 R1249D Ͻ10 Ͻ15 Ͻ10 Ͻ10 ND R1249K Ͻ10 10 Ͻ15 Ͻ10 ND a The values shown are means of duplicate or triplicate determinations and are derived from Fig. 2, 4, and 5 (see figure legends for details). Login to comment 121 ABCC1 p.Glu1204Leu sion of MRP1 in HEK cell membranes was further explored by replacing Arg1202 with the hydrophobic Leu, and Glu1204 with Leu and Asp. Login to comment 124 ABCC1 p.Arg1202Gly ABCC1 p.Arg1202Leu ABCC1 p.Glu1204Asp ABCC1 p.Glu1204Leu Immunoblots showed that expression levels of the R1202G and R1202L mutants (Fig. 3B) and the E1204L and E1204D mutants (Fig. 3C) ranged from 80 to 225% of wild-type MRP1. Login to comment 127 ABCC1 p.Arg1202Gly ABCC1 p.Arg1202Leu At 1 min, E217betaG and LTC4 uptake by the R1202G and R1202L mutants was comparable with wild-type levels (Fig. 4, A and B). Login to comment 128 ABCC1 p.Arg1202Gly ABCC1 p.Arg1202Leu Levels of E1SO4 uptake by the R1202G mutant were reduced by ϳ25%, whereas uptake by the R1202L mutant was reduced by ϳ50% (Fig. 4C). Login to comment 129 ABCC1 p.Arg1202Gly ABCC1 p.Arg1202Leu Uptake levels of MTX by the R1202G mutant was moderately reduced (by ϳ30%), whereas uptake by R1202L was comparable with wild-type MRP1 (Fig. 4D). Login to comment 130 ABCC1 p.Glu1204Leu In contrast to the neutrally substituted Arg1202 mutants, E217betaG uptake by the neutrally substituted Glu1204 mutant E1204L was Ͻ10% of wild-type MRP1 levels (Fig. 4E). Login to comment 131 ABCC1 p.Glu1204Leu In addition, LTC4 uptake by E1204L was reduced by 50% (Fig. 4F), and E1SO4 uptake was reduced by 90% (Fig. 4G). Login to comment 134 ABCC1 p.Glu1204Asp ABCC1 p.Glu1204Leu To determine whether the substrate-selective loss of transport function observed in the E1204L mutant was because of the loss of the acidic character or the change in the size of the side chain, organic anion uptake by the same-charge mutant, E1204D, was also assessed. Login to comment 135 ABCC1 p.Glu1204Asp As shown in Fig. 4, E-H, and Table I, the E1204D mutant exhibited transport levels comparable with wild-type MRP1 for all substrates tested. Login to comment 137 ABCC1 p.Glu1204Asp ABCC1 p.Glu1204Leu As shown in Fig. 4I, both E1204L and E1204D exhibited a similar and substantial decrease in GSH transport levels (Ͼ75%). Login to comment 139 ABCC1 p.Glu1204Asp In general, the neutral mutants of Arg1202 showed only moderate and substrate-specific decreases in their transport activities. On the other hand, a neutral substitution of Glu1204 reduced or eliminated transport of all organic anions except MTX, whereas substitution with Asp had no effect with the exception that E1204D no longer transported GSH (see Table I for summary). Login to comment 141 ABCC1 p.Arg1249Asp ABCC1 p.Arg1197Glu ABCC1 p.Arg1202Asp ABCC1 p.Glu1204Lys Transport Activities of TM16/17 Arg1197 and Arg1249 Mutants-Unlike the R1202D and E1204K mutants, oppositely charged substitutions of Arg1197 (R1197E) and Arg1249 (R1249D) did not adversely affect expression of MRP1 (Fig. 3A). Login to comment 143 ABCC1 p.Arg1249Asp ABCC1 p.Arg1197Glu Thus, levels of E217betaG uptake by both mutants were Ͻ10% of wild-type MRP1 levels (Fig. 5A), whereas LTC4 uptake by the R1197E and R1249D mutants was Ͻ15% of wild-type MRP1 (Fig. 5B). Login to comment 144 ABCC1 p.Arg1249Asp ABCC1 p.Arg1197Glu Uptake of E1SO4 by the R1197E mutant was Ͻ15% of wild-type MRP1, whereas uptake of this sulfated estrogen by the R1249D mutant was reduced by Ͼ90% (Fig. 5C). Login to comment 145 ABCC1 p.Arg1249Asp ABCC1 p.Arg1197Glu