ABCMdb

ABCC1 p.Glu1144Ala

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Publications

PMID: 19015228 [PubMed] Conseil G et al: "Multiple roles of charged amino acids in cytoplasmic loop 7 for expression and function of the multidrug and organic anion transporter MRP1 (ABCC1)."

No. Sentence Comment

6 The properties of R1173A and E1184A were comparable with those of wild-type MRP1, whereas the remaining mutants were either poorly expressed (R1166A, D1183A) or exhibited reduced transport of one or more organic anions (E1144A, D1179A, K1181A, 1169 AAQA). Login to comment
8 The moderate substrate-selective changes in transport activity displayed by mutants E1144A, D1179A, K1181A, and 1169 AAQA were accompanied by changes in orthovanadate-induced trapping of [␣-32 P]azidoADP by NBS2 indicating changes in ATP hydrolysis or release of ADP. Login to comment
9 In the case of E1144A, estradiol glucuronide no longer inhibited trapping of azidoADP. Login to comment
59 Mutations were first generated in the pGEM-3Z-XmaI/MRP1 plasmid according to the manufacturer`s instructions with the following mutagenic primers (Integrated DNA Technologies, Inc., Coralville, IA), which also introduced an additional restriction site (substituted nucleotides are underlined): E1144A (5Ј-G AAG CGC CTC GCG TCG GTC AGC-3Ј); R1166A (5Ј C AGC GTC ATT GCA GCA TTC GAG GAG CAG-3Ј); R1166K (5Ј C AGC GTC ATT AAG GCC TTC GAG G-3Ј); 1169 EEQE-1169 AAQA (5Ј-C ATT CGA GCC TTC GCG GCA CAG GCA CGC TTC ATC C-3Ј); R1173A (5Ј-C GAG GAG CAG GAG GCA TTC ATC CAC CAG AG-3Ј); D1179A (5Ј-C CAC CAG AGT GCC CTT AAG GTG GAC G-3Ј), K1181A (5Ј-G AGT GAC CTG GCA GTC GAC GAG AAC C-3Ј); D1183A (5Ј-CTG AAG GTG GCC GAG AAC CAG-3Ј); D1183E (5Ј- CTG AAG GTG GAA GAG AAC CAG-3Ј); and E1184A (5Ј-GT GAC CTG AAG GTA GAC GCG AAC CAG AAG GCC-3Ј). Login to comment
154 Thus, E217betaG, E13SO4, and MTX transport by E1144A was moderately reduced (by ϳ50%), whereas uptake of GSH and LTC4 was comparable with that of wild-type MRP1. Login to comment
160 Because the single E1144A and K1181A mutants and the 1169 AAQA triple mutant exhibited the most substantial changes in transport activity, their ATP binding and hydrolysis properties were examined. Login to comment
165 In contrast, increased dinucleotide trapping (1.5to 1.7-fold) was observed for the E1144A and 1169 AAQA mutants (after corrections were made to take into account differences in MRP1 expression) (Fig. 5B). Login to comment
167 To determine whether the increased photolabeling under trapping conditions was due to a change in the affinity for azidoADP, additional trapping experiments were performed on the E1144A and 1169 AAQA mutants. Login to comment
172 Effect of E217betaG on 8-Azido-[␣-32 P]ATP Photolabeling and Vanadate-Induced 8-Azido-[␣-32 P]ADP Trapping by E1144A Mutant MRP1. Login to comment
173 Because E217betaG transport by the E1144A mutant was substantially reduced, the effect of this organic anion substrate on the interactions of the mutant protein with ATP was examined. Login to comment
174 As shown in Fig. 6A, neither 25 nor 50 ␮M E217betaG caused any detectable change in 8-azido-[␣-32 P]ATP photolabeling of either the E1144A mutant or wild-type MRP1 at 4°C. Login to comment
176 In contrast, E217betaG had no effect on vanadate-induced trapping of azidoADP by the E1144A mutant (Fig. 6B). Login to comment
185 Shown is an immunoblot of membrane vesicles (1 ␮g of protein) prepared from HEK 293T cells transfected with cDNA expression vectors encoding wild-type, E1144A, 1169 AAQA, R1173A, D1179A, K1181A, and E1184A MRP1 mutants. Login to comment
192 The remaining five single mutants were either poorly expressed (R1166A, D1183A) (Fig. 2) or exhibited significantly reduced transport levels of one or more organic anions (E1144A, D1179A, K1181A) (Table 1). Login to comment
212 Percentage Wild-Type MRP1 Uptake Activity E1144A 1169 AAQA R1173A D1179A K1181A E1184A % LTC4 90 Ϯ 5 70 Ϯ 5 120 Ϯ 5 65 Ϯ 5 55 Ϯ 5 85 Ϯ 5 E217bG 55 Ϯ 5 80 Ϯ 0 130 Ϯ 10 90 Ϯ 5 65 Ϯ 5 95 Ϯ 15 E13SO4 45 Ϯ 5 45 Ϯ 5 125 Ϯ 0 65 Ϯ 0 50 Ϯ 5 90 Ϯ 10 MTX 55 Ϯ 5 105 Ϯ 5 N.D. N.D. 45 Ϯ 5 N.D. GSH 80 Ϯ 5 95 Ϯ 10 N.D. N.D. 30 Ϯ 5 N.D. N.D., not determined. Login to comment
219 8-Azido-[32 P]ATP photolabeling and vanadate-dependent 8-azido- [32 P]ADP trapping of CL7 mutants E1144A, 1169 AAQA and K1181A of MRP1. Login to comment
230 Effect of E217betaG on 8-azido-[32 P]ATP photolabeling and vanadate-dependent azido-[32 P]ADP trapping by MRP1 mutant E1144A. Login to comment
232 The relative levels of labeling were estimated by densitometric analysis of the films, and for wild-type and E1144A mutant MRP1 are expressed relative to labeling in the absence of substrate. Login to comment
233 In the lower part of B, the relative levels of vanadate-induced azidoADP trapping by wild-type (F) and E1144A (E) MRP1 have also been plotted against concentration of E217betaG. Login to comment
244 Unlike the R1166A and D1183A mutants, the second group of functionally altered single Ala-substituted mutants (E1144A, D1179A, K1181A) and the 1169 AAQA triple mutant were all expressed at levels comparable with or greater than that of wild-type MRP1 (Fig. 4). Login to comment
255 The altered transport activity and nucleotide interactions of the E1144A, K1181A, and 1169 AAQA mutants may thus result from perturbation of this signaling (Table 1, Fig. 5). Login to comment
256 The observation that E217betaG failed to inhibit vanadate-induced trapping of azidoADP by the E1144A mutant, as it does with wild-type MRP1 (Fig. 6), suggests that the signaling that occurs after E217betaG interacts with its binding site in the MSDs of E1144A has become at least partially uncoupled from the hydrolysis of ATP (or release of ADP) at NBD2/ NBS2. Login to comment

