ABCMdb

PMID: 12186871

Haimeur A, Deeley RG, Cole SP
Charged amino acids in the sixth transmembrane helix of multidrug resistance protein 1 (MRP1/ABCC1) are critical determinants of transport activity.
J Biol Chem. 2002 Nov 1;277(44):41326-33. Epub 2002 Aug 18., 2002-11-01 [PubMed]

Sentences

No. Mutations Sentence Comment

47 ABCC1 p.His335Glu ABCC1 p.Lys332Asp ABCC1 p.His335Leu ABCC1 p.Lys332Leu ABCC1 p.His335Gln ABCC1 p.Asp336Arg ABCC1 p.Asp336Leu The sequences of the individual sense strands, with the altered codons underlined and the corresponding changes in amino acids indicated in parentheses were as follows: (K332D) 5Ј-CTC ATG AGC TTC TTC TTC GAC GCC ATC CAC GAC CTG-3Ј; (K332L) 5Ј-CTC ATG AGC TTC TTC TTC CTG GCC ATC CAC GAC CTG-3Ј; (H335E) 5Ј-GC TTC TTC TTC AAG GCC ATC GAG GAC CTG ATG ATG-3Ј; (H335L) 5Ј-GC TTC TTC TTC AAG GCC ATC TTG GAC CTG ATG ATG-3Ј; (H335Q) 5Ј-GC TTC TTC TTC AAG GCC ATC CAG GAC CTG ATG ATG-3Ј; (D336R) 5Ј-C AAG GCC ATC CAC CGG CTG ATG ATG TTT TCG-3Ј; (D336L) 5Ј-C AAG GCC ATC CAC CTG CTG ATG ATG TTT TCG-3Ј. Login to comment 94 ABCC1 p.Asp336Leu Similarly, Asp336 was replaced with Leu as well as Arg. Login to comment 96 ABCC1 p.His335Leu Consequently, in addition to substituting His335 with Leu, it was replaced with a polar neutral amino acid (Gln) and an acidic Glu residue. Login to comment 104 ABCC1 p.His335Glu ABCC1 p.Lys332Asp ABCC1 p.Asp336Arg Representative confocal micrographs of cells expressing GFP-tagged wild-type MRP1 and mutants K332D, H335E, and D336R are shown in Fig. 2. Login to comment 106 ABCC1 p.Lys332Asp ABCC1 p.Lys332Leu ABCC1 p.Asp336Arg ABCC1 p.Asp336Leu In the case of the MRP1-Lys332 mutants K332D and K332L (Fig. 3B) and the MRP1-Asp336 mutants D336L and D336R (Fig. 3D), LTC4 uptake was reduced to levels that were indistinguishable from those observed with vesicles prepared from empty vector-transfected control cells. Login to comment 108 ABCC1 p.His335Glu ABCC1 p.His335Leu ABCC1 p.His335Gln Thus H335E and H335L and H335Q all transported LTC4 at levels that were ϳ50-60% of wild-type MRP1 uptake levels (Fig. 3C). Login to comment 110 ABCC1 p.His335Glu ABCC1 p.His335Leu ABCC1 p.His335Gln Kinetic Analysis of [3 H]LTC4 Uptake in His335 Mutant MRP1-enriched Membrane Vesicles-To further investigate the effect of the His335 substitutions on the reduced ability of MRP1 to transport LTC4, the kinetic parameters of [3 H]LTC4 uptake by the H335E, H335L, and H335Q MRP1 mutants were determined (Fig. 4). Login to comment 113 ABCC1 p.His335Glu ABCC1 p.His335Leu Thus, for wild-type MRP1, the Vmax (LTC4) was 101 pmol-1 mg-1 min-1 compared with 69, 63, and 48 pmol-1 mg-1 min-1 for the H335E, H335L, FIG. 1. Login to comment 122 ABCC1 p.His335Gln and H335Q mutants, respectively. Login to comment 125 ABCC1 p.Asp336Arg ABCC1 p.Asp336Leu In contrast, substitution of Asp336 with either Leu or Arg led to a complete loss of E217betaG uptake. Login to comment 126 ABCC1 p.Asp336Arg ABCC1 p.Asp336Leu Thus, as shown in Fig. 5C, MRP1-Asp336 mutants D336L and D336R exhibited the same E217betaG uptake activity as the empty vector control. Login to comment 131 ABCC1 p.His335Glu ABCC1 p.Lys332Asp ABCC1 p.His335Leu ABCC1 p.Lys332Leu ABCC1 p.His335Gln In contrast, LTC4 had very little effect (Ͻ15%) on E217betaG uptake by MRP1 