Finally, the R1197E and R1249D mutants showed MTX transport activity that was not significantly different from the empty vector control (Fig. 5D). Login to comment 146 ABCC1 p.Arg1249Lys ABCC1 p.Arg1197Lys To determine whether the charge or size of the Arg1197 and Arg1249 side chains were important for MRP1 transport function, the like-charge substituted mutants R1197K and R1249K were created. Login to comment 148 ABCC1 p.Arg1249Lys ABCC1 p.Arg1197Lys Nevertheless, ATP-dependent E217betaG uptake by the R1197K and R1249K mutants was 20 and Ͻ10% of wild-type MRP1, respectively (Fig. 5A). Login to comment 149 ABCC1 p.Arg1249Lys ABCC1 p.Arg1197Lys Similarly, uptake levels of LTC4 (Fig. 5B), E1SO4 (Fig. 5C), and MTX (Fig. 5D) by the R1197K and R1249K mutants were reduced to Յ10% of wild-type MRP1 uptake levels. Login to comment 151 ABCC1 p.Glu1204Leu ABCC1 p.Arg1249Lys ABCC1 p.Arg1197Lys Effect of Glu1204 , Arg1197 , and Arg1249 Mutations on Photolabeling with [3 H]LTC4 and 8-Azido-[␣-32 P]ATP-In the next series of experiments, those same-charge or neutrally substituted mutants that showed substantially reduced transport activities (R1197K, E1204L, and R1249K) were further examined to determine whether their loss of transport activity was accompanied by a decrease in substrate binding. Login to comment 152 ABCC1 p.Arg1249Lys ABCC1 p.Arg1197Lys As shown in Fig. 6A, [3 H]LTC4 photolabeling of the R1197K and R1249K mutants was completely abrogated, indicating that the reduced LTC4 transport activity of these mutants was associated with decreased binding of this substrate. Login to comment 153 ABCC1 p.Glu1204Leu In contrast, [3 H]LTC4 labeling of the E1204L mutant was comparable with wild-type MRP1, despite the fact that transport of this organic anion by this mutant was substantially reduced. Login to comment 154 ABCC1 p.Glu1204Leu ABCC1 p.Arg1249Lys ABCC1 p.Arg1197Lys To determine whether the mutations of Arg1197 , Glu1204 , and Arg1249 that altered the transport properties of MRP1 also affected the interaction of the transporter with nucleotide, the ability of the R1197K, E1204L, and R1249K mutants to be photolabeled with 8-azido-[␣-32 P]ATP, both at 4 °C to minimize hydrolysis and at 37 °C in the presence of sodium vanadate to trap azido-ADP after hydrolysis, was examined (31, 32). Login to comment 155 ABCC1 p.Arg1249Lys ABCC1 p.Arg1197Lys As shown in Fig. 6B, 8-azido-[␣-32 P]ATP labeling of the R1197K and R1249K mutants was comparable with wild-type MRP1. Login to comment 157 ABCC1 p.Arg1249Lys ABCC1 p.Arg1197Lys Furthermore, orthovanadate-induced trapping of 8-azido-[␣- 32 P]ADP of the R1197K and R1249K mutants was also comparable with wild-type MRP1 (Fig. 6C). Login to comment 159 ABCC1 p.Glu1204Leu 8-Azido-[␣-32 P]ATP labeling of the transport-compromised E1204L mutant was also comparable with wild-type MRP1 (Fig. 6B). Login to comment 161 ABCC1 p.Glu1204Leu ABCC1 p.Glu1204Leu Because E1204L could still be photolabeled with LTC4 despite substantially reduced transport of this organic anion, [3 H]LTC4 photolabeling of the mutant protein after prior incubation with ATP and vanadate was examined to determine whether the increased trapping of azido-ADP by E1204L altered the substrate binding properties of MRP1. Login to comment 163 ABCC1 p.Glu1204Leu A similar decrease in [3 H]LTC4 labeling of the E1204L mutant was observed under the same conditions. Login to comment 169 ABCC1 p.Arg1202Gly ABCC1 p.Arg1202Leu A-D, uptake of 3 H-labeled organic anions by the membrane