ABCMdb

ABCC1 p.Asn1100Ala

Predicted by SNAP2:	A: D (53%), C: D (75%), D: D (63%), E: D (63%), F: D (91%), G: D (53%), H: D (71%), I: D (75%), K: D (63%), L: D (75%), M: D (66%), P: D (75%), Q: N (61%), R: D (71%), S: N (72%), T: N (61%), V: D (71%), W: D (85%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: N, L: D, M: D, P: D, Q: D, R: D, S: N, T: N, V: D, W: D, Y: D,

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[hide] Functional importance of polar and charged amino a... J Biol Chem. 2003 Nov 14;278(46):46052-63. Epub 2003 Sep 3. Zhang DW, Gu HM, Situ D, Haimeur A, Cole SP, Deeley RG
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.
J Biol Chem. 2003 Nov 14;278(46):46052-63. Epub 2003 Sep 3., 2003-11-14 [PMID:12954620]

Abstract [show]
Human multidrug resistance protein 1 (MRP1) confers resistance to many chemotherapeutic agents and transports diverse conjugated organic anions. We previously demonstrated that Glu1089 in transmembrane (TM) 14 is critical for the protein to confer anthracycline resistance. We have now assessed the functional importance of all polar and charged amino acids in this TM helix. Asn1100, Ser1097, and Lys1092, which are all predicted to be on the same face of the helix as to Glu1089, are involved in determining the substrate specificity of the protein. Notably, elimination of the positively charged side chain of Lys1092, increased resistance to the cationic drugs vincristine and doxorubicin, but not the electroneutral drug etoposide (VP-16). In addition, mutations S1097A and N1100A selectively decreased transport of 17beta-estradiol 17-(beta-d-glucuronide) (E217betaG) but not cysteinyl leukotriene 4 (LTC4), demonstrating the importance of multiple residues in this helix in determining substrate specificity. In contrast, mutations of Asp1084 that eliminate the carboxylate side chain markedly decreased resistance to all drugs tested, as well as transport of both E217betaG and LTC4, despite the fact that LTC4 binding was unaffected. We show that these mutations prevent the ATP-dependent transition of the protein from a high to low affinity substrate binding state and drastically diminish ADP trapping at nucleotide binding domain 2. Based on results presented here and crystal structures of prokaryotic ATP binding cassette transporters, Asp1084 may be critical for interaction between the cytoplasmic loop connecting TM13 and TM14 and a region of nucleotide binding domain 2 between the conserved Walker A and ABC signature motifs.