mutants K332D and K332L, indicating that loss of LTC4 transport in these mutants is associated with a loss of binding of this substrate. On the other hand, LTC4 was still able to inhibit E217betaG uptake by MRP1 mutants H335E, H335L, and H335Q, which is consistent with only a partial reduction in LTC4 transport activity observed with these mutants (Fig. 6B). Login to comment 140 ABCC1 p.Lys332Asp ABCC1 p.Lys332Leu B, wild-type MRP1 (f), MRP1 mutants K332D (Œ) and K332L (‚), and control empty pcDNA3.1(-) vector (E). Login to comment 141 ABCC1 p.His335Glu ABCC1 p.His335Leu ABCC1 p.His335Gln C, wild-type MRP1 (f); MRP1 mutants H335E (ƒ), H335L (Ⅺ), and H335Q (q); and control pcDNA3.1(-) vector (E). Login to comment 142 ABCC1 p.Asp336Arg ABCC1 p.Asp336Leu D, wild-type MRP1 (f), MRP1 mutants D336L (Œ) and D336R (‚), and control empty pcDNA3.1(-) vector (E). Login to comment 145 ABCC1 p.His335Glu ABCC1 p.His335Leu ABCC1 p.His335Gln Kinetics of [3 H]LTC4 uptake by wild-type MRP1 and MRP1 TM6 mutants H335E, H335L, and H335Q. Login to comment 146 ABCC1 p.His335Glu ABCC1 p.His335Leu ABCC1 p.His335Gln A, shown is the Michaelis-Menten plot of the initial rate of ATP-dependent [3 H]LTC4 uptake by membrane vesicles prepared from HEK293T cells transfected with wild-type MRP1 (f) or MRP1 His335 mutants H335E (Ⅺ), H335L (ƒ), and H335Q (q). Login to comment 152 ABCC1 p.Lys332Asp ABCC1 p.Lys332Leu ABCC1 p.Asp336Arg ABCC1 p.Asp336Leu However, this band is not detectable in [3 H]LTC4 photolabeled proteins from cells expressing comparable levels of the MRP1 mutants K332D and K332L or mutants D336L and D336R, indicating that these mutations abrogate photolabeling and hence binding of this compound. Login to comment 154 ABCC1 p.His335Leu ABCC1 p.His335Gln ABCC1 p.His335Asp For the H335D, H335L, and H335Q MRP1 mutants, photolabeling was decreased by 40-45%. Login to comment 160 ABCC1 p.Lys332Asp ABCC1 p.Lys332Leu The relative levels of [3 H]GSH uptake by the MRP1 K332D and K332L mutants were less than 15% of wild-type MRP1 (Fig. 7A) after subtracting basal [3 H]GSH transport by membrane vesicles from the empty vector-transfected control cells. Login to comment 163 ABCC1 p.Lys332Asp ABCC1 p.Lys332Leu Membrane vesicles prepared from transfected cells were incubated at 37 °C with 400 nM [3 H]E217betaG in transport buffer for the times indicated. A, time courses of ATP-dependent [3 H]E217betaG uptake by membrane vesicles prepared from HEK293T cells transfected with wild-type MRP1 (f), TM6 mutants K332D (Œ) and K332L (‚), and the empty pcDNA3.1(-) vector (E). Login to comment 164 ABCC1 p.His335Glu ABCC1 p.His335Leu ABCC1 p.His335Gln B, time courses of [3 H]E217betaG uptake by wild-type MRP1 (f); TM6 mutants H335E (ƒ), H335L (Ⅺ), and H335Q (q); and the empty pcDNA3.1(-) vector control (E). Login to comment 165 ABCC1 p.Asp336Arg ABCC1 p.Asp336Leu C, time courses of [3 H]E217betaG uptake by membrane vesicles prepared from HEK293T cells transfected with cDNAs encoding wild-type MRP1 (f), TM6 mutants D336L (Œ), and D336R (‚), and the empty pcDNA3.1(-) vector control (E). Login to comment 170 ABCC1 p.Lys332Asp ABCC1 p.Lys332Leu A, ATP-dependent [3 H]E217betaG uptake in membrane vesicles from cells expressing wild-type MRP1 (WT-MRP1) (open bars), TM6 mutants K332D and K332L (shaded bars), and the empty pcDNA3.1(-) vector control (solid bars). Login to comment 171 ABCC1 p.His335Glu ABCC1 p.His335Leu ABCC1 p.His335Gln B, [3 H]E217betaG uptake by wild-type MRP1 (WT-MRP1) (open bars); TM6 mutants H335E, H335L, and H335Q (shaded bars); and the empty pcDNA3.1(-) vector control (solid bars). Login to comment 177 ABCC1 p.Asp336Arg ABCC1 p.Asp336Leu ABCC1 p.Asp336Leu Substitution of Asp336 with Leu (D336L) reduced GSH uptake to ϳ25% of wild-type MRP1 levels, whereas substitution of this negatively charged residue with a positively charged residue (D336R) further reduced uptake of this tripeptide to just above basal levels observed with vesicles from the empty vector control (Fig. 7C). Login to comment 183 ABCC1 p.Lys332Asp ABCC1 p.Lys332Leu Substitution of Lys332 with a neutral (K332L) or negatively charged (K332D) amino acid had no effect on MTX uptake by MRP1. Login to comment 184 ABCC1 p.His335Glu ABCC1 p.His335Leu ABCC1 p.His335Gln Similarly, MTX uptake by the H335E, H335L, and H335Q MRP1 mutants was comparable with wild-type MRP1. Login to comment 185 ABCC1 p.Asp336Arg ABCC1 p.Asp336Leu ABCC1 p.Asp336Leu In contrast, MTX uptake was reduced by ϳ50% when Asp336 was substituted with Leu (D336L) and by ϳ65% when substituted with Arg (D336R) (not shown). Login to comment 190 ABCC1 p.Lys332Asp ABCC1 p.Lys332Leu Thus, replacing Lys332 with either Leu or Asp eliminated the ability of MRP1 to transport LTC4 (and markedly reduced GSH transport) without affecting the transport of other organic anions including E217betaG, estrone 3-sulfate, and MTX. Login to comment 204 ABCC1 p.His335Glu ABCC1 p.Lys332Asp ABCC1 p.His335Leu ABCC1 p.Lys332Leu ABCC1 p.His335Gln ABCC1 p.Asp336Arg ABCC1 p.Asp336Leu Membrane vesicles were preincubated with acivicin and then incubated with [3 H]GSH in the presence of 30 ␮M apigenin in transport buffer for 20 min at 37 °C. A, K332D and K332L; B, H335E, H335L, and H335Q; C, D336L and D336R. Login to comment 206 ABCC1 p.Lys332Asp ABCC1 p.Lys332Leu D, WT-MRP1 (f); K332D (Œ); K332L (‚); vector control (E). Login to comment 207 ABCC1 p.His335Glu ABCC1 p.His335Leu ABCC1 p.His335Gln E, WT-MRP1 (f); H335E (ƒ); H335L (Ⅺ); H335Q (q); vector control (E). Login to comment 208 ABCC1 p.Asp336Arg ABCC1 p.Asp336Leu F, WT-MRP1 (f); D336L (Œ); D336R (‚); vector control (E). Login to comment 217 ABCC1 p.Asp336Arg ABCC1 p.Asp336Leu Substitutions of MRP1-Asp336 had a more dramatic effect on MRP1 transport activity than substitutions of either Lys332 or His335 in that the D336L and D336R mutants did not transport four of the five organic anion substrates tested, and transport of the fifth, MTX, was reduced by at least 50%. Login to comment 221 ABCC7 p.Glu92Lys Interestingly, a missense mutation of the analogous acidic residue in CFTR, E92K, has been associated with a benign cystic fibrosis phenotype (45), and in vitro studies indicate that it is not critical for the chloride conducting-activity of CFTR (46). Login to comment 238 ABCC1 p.Arg433Ser We previously showed that the naturally occurring mutation of MRP1-Arg433 to Ser predicted to be located in the fourth cytoplasmic loop in close proximity to the membrane interface of TM8 results in a 2-fold decrease in LTC4 and estrone 3-sulfate transport efficiency. Login to comment