ABCMdb

PMID: 12954620

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 [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC1 p.Asn1100Ala ABCC1 p.Ser1097Ala 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 ABCC1 p.Asn1100Ala ABCC1 p.Ser1097Ala ABCC1 p.Thr1082Ala ABCC1 p.Ser1085Ala ABCC1 p.Asp1084Val ABCC1 p.Lys1092Arg ABCC1 p.Lys1092Met ABCC1 p.Asp1084Asn ABCC1 p.Lys1092Glu ABCC1 p.Asp1084Ala ABCC1 p.Lys1092Ala ABCC1 p.Asp1084Glu ABCC1 p.Asn1100Ser 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 54 ABCC1 p.Asp1084Arg Mutation D1084R was generated using the QuikChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA). Login to comment 58 ABCC1 p.Asp1084Arg It is as follows: D1084R (5Ј-CTG GAC ACA GTG CGG TCC ATG ATC CCG-3Ј). Login to comment 81 ABCC1 p.Asp1084Asn To generate MRP1D1084N -pFASTBAC Dual vector, pCEBV7-MRP1 containing mutation D1084N was digested with BstEII, and an ϳ600-bp fragment comprising nucleotides 3369-3913 of MRP1D1084N was isolated. Login to comment 118 ABCC1 p.Asn1100Ala ABCC1 p.Lys1092Met ABCC1 p.Asp1084Asn 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 ABCC1 p.Asn1100Ala ABCC1 p.Ser1097Ala ABCC1 p.Thr1082Ala ABCC1 p.Ser1085Ala ABCC1 p.Lys1092Met 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 137 ABCC1 p.Asp1084Asn The only mutation that affected LTC4 transport was replacement of Asp1084 by Asn, which almost completely eliminated transport (Fig. 3, A-C). Login to comment 138 ABCC1 p.Ser1097Ala ABCC1 p.Thr1082Ala ABCC1 p.Ser1085Ala ABCC1 p.Lys1092Met ABCC1 p.Asp1084Asn ATP-dependent transport of [3 H]E217betaG was unaffected by the T1082A, S1085A and K1092M mutations (Fig. 3, D-F), but substitution of Ser1097 with Ala and conversion of Asp1084 to Asn both dramatically decreased transport. Login to comment 139 ABCC1 p.Asn1100Ala Replacement of Asn1100 with Ala also decreased transport efficiency by 30-40%. Login to comment 140 ABCC1 p.Asn1100Ala ABCC1 p.Ser1097Ala ABCC1 p.Asp1084Asn 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 ABCC1 p.Asn1100Ala ABCC1 p.Thr1082Ala ABCC1 p.Ser1085Ala 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 143 ABCC1 p.Asp1084Asn Similarly, mutation D1084N also affected the drug resistance profile of MRP1. Login to comment 144 ABCC1 p.Asp1084Asn The D1084N mutant protein essentially lost its ability to confer resistance to vincristine and doxorubicin, and retained only ϳ30% of the ability of the wild type protein to increase VP-16 resistance. Login to comment 145 ABCC1 p.Lys1092Met In contrast, although substitution of Lys1092 with Met had no detectable effect on resistance to the electroneutral drug VP-16, the mutation increased resistance to the cationic drugs, vincristine and doxorubicin. Login to comment 147 ABCC1 p.Ser1097Ala ABCC1 p.Lys1092Met ABCC1 p.Asp1084Asn Transport of [3 H]GSH by Wild Type and Mutant MRP1-The studies described above indicated that mutations S1097A and D1084N affected the ability of MRP1 to confer drug resistance and to transport conjugated organic anions, whereas the K1092M mutation influenced only the drug resistance profile of MRP1. Login to comment 163 ABCC1 p.Asp1084Asn As observed when examining LTC4 transport, only replacement of Asp1084 by Asn influenced the ability of MRP1 to transport GSH, and this mutation essentially eliminated transport activity. Login to comment 164 ABCC1 p.Ser1097Ala ABCC1 p.Lys1092Met Other mutations, including mutations S1097A and K1092M that altered the drug resistance profile of MRP1, had no effect. Login to comment 165 ABCC1 p.Ser1097Ala ABCC1 p.Lys1092Met These findings suggest that the effects of mutations S1097A and K1092M on MRP1-mediated drug resistance appear to result primarily from changes in the ability to interact with the drug substrate rather than GSH. Login to comment 166 ABCC1 p.Asp1084Asn Kinetic Parameters of [3 H]LTC4 and [3 H]E217betaG Transport by Wild Type and Mutant MRP1-Because mutation D1084N FIG. 3. Login to comment 176 ABCC1 p.Asn1100Ala ABCC1 p.Ser1097Ala 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 ABCC1 p.Asn1100Ala ABCC1 p.Asn1100Ala ABCC1 p.Ser1097Ala ABCC1 p.Ser1097Ala ABCC1 p.Asp1084Asn ABCC1 p.Asp1084Asn 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 