ABCMdb

ABCC1 p.Asp1084Arg

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Publications

PMID: 12954620 [PubMed] Zhang DW et al: "Functional importance of polar and charged amino acid residues in transmembrane helix 14 of multidrug resistance protein 1 (MRP1/ABCC1): identification of an aspartate residue critical for conversion from a high to low affinity substrate binding state."

No. Sentence Comment

54 Mutation D1084R was generated using the QuikChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA). Login to comment
58 It is as follows: D1084R (5Ј-CTG GAC ACA GTG CGG TCC ATG ATC CCG-3Ј). Login to comment
218 Effects of Mutations D1084A, D1084E, D1084V, D1084R, K1092A, K1092E, K1092R, and N1100S on Transport of [3 H]LTC4 and [3 H]E217betaG by Wild Type MRP1-Because mutation D1084N dramatically affected all of the MRP1 functions tested, we also mutated Asp1084 to Ala, Glu, Arg, and Val. Login to comment
242 We also examined the ability of the mutants D1084E and D1084R to bind [3 H]LTC4 by photolabeling studies. Login to comment
243 As shown in Fig. 8E, the relative normalized densitometry values of photolabeling of D1084E and D1084R with [3 H]LTC4 were 120 and 100, respectively. Login to comment
244 Thus, similar to the results obtained with photolabeling of mutant D1084N with LTC4, substitution of Asp1084 by Arg or Glu had no significant effect on the photolabeling. Login to comment

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

84 The TM14-associated D1084R mutant described previously (27) was included for comparison. Login to comment
87 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 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 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 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 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
101 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 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 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 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
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
185 Similarly, D1084E and D1084R can still be photolabeled with LTC4 as well as wild-type MRP1. Login to comment