vesicles shown in Fig. 3B which were prepared from cells transfected with empty pcDNA3.1 vector (open bars), and vector containing wild-type MRP1 cDNA (black bars), and the neutrally substituted Arg1202 mutant R1202G and R1202L cDNAs (gray bars). Login to comment 172 ABCC1 p.Glu1204Asp ABCC1 p.Glu1204Leu E-H, uptake of 3 H-labeled organic anions by the membrane vesicles shown in Fig. 3C which were prepared from cells transfected with empty pcDNA3.1 vector (open bars), vector containing wild-type MRP1 cDNA (black bars), and the Glu1204 mutant E1204L and E1204D cDNAs (gray bars). Login to comment 177 ABCC1 p.Arg1249Asp ABCC1 p.Arg1197Glu ABCC1 p.Arg1249Lys ABCC1 p.Arg1197Lys Levels of 3 H-labeled organic anion uptake by membrane vesicles were prepared from cells expressing wild-type MRP1 (black bars), mutants R1197E, R1197K, R1249D, and R1249K (gray bars), and empty pcDNA3.1 vector control vesicles (open bars). Login to comment 182 ABCC1 p.Asp1084Glu We reported previously (27) that nonconservative substitutions of Asp1084 proximal to TM14 caused a substantial reduction in E217betaG, LTC4, and GSH transport and drug resistance, and we have now shown that these mutations also reduce MTX and E1SO4 transport activity, demonstrating a global disruption of MRP1 activity. We have also found that the same-charge mutant, D1084E, has significant transport activity with respect to E217betaG, MTX, and E1SO4 whereas transport of GSH and the glutathione conjugate LTC4 remains quite low. Login to comment 184 ABCC1 p.Asp1084Glu However, neither physical property of Asp1084 appears necessary for substrate binding, because GSH is still able to stimulate E1SO4 transport indicating that GSH binding to D1084E is intact. Login to comment 185 ABCC1 p.Asp1084Glu ABCC1 p.Asp1084Arg Similarly, D1084E and D1084R can still be photolabeled with LTC4 as well as wild-type MRP1. Login to comment 189 ABCC1 p.Arg1202Asp ABCC1 p.Glu1204Lys This idea is supported by the observation that membrane expression of the R1202D and E1204K mutants in mammalian cells can be substantially increased when transfected cells are grown at lower temperatures (30 °C) where the stringency of the proofreading machinery for monitoring protein folding is diminished.2 Because neutral (Leu, Gly) substitutions of TM16 Arg1202 and Glu1204 did not affect MRP1 expression in any significant way, it may be concluded that it is the opposite charge of the side chains of the substituting amino acids in the nonexpress- 2 A. Haimeur, R. G. Deeley, and S. P. C. Cole, unpublished observations. Login to comment 192 ABCC1 p.Glu1204Leu ABCC1 p.Arg1249Lys ABCC1 p.Arg1197Lys A, membrane vesicle proteins (50 ␮g) prepared from cells expressing wild-type (WT-MRP1) and transport-compromised mutant MRP1 proteins (R1197K, E1204L, and R1249K) were incubated with [3 H]LTC4 (200 nM; 250 nCi) followed by UV cross-linking, SDS-PAGE, and fluorography. Login to comment 197 ABCC1 p.Glu1204Leu D, wild-type and E1204L mutant membrane vesicles (50 ␮g) were incubated in transport buffer containing 5 mM MgCl2 for 20 min in the absence (-) or presence (ϩ) of ATP (5 mM) and vanadate (1 mM), alone or in combination, and then incubated with [3 H]LTC4 (200 nM, 110 nCi) for a further 30 min followed by UV cross-linking, SDS-PAGE, and fluorography. Login to comment 199 ABCC1 p.Arg1202Asp ABCC1 p.Glu1204Lys ing R1202D and E1204K mutants that is key to MRP1 protein destabilization. Login to comment 200 ABCC1 p.Arg1202Asp ABCC1 p.Glu1204Lys In this regard, it is