PMID: 21143116 [PubMed] He SM et al: "Structural and functional properties of human multidrug resistance protein 1 (MRP1/ABCC1)."

No. Sentence Comment

726 These include Cys43 in TM1 [171], Thr73 in CL1 [366], Trp222 in L0 [279], Trp223 in L0 [279], Arg230 in L0 [279], Trp261 in L0 [271, 302, 338], Lys267 in L0 [271, 302, 338], Lys319 in TM6 [339], Tyr324 in TM6 [301], Lys332 in TM6 [166, 339-341], His335 in TM6, Asp336 in TM6 [339], Lys347 in TM6 [339], Lys396 in TM7 [339], Arg433 in TM8 [363], Asp436 in TM8 [339], Trp445 in TM8, Trp459 in TM8, Pro478 in TM9, Thr550 in TM10 [343], Trp553 in TM10 [344], Thr556 in TM10 [343], Pro557 in TM10 [345], Tyr568 in TM10 [343], Arg593 in TM11 [339], Phe594 in TM11 [300], Asn597 in TM11, Ser604 in TM11, Ser605 in TM11, Trp653 in NBD1 [351], Lys684 in Walker A motif of NBD1 [350, 364], Ser685 in Walker A motif of NBD1 [353], Arg723 in NBD1 [366], Gly771 in the ABC signature (C motif) of NBD1 [364], Asp792 in Walker B motif of NBD1 [361], Asp793 in Walker B motif of NBD1 [353], Ala989 in TM12 [367], Pro1120 in TM13, Pro1121 in TM13, Arg1058 at TM13/CL6 interface [366], Glu1089 in TM14, Lys1092 in TM14, Ser1097 in TM14, Asn1100 in TM14, Arg1138 in TM15 [354], Lys1141 in CL7 [354], Arg1142 in CL7 [354], Glu1144Ala in CL7 [355], Pro1150 in CL7 [345, 347, 348], Arg1166Ala in CL7 [355], Asp1179Ala in CL7 [355], Lys1181Ala in CL7 [355], Asp1183 in CL7 [355], Tyr1189 in CL7 [368], Tyr1190 in CL7 [368], Arg1197 in TM16 [357], Trp1198 in TM16 [344], Arg1202 in TM16 [357], Glu1204 in TM16 [357], Tyr1236 in TM17, Thr1241 in TM17, Thr1242 in TM17, Tyr1243 in TM17, Asn1245 in TM17, Trp1246 in TM17 [166, 339-341], Arg1249 in TM17 [342], Tyr1302 in NBD2 [351], Lys1333 in Walker A motif of NBD2 [350], Gly1433 in the ABC signature motif of NBD2 [352, 364], Glu1455 in Walker B motif of NBD2 [322], and His1486 in NBD2 [349]. Login to comment
813 The single mutants Glu1144Ala, Asp1179Ala, and Lys1181Ala) and the triple mutant 1169AAQA showed only moderate and substrate-selective changes in transport activity [355]. Login to comment