ABCMdb

PMID: 11925441

Zhang DW, Cole SP, Deeley RG
Determinants of the substrate specificity of multidrug resistance protein 1: role of amino acid residues with hydrogen bonding potential in predicted transmembrane helix 17.
J Biol Chem. 2002 Jun 7;277(23):20934-41. Epub 2002 Mar 29., 2002-06-07 [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC1 p.Asn1245Ala ABCC1 p.Tyr1243Phe ABCC1 p.Tyr1236Phe To determine whether other residues with hydrogen bonding potential within TM17 influence substrate specificity, we replaced Ser1233 , Ser1235 , Ser1237 , Gln1239 , Thr1241 , and Asn1245 with Ala and Tyr1236 and Tyr1243 with Phe. Login to comment 3 ABCC1 p.Ser1237Ala ABCC1 p.Gln1239Ala ABCC1 p.Ser1235Ala ABCC1 p.Ser1233Ala Mutations S1233A, S1235A, S1237A, and Q1239A had no effect on any substrate tested. Login to comment 4 ABCC1 p.Tyr1236Phe ABCC1 p.Thr1241Ala In contrast, mutations Y1236F and T1241A decreased resistance to vincristine but not to VP-16, doxorubicin, and epirubicin. Login to comment 5 ABCC1 p.Tyr1243Phe Mutation Y1243F reduced resistance to all drugs tested by 2-3-fold. Login to comment 6 ABCC1 p.Asn1245Ala Replacement of Asn1245 with Ala also decreased resistance to VP-16, doxorubicin, and epirubicin but increased resistance to vincristine. Login to comment 8 ABCC1 p.Tyr1243Phe Only mutation Y1243F altered the ability of MRP1 to transport both leukotriene 4 and E217betaG. Login to comment 50 ABCC1 p.Asn1245Ala We have systematically mutated the amino acids Ser1233 , Ser1235 , Ser1237 , Gln1239 , Thr1241 , and Asn1245 to alanine and each of two tyrosines, Tyr1236 and Tyr1243 , to Phe. Login to comment 104 ABCC1 p.Asn1245Ala To examine their possible importance in determining the substrate specificity of MRP1, we generated a series of eight mutant proteins in which Ser1233 , Ser1235 , Ser1237 , Gln1239 , Thr1241 , and Asn1245 were replaced with Ala, and Tyr1236 and Tyr1243 were substituted with Phe (Fig. 1). Login to comment 112 ABCC1 p.Ser1237Ala ABCC1 p.Gln1239Ala ABCC1 p.Ser1235Ala ABCC1 p.Ser1233Ala Substitution of four hydrophilic amino acid residues within TM17 with Ala (S1233A, S1235A, S1237A, Q1239A) had no significant effect on the ability of MRP1 to confer resistance to any drug tested. Login to comment 113 ABCC1 p.Asn1245Ala ABCC1 p.Tyr1243Phe ABCC1 p.Tyr1236Phe ABCC1 p.Thr1241Ala Mutation of one polar-aromatic residue, Y1243F, caused an approximately 2-3-fold reduction of resistance to all four drugs, whereas three mutations, Y1236F, T1241A, and N1245A, resulted in a 2-3-fold loss of resistance to only certain drugs. Login to comment 114 ABCC1 p.Tyr1236Phe ABCC1 p.Thr1241Ala Interestingly, both mutation Y1236F and mutation T1241A decreased vincristine resistance ϳ3-fold without a significant effect on the resistance to VP-16, doxorubicin, and epirubicin. Login to comment 115 ABCC1 p.Asn1245Ala In contrast, conversion of Asn1245 to Ala resulted in a mutant protein with enhanced resistance to vincristine (1.6-fold) but a 2-3-fold partial decrease in the ability to confer resistance to VP-16, doxorubicin, and epirubicin. Login to comment 117 ABCC1 p.Asn1245Ala ABCC1 p.Tyr1243Phe ABCC1 p.Tyr1236Phe ABCC1 p.Thr1241Ala Subcellular Localization of Mutant and Wild Type MRP1 in Transfected HEK293 Cells-To determine whether effects of mutations Y1236F, T1241A, Y1243F, and N1245A on the drug FIG. 1. Login to comment 136 ABCC1 p.Thr1242Ala In contrast, the T1242A mutation had no significant effect on LTC4 transport, and the effect of the Trp1246 mutations was relatively minor (a 2-fold increase in Km) (38, 39).2 To determine whether any of the other mutations in TM17 of MRP1 altered the efficiency with which the protein transported either LTC4 or E217betaG, we examined ATP-dependent uptake of these compounds by membrane vesicles prepared from HEK transfectants expressing each of these mutant proteins (Figs. Login to comment 140 ABCC1 p.Tyr1243Phe The only mutation that affected LTC4 transport was conversion of Tyr1243 to Phe, which decreased transport of LTC4 by ϳ30% (Fig. 4). Login to comment 141 ABCC1 p.Tyr1236Phe ABCC1 p.Ser1237Ala ABCC1 p.Gln1239Ala ABCC1 p.Ser1235Ala ABCC1 p.Ser1233Ala ABCC1 p.Thr1241Ala ATP-dependent transport of [3 H]E217betaG was also examined (Fig. 5), but none of the mutations S1233A, S1235A, Y1236F, S1237A, Q1239A, and T1241A had any effect. Login to comment 142 ABCC1 p.Asn1245Ala ABCC1 p.Tyr1243Phe However, mutations Y1243F and N1245A both decreased the levels of E217betaG transport ϳ5-6-fold (Fig. 5). Login to comment 144 ABCC1 p.Asn1245Ala ABCC1 p.Tyr1243Phe