worth noting that replacing Arg1202 with an Asp (or Glu1204 with a Lys) results in a potential net gain of two charges in the cytoplasmic half of TM16 as well as placing two same-charge ionizable residues in relatively close proximity to one another which could well perturb the ␣-helical geometry of TM16 and contribute to misfolding or aberrant TM helix-packing. Login to comment 201 ABCC1 p.Arg1202Gly ABCC1 p.Arg1202Leu In addition to being readily expressed, the neutrally substituted mutants of TM16 Arg1202 (R1202G and R1202L) exhibited transport activities that were, in the case of most substrates, similar to those of wild-type MRP1. Login to comment 202 ABCC1 p.Arg1202Gly Our findings are consistent with a recent report that a neutrally substituted Arg1202 mutant (R1202G) could still transport LTC4 (28). Login to comment 205 ABCC1 p.Glu1204Leu Unlike the neutrally substituted Arg1202 mutants, transport of organic anions by the neutrally substituted Glu1204 mutant E1204L was substantially reduced or eliminated with the exception of MTX. Login to comment 206 ABCC1 p.Glu1204Asp ABCC1 p.Glu1204Leu Nevertheless, the substrate (LTC4)-binding site of E1204L remained intact. Furthermore, GSH transport remained very low, although other MRP1 transport activities of the same-charge E1204D mutant were comparable with wild-type MRP1. Login to comment 211 ABCC1 p.Glu1204Leu This was shown to be the case when the nucleotide interactions of the transport-compromised E1204L mutant were examined. Login to comment 223 ABCC1 p.Glu1204Leu Thus, inactivation of NBD2 abolishes transport by MRP1, but inactivation of NBD1 results in only a partial loss of activity. Our demonstration that vanadate-induced trapping of azido-ADP by the mutant E1204L protein (and specifically by NBD2) was substantially increased suggests that the mutation may impair the ability of NBD2 to release ADP after hydrolysis of ATP, which could in turn impair substrate translocation and/or release. Login to comment 224 ABCC1 p.Glu1204Leu Alternatively, the E1204L mutant may hydrolyze ATP and release ADP very rapidly in the absence of vanadate but be unable to proceed through a second catalytic cycle (36). Login to comment 228 ABCC1 p.Glu1204Leu Thus, the altered catalytic activity and impaired transport of the E1204L mutant suggests that Glu1204 (or at least the region in which it resides) could play a role in the signaling between the substrate translocation pathway and NBD2. Login to comment 229 ABCC1 p.Arg1249Ala ABCC1 p.Arg1249Asp Unlike Arg1202 and Glu1204 , substitution of Arg1197 and Arg1249 with oppositely charged residues did not adversely affect expression of MRP1 but instead caused a substantial reduction in transport activity. Our observations with respect to the R1249D mutant are consistent with those of Ren et al. (19) who reported that Ala substitution of Arg1249 impaired MRP1-mediated LTC4 transport and reduced vincristine resistance. Login to comment 232 ABCC1 p.Arg1249Lys ABCC1 p.Arg1197Lys The lack of LTC4 labeling of the R1197K and R1249K mutants indicates that their binding site for LTC4 (and likely other organic anions) has been disrupted. Login to comment 234 ABCC1 p.Arg1249Lys ABCC1 p.Arg1197Lys The bulkier, less ionizable Lys side chains in the R1197K and R1249K mutants presumably either cannot form or are prevented from forming the interhelical and/or intrahelical interactions that are established by Arg in wild-type MRP1 for proper folding into a functional transporter. Login to comment