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5 In addition, mutations S1097A and N1100A selectively decreased transport of 17beta-estradiol 17-(beta-D-glucuronide) (E217betaG) but not cysteinyl leukotriene 4 (LTC4), demonstrating the importance of multiple residues in this helix in determining substrate specificity. Login to comment
51 They are as follows: T1082A (5Ј-C TCC AAG GAG CTC GAC GCA GTG GAC TCC-3Ј), D1084N (5Ј-CTG GAC ACA GTG AAT TCC ATG ATC CCG-3Ј), D1084A (5Ј-CTG GAC ACA GTG AAA TCG ATG ATC CCG-3Ј), D1084E (5Ј-CTG GAC ACA GTG GAC TCG ATG ATC CCG-3Ј), D1084V (5Ј-CTG GAC ACA GTG GTA TCG ATG ATC CCG-3Ј), S1085A (5Ј-CTG GAC ACA GTC GAC GCC ATG ATC CCG G-3Ј), K1092M (5Ј-C CCG GAG GTC ATC ATG ATG TTC ATG GGC-3Ј), K1092A (5Ј-C CCG GAG GTC ATC GCG ATG TTC ATG GGC-3Ј), K1092E (5Ј-C CCG GAG GTC ATC GAG ATG TTC ATG GGC-3Ј), K1092R (5Ј-C CCG GAG GTC ATC AGG ATG TTC ATG GGC-3Ј), S1097A (5Ј-G ATG TTC ATG GGC GCC CTG TTC AAC-3Ј), N1100A (5Ј-TTC ATG GGC TCG CTC TTC GCC GTC ATT GGT G-3Ј), N1100S (5Ј-TTC ATG GGC TCG CTC TTC AGT GTC ATT GGT G-3Ј). Login to comment
118 RESULTS Expression of Mutant MRP1 in Stably Transfected HEK293 Cells-To examine the functional importance of charged and polar residues in TM14 of MRP1, we generated a series of six mutant proteins in which Thr1082 , Ser1085 , Ser1097 , and Asn1100 were replaced with Ala, Asp1084 was replaced with Asn, and Lys1092 was mutated to Met (Fig. 1). Login to comment
136 The levels of LTC4 uptake by vesicles prepared from HEK transfectants expressing either wild type MRP1 or mutations T1082A, S1085A, K1092M, S1097A, and N1100A were proportional to the relative expression levels of the wild type and mutant proteins. Login to comment
139 Replacement of Asn1100 with Ala also decreased transport efficiency by 30-40%. Login to comment
140 Thus, mutations S1097A and N1100A only affected transport of the estrogen conjugate, whereas mutation D1084N decreased the ability of MRP1 to transport both LTC4 and E217betaG. Login to comment
142 The results are presented as relative drug resistance factors in Table I. Mutations T1082A, S1085A, and N1100A had no significant effect on drug resistance, but conversion of one polar residue, Ser1097 , to Ala caused a 2-3-fold decrease in resistance to vincristine, VP-16, and doxorubicin. Login to comment
176 The values shown represent the mean (ϮS.D.) of relative resistance values determined from three independent experiments. Resistance factors normalized for differences in the levels of mutant proteins expressed in the transfectant populations used are shown in parentheses. Transfectant Drug (relative resistance factor) Vincristine VP-16 Doxorubicin HEKMRP1 15.9 Ϯ 2.0 (15.9) 15.9 Ϯ 3.1 (15.9) 7.4 Ϯ 1.3 (7.4) HEKMRP1T082A 16.6 Ϯ 2.9 (19.5) 14.3 Ϯ 2.7 (16.8) 7.1 Ϯ 2.8 (8.4) HEKMRP1D1084N 1.4 Ϯ 0.4 (1.4) 4.4 Ϯ 0.6 (4.4) 1.1 Ϯ 0.1 (1.1) HEKMRP1S1085A 13.5 Ϯ 1.5 (17.5) 15.4 Ϯ 2.3 (20.0) 6.2 Ϯ 1.3 (8.1) HEKMRP1K1092M 40.1 Ϯ 8.1 (50.1) 12.5 Ϯ 3.0 (15.6) 15.3 Ϯ 3.2 (19.1) HEKMRP1S1097A 7.9 Ϯ 1.1 (9.9) 3.7 Ϯ 0.9 (4.6) 1.8 Ϯ 0.2 (2.3) HEKMRP1N1100A 14.6 Ϯ 2.1 (19.5) 11.4 Ϯ 4.0 (15.2) 6.7 Ϯ 1.5 (8.9) Mutational and Functional Analysis of Mutant Human MRP146056 affected the ability of the protein to transport both LTC4 and E217betaG whereas mutations S1097A and N1100A selectively decreased the transport of only E217betaG, we compared their effect on the kinetic parameters of transport of both substrates (Fig. 5). Login to comment
177 For E217betaG transport, the normalized Vmax values for mutations D1084N, S1097A, and N1100A were lower than that for wild type MRP1 (3207 pmol/mg/min for wild type MRP1, versus 151, 2279, and 1852 pmol/mg/min for mutations D1084N, S1097A and N1100A, respectively) (Fig. 5A and Table II). Login to comment
178 The apparent Km value for mutation N1100A (1.9 ␮M) was comparable with that of the wild type protein (1.6 ␮M). Login to comment
181 Thus, although mutations D1084N, S1097A, and N1100A all decreased the conjugated estrogen transport, the three mutations had different effects on the apparent Km values for E217betaG uptake. Login to comment
182 For LTC4 uptake, the Km values for wild type MRP1 and mutant MRP1S1097A and MRP1N1100A were essentially identical (146, 141, and 138 nM for wild type MRP1 and mutations S1097A and N1100A, respectively) and the normalized Vmax values for mutations S1097A and N1100A were also very similar with that of wild type MRP1 (223, 208, and 242 pmol/mg/ min for mutations S1097A, N1100A, and wild type MRP1, respectively) (Table II). Login to comment
230 Replacement of Asn1100 with Ala selectively decreased the transport of E217betaG. Login to comment
240 Mutation N1100S, like N1100A, decreased only E217betaG transport. Login to comment
278 On the other hand, mutations S1097A, N1100A, and N1100S reduced E217betaG but not LTC4 or GSH transport activity. Login to comment
296 In addition, in contrast to the effect of mutation S1097A on E217betaG transport, mutation N1100A, which only affected the transport of E217betaG, decreased the Vmax without significantly affecting Km, suggesting that the mutation affects a step in the transport process subsequent to initial binding of this substrate. Login to comment

[hide] A molecular understanding of ATP-dependent solute ... Cancer Metastasis Rev. 2007 Mar;26(1):15-37. Chang XB
A molecular understanding of ATP-dependent solute transport by multidrug resistance-associated protein MRP1.
Cancer Metastasis Rev. 2007 Mar;26(1):15-37., [PMID:17295059]

Abstract [show]
Over a million new cases of cancers are diagnosed each year in the United States and over half of these patients die from these devastating diseases. Thus, cancers cause a major public health problem in the United States and worldwide. Chemotherapy remains the principal mode to treat many metastatic cancers. However, occurrence of cellular multidrug resistance (MDR) prevents efficient killing of cancer cells, leading to chemotherapeutic treatment failure. Numerous mechanisms of MDR exist in cancer cells, such as intrinsic or acquired MDR. Overexpression of ATP-binding cassette (ABC) drug transporters, such as P-glycoprotein (P-gp or ABCB1), breast cancer resistance protein (BCRP or ABCG2) and/or multidrug resistance-associated protein (MRP1 or ABCC1), confers an acquired MDR due to their capabilities of transporting a broad range of chemically diverse anticancer drugs. In addition to their roles in MDR, there is substantial evidence suggesting that these drug transporters have functions in tissue defense. Basically, these drug transporters are expressed in tissues important for absorption, such as in lung and gut, and for metabolism and elimination, such as in liver and kidney. In addition, these drug transporters play an important role in maintaining the barrier function of many tissues including blood-brain barrier, blood-cerebral spinal fluid barrier, blood-testis barrier and the maternal-fetal barrier. Thus, these ATP-dependent drug transporters play an important role in the absorption, disposition and elimination of the structurally diverse array of the endobiotics and xenobiotics. In this review, the molecular mechanism of ATP-dependent solute transport by MRP1 will be addressed.

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No. Sentence Comment
153 In addition, S1097A and N1100A selectively decreased the transport of E217βG but not to LTC4 [82]. Login to comment