ABCC1 p.Asn1100Ala 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 179 ABCC1 p.Asp1084Asn However, replacement of Asp1084 with Asn decreased the Km value ϳ3-fold (0.5 ␮M). Login to comment 180 ABCC1 p.Ser1097Ala On the other hand, mutation S1097A significantly increased the Km value (28.4 ␮M) (Fig. 5A and Table II). Login to comment 181 ABCC1 p.Asn1100Ala ABCC1 p.Ser1097Ala ABCC1 p.Asp1084Asn 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 ABCC1 p.Asn1100Ala ABCC1 p.Asn1100Ala ABCC1 p.Asn1100Ala ABCC1 p.Ser1097Ala ABCC1 p.Ser1097Ala ABCC1 p.Ser1097Ala 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 183 ABCC1 p.Asp1084Asn ABCC1 p.Asp1084Asn However, replacement of Asp1084 with Asn decreased the values of normalized Vmax and the apparent Km for LTC4 transport ϳ8- and 2-fold, respectively (30 pmol/mg/min, and 77 nM for mutation D1084N) (Fig. 5B and Table II). Login to comment 184 ABCC1 p.Asp1084Asn Thus, similar to the results obtained with E217betaG as a substrate, the D1084N mutation appeared to increase the affinity of MRP1 for substrate while decreasing the Vmax. Login to comment 201 ABCC1 p.Asp1084Asn ABCC1 p.Asp1084Asn Effect of Mutation D1084N on Photolabeling of MRP1 with [3 H]LTC4 and 8-Azido-[␣-32 P]ATP-Because transport studies indicated that mutation D1084N markedly decreased the Vmax for LTC4 transport while modestly decreasing the apparent Km, we confirmed the ability of the mutant protein to bind [3 H]LTC4 by photolabeling studies. Login to comment 202 ABCC1 p.Asp1084Asn Based on densitometry of several preparations of membrane proteins, substitution of Asp1084 with Asn had no significant effect on photolabeling with [3 H]LTC4 (Fig. 6B). Login to comment 206 ABCC1 p.Asp1084Asn ABCC1 p.Asp1084Asn ABCC1 p.Asp1084Asn To investigate how mutation D1084N abrogated the effect of ATP on the binding of LTC4, we examined the ability of the wild type and mutant proteins to be photolabeled with 8-azido-[␣-32 P]ATP both at 4 °C to minimize hydrolysis and under vanadate-induced ADP trapping conditions at 37 °C. As shown in Fig. 6C, mutation D1084N had no marked effect on the binding of the ATP analog at 4 °C. However, the D1084N mutation virtually abolished the vanadate-dependent trapping of 8-azido-[␣-32 P]ADP by MRP1 at 37 °C (Fig. 6D), suggesting that it substantially decreased ATP hydrolysis by MRP1. Login to comment 208 ABCC1 p.Asp1084Asn ABCC1 p.Asp1084Asn To investigate the effect of mutation D1084N on the trapping of ATP more precisely, we took advantage of a pFASTBAC Dual vector, in which the COOH-proximal MRP1 fragment (amino acids 932-1531) was modified to contain mutation D1084N, and co-expressed with a wild type NH2-proximal fragment (amino acids 1-932). Login to comment 212 ABCC1 p.Asp1084Asn As observed with results obtained from analyses of membrane vesicles prepared from HEK293 cells stably transfected with full-length mutant MRP1D1084N , replacement of Asp1084 by Asn essentially eliminated the transport activity. Login to comment 217 ABCC1 p.Asp1084Asn Vanadate-induced trapping experiments revealed that replacement of Asp1084 by Asn dramatically reduced trapping of 8-azido-ADP by both NBD1 and NBD2 of MRP1 (Fig. 7E), strongly suggesting that it decreased the ability of both NBDs to hydrolyze ATP. Login to comment 218 ABCC1 p.Asp1084Val ABCC1 p.Asp1084Val ABCC1 p.Lys1092Arg ABCC1 p.Asp1084Asn ABCC1 p.Lys1092Glu ABCC1 p.Asp1084Ala ABCC1 p.Asp1084Ala ABCC1 p.Lys1092Ala ABCC1 p.Asp1084Glu ABCC1 p.Asp1084Glu ABCC1 p.Asn1100Ser ABCC1 p.Asp1084Arg ABCC1 p.Asp1084Arg 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 229 ABCC1 p.Lys1092Met ABCC1 p.Lys1092Ala Mutational and Functional Analysis of Mutant Human MRP146058 addition, because of the effect of the K1092M mutation on the drug resistance profile of MRP1, Lys1092 was converted to Ala, Glu, and Arg. Login to comment 230 ABCC1 p.Asn1100Ala Replacement of Asn1100 with Ala selectively decreased the transport of E217betaG. Login to comment 235 ABCC1 p.Asn1100Ser Consequently, we also mutated Asn1100 to Ser, as it is in the rodent proteins. Login to comment 239 ABCC1 p.Lys1092Arg ABCC1 p.Lys1092Glu ABCC1 p.Lys1092Ala Mutations