Kinetic Parameters of [3 H]LTC4 and [3 H]E217betaG Transport-We have shown that mutation Y1243F affected the ability of the protein to transport both LTC4 and E217betaG and that mutation N1245A decreased the transport of only E217betaG. Login to comment 158 ABCC1 p.Asn1245Ala ABCC1 p.Asn1245Ala For wild type MRP1 and mutant MRP1N1245A, the Km values for LTC4 uptake were identical (90 nM), and the normalized Vmax value for N1245A was also very similar to that of wild type MRP1 (Vmax ϭ 100 pmol/mg/ min for mutation N1245A; 117 pmol/mg/min for wild type MRP1) (Table II). Login to comment 159 ABCC1 p.Tyr1243Phe ABCC1 p.Tyr1243Phe However, replacement of Tyr1243 with Phe decreased the normalized Vmax value for LTC4 transport ϳ30% relative to wild type MRP1 (Vmax ϭ 75 pmol/mg/min for mutation Y1243F). Login to comment 160 ABCC1 p.Tyr1243Phe The apparent Km value for mutation Y1243F was 112 nM (Fig. 6 and Table II). Login to comment 161 ABCC1 p.Tyr1243Phe Thus, the Y1243F mutation decreased the Vmax/Km ratio for LTC4 ϳ2-fold. Login to comment 162 ABCC1 p.Asn1245Ala ABCC1 p.Tyr1243Phe For E217betaG transport, a nonlinear regression analysis of the data generated a Km value of 1.4 ␮M for wild type MRP1, consistent with previous estimates (10), compared with 5.4 and 10.9 ␮M for mutations Y1243F and N1245A, respectively (Fig. 6 and Table II). Login to comment 163 ABCC1 p.Asn1245Ala ABCC1 p.Asn1245Ala ABCC1 p.Tyr1243Phe ABCC1 p.Tyr1243Phe The normalized Vmax values for mutations Y1243F and N1245A were lower than that for wild type MRP1 (Vmax ϭ 403 pmol/mg/min for wild type MRP1 versus 316 pmol/ mg/min for mutation Y1243F and 288 pmol/mg/min for mutation N1245A) (Fig. 6 and Table II). Login to comment 164 ABCC1 p.Asn1245Ala ABCC1 p.Tyr1243Phe Thus mutations Y1243F and N1245A decreased the Vmax/Km ratio for E217betaG ϳ5-and 11-fold, respectively. Login to comment 165 ABCC1 p.Asn1245Ala ABCC1 p.Tyr1243Phe Effect of Mutations Y1243F and N1245A on the Inhibition of MRP1-mediated E217betaG/LTC4 Transport by LTC4/ E217betaG-As an alternative means of assessing the effects of the TM17 mutations on the interaction between LTC4 and the human protein, we examined the ability of LTC4 to inhibit transport of E217betaG. Login to comment 168 ABCC1 p.Asn1245Ala ABCC1 p.Tyr1243Phe Converting Asn1245 to Ala reproducibly decreased the IC50 value (207 nM), whereas mutation of Tyr1243 to Phe resulted in a slight, reproducible increase (407 nM). Login to comment 170 ABCC1 p.Asn1245Ala ABCC1 p.Tyr1243Phe For the wild type protein, the IC50 value for E217betaG was 3.6 ␮M compared with 11.8 ␮M for mutation Y1243F and 15.1 ␮M for mutation N1245A. Login to comment 171 ABCC1 p.Asn1245Ala ABCC1 p.Tyr1243Phe Since these results are independent of protein expression levels, they provide additional evidence that the observed decrease in transport of mutations Y1243F and N1245A at nonsaturating concentrations of E217betaG is primarily attributable to changes in the affinity of the proteins for this substrate. Login to comment 191 ABCC2 p.Trp1254Tyr However, when the corresponding residue, Trp1254 , is mutated in MRP2, only nonconserved substitutions eliminate E217betaG transport, and in contrast to MRP1, only the most conservatively substituted MRP2 Trp1254 mutant, W1254Y, transports LTC4 (45). Login to comment 199 ABCC1 p.Ser1237Ala ABCC1 p.Gln1239Ala ABCC1 p.Ser1235Ala ABCC1 p.Ser1233Ala With the exception of Tyr1236 , mutation of the amino acids with polar substituents predicted to be most distant from the membrane/cytoplasm interface (S1233A, S1235A, S1237A, and Q1239A) to Ala had no effect on the ability of MRP1 to confer resistance to any of the drugs tested. Login to comment 205 ABCC1 p.Gln1239Ala Based on the substrates we have analyzed, mutation of Gln1239 to Ala had no detectable effect on MRP1 function. Login to comment 211 ABCC1 p.Thr1241Ala The similar effect of the mutation, T1241A, on the resistance to vincristine might be also due to the elimination of the hydrogen bonding capability. Login to comment 218 ABCC1 p.Asn1245Ala Substitution of Asn1245 with Ala decreased the resistance to VP-16 and the two anthracyclines tested, consistent with the involvement of hydrogen bonding in the interaction with these drugs. Login to comment 230 ABCC1 p.Asn1245Ala By analogy, conversion of Asn1245 to Ala in MRP1 may increase accessibility to a relatively large substrate, such as vincristine, whereas the loss of hydrogen bonding may contribute to the decrease in resistance to other drugs, such as VP-16, doxorubicin, and epirubicin. Login to comment