ABCMdb

ABCC1 p.Arg1249Lys

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Publications

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

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
146 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 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 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 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 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
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
155 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 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
177 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
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
232 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 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

PMID: 17295059 [PubMed] Chang XB et al: "A molecular understanding of ATP-dependent solute transport by multidrug resistance-associated protein MRP1."

No. Sentence Comment

117 Many mutations in TM17, such as Y1236F, T1241A, T1242A, T1242C, T1242S, T1242L, Y1243F, N1245A, W1246C, W1246A, W1246F, W1246Y, or R1249K, significantly affect MRP1 function [83-86]. Login to comment

PMID: 19949927 [PubMed] Chang XB et al: "Molecular mechanism of ATP-dependent solute transport by multidrug resistance-associated protein 1."

No. Sentence Comment

104 Mutations of C43S in TM1 (112); P343A, K332L and K332D in TM6 (113, 114); W445A and P448A in TM8 (113, 115); T550A, T556A and P557A in TM10 (113, 116); N590A, F594A, P595A, N597A, S604A and S605A in TM11 (113, 117, 118); E1089Q, E1089A, E1089L, E1089N, K1092, S1097 and N1100 in TM14 (119, 120); R1197K in TM16 (121); Y1236F, T1241A, T1242A, T1242C, T1242S, T1242L, Y1243F, N1245A, W1246C, W1246A, W1246F, W1246Y or R1249K in TM17 (121-124) significantly affect MRP1 function. Login to comment