K1092A, K1092R, and K1092E had no effect on transport of either LTC4 or E217betaG. Login to comment 240 ABCC1 p.Asn1100Ala ABCC1 p.Asn1100Ser Mutation N1100S, like N1100A, decreased only E217betaG transport. Login to comment 241 ABCC1 p.Asp1084Ala In contrast, replacement of Asp1084 with Ala, Arg, and Val dramatically decreased the ability of MRP1 to transport both LTC4 and E217betaG, whereas conversion to Glu also resulted in an approximate 50% decrease in transport of both substrates. Login to comment 242 ABCC1 p.Asp1084Glu ABCC1 p.Asp1084Arg We also examined the ability of the mutants D1084E and D1084R to bind [3 H]LTC4 by photolabeling studies. Login to comment 243 ABCC1 p.Asp1084Glu ABCC1 p.Asp1084Arg 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 ABCC1 p.Asp1084Asn ABCC1 p.Asp1084Glu ABCC1 p.Asp1084Arg 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 247 ABCC1 p.Asn1100Ser Mutation N1100S had no effect on the drug profile of MRP1. Login to comment 248 ABCC1 p.Lys1092Arg ABCC1 p.Lys1092Met ABCC1 p.Lys1092Glu ABCC1 p.Lys1092Ala The mutation K1092R also showed the same phenotype as wild type MRP1, whereas mutations K1092A and K1092E, like K1092M, increased the ability of MRP1 to confer resistance to vincristine and doxorubicin but not to VP-16. Login to comment 249 ABCC1 p.Asp1084Val ABCC1 p.Asp1084Asn ABCC1 p.Asp1084Ala Substitution of Asp1084 by Ala and Val, as observed with mutation D1084N, significantly reduced resistance to vincristine, VP-16, and doxorubicin. Login to comment 273 ABCC1 p.Asp1084Glu In contrast, conservative substitution of Asp1084 with Glu resulted in a mutant protein which retained ϳ50% of the ability of the wild type protein to transport LTC4 and E217betaG, and to confer vincristine and doxorubicin resistance, while having no effect on VP-16 resistance. Login to comment 278 ABCC1 p.Asn1100Ala ABCC1 p.Ser1097Ala ABCC1 p.Asn1100Ser On the other hand, mutations S1097A, N1100A, and N1100S reduced E217betaG but not LTC4 or GSH transport activity. Login to comment 279 ABCC1 p.Ser1097Ala Mutation S1097A also reduced resistance to all three classes of drugs tested. Login to comment 283 ABCC1 p.Ser1097Ala Consistent with this possibility, mutation S1097A significantly increased the Km value for E217betaG transport without affecting transport of LTC4, suggesting that the hydrogen bonding capability of Ser1097 may be involved specifically in the interac- FIG. 8. Login to comment 292 ABCC1 p.Ser1097Ala Substitution of Ser1097 with Ala also dramatically decreased resistance to all three drugs tested. Login to comment 294 ABCC1 p.Ser1097Ala However, in the present study, we did not observed any effect of mutation S1097A on verapamil-stimulated GSH transport by MRP1. Login to comment 296 ABCC1 p.Asn1100Ala ABCC1 p.Ser1097Ala 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 322 ABCC1 p.Asp1084Asn ABCC1 p.Asp1084Glu Photolabeling studies with [3 H]LTC4 also showed that the replacement of Asp1084 by Asn and Glu had no effect on binding of the substrate to the protein. Login to comment 326 ABCC1 p.Asp1084Asn In the present study, kinetic analyses of the residual transport activity of the D1084N mutant protein yielded a lower Km for both LTC4 and E217betaG than the wild type protein, suggesting that the affinity for both substrates was actually increased. Login to comment 330 ABCC1 p.Asp1084Asn Consistent with this suggestion, we found that LTC4 binding by the D1084N mutation did not decrease in the presence of ATP even with the addition of vanadate. Login to comment 331 ABCC1 p.Asp1084Asn This finding together with the effect of mutation D1084N on the overall activity of MRP1 raised the interesting possibility that the mutation might affect the binding and/or hydrolysis of ATP, so that substrate could not be translocated and/or released from the mutant protein. Login to comment 333 ABCC1 p.Asp1084Asn At 37 °C, the D1084N mutation drastically decreased trapping of 8-azido-ADP at NBD2 when photolabeling was carried out in the absence or presence of vanadate. Login to comment 334 ABCC1 p.Asp1084Asn These findings indicate that the mutation D1084N decreased the ability of NBD2 to hydrolyze ATP. Login to comment