ABCMdb

ABCC1 p.Asp1084Asn

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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

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
81 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 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
137 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 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
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
143 Similarly, mutation D1084N also affected the drug resistance profile of MRP1. Login to comment
144 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
147 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 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
166 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
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
179 However, replacement of Asp1084 with Asn decreased the Km value ϳ3-fold (0.5 ␮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
183 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 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 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 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 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 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 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 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 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
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
249 Substitution of Asp1084 by Ala and Val, as observed with mutation D1084N, significantly reduced resistance to vincristine, VP-16, and doxorubicin. Login to comment
322 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 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 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 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 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 These findings indicate that the mutation D1084N decreased the ability of NBD2 to hydrolyze ATP. 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

98 sitely charged amino acid caused a global and almost complete loss of transport activity. We reported previously (27) that substitution of Asp1084 with Asn, Ala, or Val also eliminated LTC4 and E217betaG transport by MRP1. Login to comment

PMID: 15260484 [PubMed] Zhang DW et al: "Transmembrane helix 11 of multidrug resistance protein 1 (MRP1/ABCC1): identification of polar amino acids important for substrate specificity and binding of ATP at nucleotide binding domain 1."

No. Sentence Comment

312 The D1084N mutation essentially eliminated the transport of LTC4 and prevented the transition of the protein from a high- to low-affinity substrate binding state in the presence of ATP (33). Login to comment