ABCMdb

ABCC1 p.Glu1204Leu

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PMID: 15208328 [PubMed] Situ D et al: "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."

No. Sentence Comment

5 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 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
118 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 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 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
130 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 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 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
137 As shown in Fig. 4I, both E1204L and E1204D exhibited a similar and substantial decrease in GSH transport levels (Ͼ75%). Login to comment
151 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
153 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 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
159 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 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 A similar decrease in [3 H]LTC4 labeling of the E1204L mutant was observed under the same conditions. Login to comment
172 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
192 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 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
205 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 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 This was shown to be the case when the nucleotide interactions of the transport-compromised E1204L mutant were examined. Login to comment
223 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 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 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

PMID: 16415113 [PubMed] Zhang DW et al: "Mutational analysis of polar amino acid residues within predicted transmembrane helices 10 and 16 of multidrug resistance protein 1 (ABCC1): effect on substrate specificity."

No. Sentence Comment

164 Conversely, replacement of Glu1204 with Leu or Arg1197 with Glu or Lys affected either substrate specificity or overall transport activity of MRP1 (Situ et al., 2004). Login to comment

PMID: 16816140 [PubMed] Deeley RG et al: "Transmembrane transport of endo- and xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins."

No. Sentence Comment

834 The distinct phenotypes associated with mutations of the highly conserved Arg1202 and Glu1204 are presumably caused by perturbations in the ␣-helical geometry of TM16 that contribute (depending on the substituting amino acid) to misfolding of MRP1 and, in the case of the neutral Glu1204 Leu mutant, disruption of the signaling between the TMs that comprise the substrate translocation pathway through the membrane and NBD2. Login to comment
833 The distinct phenotypes associated with mutations of the highly conserved Arg1202 and Glu1204 are presumably caused by perturbations in the ␣-helical geometry of TM16 that contribute (depending on the substituting amino acid) to misfolding of MRP1 and, in the case of the neutral Glu1204 Leu mutant, disruption of the signaling between the TMs that comprise the substrate translocation pathway through the membrane and NBD2. Login to comment
835 The distinct phenotypes associated with mutations of the highly conserved Arg1202 and Glu1204 are presumably caused by perturbations in the ␣-helical geometry of TM16 that contribute (depending on the substituting amino acid) to misfolding of MRP1 and, in the case of the neutral Glu1204 Leu mutant, disruption of the signaling between the TMs that comprise the substrate translocation pathway through the membrane and NBD2. Login to comment

PMID: 16959783 [PubMed] Matsumura Y et al: "Characterization and classification of ATP-binding cassette transporter ABCA3 mutants in fatal surfactant deficiency."

No. Sentence Comment

251 For example, the E1204L mutation in TM-16 of MRP1(multidrug resistance associated protein 1) affects vanadate-induced nucleotide trapping as well as transport activity of the protein (41). Login to comment

PMID: 16815813 [PubMed] Choudhuri S et al: "Structure, function, expression, genomic organization, and single nucleotide polymorphisms of human ABCB1 (MDR1), ABCC (MRP), and ABCG2 (BCRP) efflux transporters."

No. Sentence Comment

410 Organic anion transport by Glu1204Leu substitution was substantially reduced although substrate binding by the transport- compromised Glu1204Leu mutant remained intact. Login to comment

PMID: 24157718 [PubMed] Abel S et al: "Bindings of hMRP1 transmembrane peptides with dodecylphosphocholine and dodecyl-beta-d-maltoside micelles: a molecular dynamics simulation study."

No. Sentence Comment

39 Indeed, mutations of the ionizable residues (for example, R1197E, R1202(G,L) and E1204L) have impact on protein expression, substrate binding and/or transport [37], whereas the mutation of a single tryptophan W1246A in TM17 affects the estradiol 17-b2;-D-glucuronide transport [36]. Login to comment