ABCMdb

ABCC1 p.His335Gln

Predicted by SNAP2:	A: D (53%), C: N (66%), D: D (71%), E: D (66%), F: N (53%), G: N (53%), I: D (59%), K: D (66%), L: D (63%), M: N (53%), N: N (66%), P: D (80%), Q: N (87%), R: D (59%), S: N (66%), T: N (61%), V: N (53%), W: D (85%), Y: N (57%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: N, R: D, S: D, T: D, V: D, W: D, Y: N,

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Publications
[hide] Charged amino acids in the sixth transmembrane hel... J Biol Chem. 2002 Nov 1;277(44):41326-33. Epub 2002 Aug 18. 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 [PMID:12186871]

Abstract [show]
The multidrug resistance protein, MRP1 (ABCC1), is an ATP-binding cassette transporter that confers resistance to chemotherapeutic agents. MRP1 also mediates transport of organic anions such as leukotriene C(4) (LTC(4)), 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG), estrone 3-sulfate, methotrexate (MTX), and GSH. We replaced three charged amino acids, Lys(332), His(335), and Asp(336), predicted to be in the sixth transmembrane (TM6) helix of MRP1 with neutral and oppositely charged amino acids and determined the effect on substrate specificity and transport activity. All mutants were expressed in transfected human embryonic kidney cells at levels comparable with wild-type MRP1, and confocal microscopy showed that they were correctly routed to the plasma membrane. Vesicular transport studies revealed that the MRP1-Lys(332) mutants had lost the ability to transport LTC(4), and GSH transport was reduced; whereas E(2)17betaG, estrone 3-sulfate, and MTX transport were unaffected. E(2)17betaG transport was not inhibited by LTC(4) and could not be photolabeled with [(3)H]LTC(4), indicating that the MRP1-Lys(332) mutants no longer bound this substrate. Substitutions of MRP1-His(335) also selectively diminished LTC(4) transport and photolabeling but to a lesser extent. Kinetic analyses showed that V(max) (LTC(4)) of these mutants was decreased but K(m) was unchanged. In contrast to the selective loss of LTC(4) transport in the Lys(332) and His(335) mutants, the MRP1-Asp(336) mutants no longer transported LTC(4), E(2)17betaG, estrone 3-sulfate, or GSH, and transport of MTX was reduced by >50%. Lys(332), His(335), and Asp(336) of TM6 are predicted to be in the outer leaflet of the membrane and are all capable of forming intrahelical and interhelical ion pairs and hydrogen bonds. The importance of Lys(332) and His(335) in determining substrate specificity and of Asp(336) in overall transport activity suggests that such interactions are critical for the binding and transport of LTC(4) and other substrates of MRP1.

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No. Sentence Comment
47 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
108 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 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
122 and H335Q mutants, respectively. Login to comment
131 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
141 C, wild-type MRP1 (f); MRP1 mutants H335E (ƒ), H335L (Ⅺ), and H335Q (q); and control pcDNA3.1(-) vector (E). Login to comment
145 Kinetics of [3 H]LTC4 uptake by wild-type MRP1 and MRP1 TM6 mutants H335E, H335L, and H335Q. Login to comment
146 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
154 For the H335D, H335L, and H335Q MRP1 mutants, photolabeling was decreased by 40-45%. Login to comment
164 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
171 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
184 Similarly, MTX uptake by the H335E, H335L, and H335Q MRP1 mutants was comparable with wild-type MRP1. Login to comment
204 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
207 E, WT-MRP1 (f); H335E (ƒ); H335L (Ⅺ); H335Q (q); vector control (E). Login to comment