ABCMdb

ABCC2 p.Lys578Ala

Predicted by SNAP2:	A: D (66%), C: D (66%), D: D (80%), E: D (80%), F: D (80%), G: D (75%), H: D (71%), I: D (53%), L: D (63%), M: D (66%), N: D (66%), P: D (85%), Q: D (66%), R: N (61%), S: D (63%), T: N (72%), V: D (63%), W: D (75%), Y: D (80%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, L: D, M: D, N: D, P: D, Q: D, R: N, S: D, T: D, V: D, W: D, Y: D,

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Publications
[hide] Insight in eukaryotic ABC transporter function by ... FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19. Frelet A, Klein M
Insight in eukaryotic ABC transporter function by mutation analysis.
FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19., 2006-02-13 [PMID:16442101]

Abstract [show]
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.

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Sentences [show]
No. Sentence Comment
491 HsMRP2 Replacement of basic residues (K324 (TM6), K483 (TM9), R1210 (TM16) and R1257 (TM17)) with alanine decreased the ability to transport an organic anion substrate, glutathione-methlyfluorescein and K578A resulted in decreased protein expression. Login to comment

[hide] Identification of basic residues involved in drug ... J Biol Chem. 2000 Dec 15;275(50):39617-24. Ryu S, Kawabe T, Nada S, Yamaguchi A
Identification of basic residues involved in drug export function of human multidrug resistance-associated protein 2.
J Biol Chem. 2000 Dec 15;275(50):39617-24., [PMID:10978330]

Abstract [show]
Multidrurg resistance-associated protein 2 (MRP2)/canalicular multispecific organic anion transporter (cMOAT) is involved in the ATP-dependent export of organic anions across the bile canalicular membrane. To identify functional amino acid residues that play essential roles in the substrate transport, each of 13 basic residues around transmembrane regions (TMs) 6-17 were replaced with alanine. Wild type and mutant proteins were expressed in COS-7 cells, and the transport activity was measured as the excretion of glutathione-methylfluorescein. Four mutants, K324A (TM6), K483A (TM9), R1210A (TM16), and R1257A (TM17), showed decreased transport activity, and another mutant, K578A (TM11), showed decreased protein expression. These five mutants were normally delivered to the cell surface similar to the other fully active mutants and wild type MRP2. The importance of TM6, TM16, and TM17 in the transport function of MRP2 is consistent with the previous observation indicating the importance of the corresponding TM1, TM11, and TM12 on P-glycoprotein (Loo, T. W., and Clarke, D. M. (1999) J. Biol. Chem. 274, 35388-35392). Another observation that MRP2 inhibitor, cyclosporine A, failed to inhibit R1230A specifically, indicated the existence of its binding site within TM16.

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Sentences [show]
No. Sentence Comment
3 Four mutants, K324A (TM6), K483A (TM9), R1210A (TM16), and R1257A (TM17), showed decreased transport activity, and another mutant, K578A (TM11), showed decreased protein expression. Login to comment
42 Site-directed Mutagenesis-The seven mutants of MRP2, K316A, K324A, K329A, H439A, K483A, K578A, and R590A, were generated by the overlapping polymerase chain reaction method. Login to comment
96 Except for K578A, all of the mutants were expressed at nearly the same level when compared with wild type MRP2. Login to comment
112 The experiments Only the expression of K578A was faint in both immunoblot and immunocytochemistry. Login to comment
118 As to K578A mutant, GS-MF excretion decreased to the level of control (Fig. 8); however, expression level of this mutant also decreased (Figs. 5 and 6). Login to comment
121 As shown in Fig. 10, we observed with confocal fluorescence microscopy that these mutant MRP2s including K578A were expressed at the cell surface. Login to comment
122 Therefore, the defect in the transport function of these mutants was clearly due to the functional defect except for K578A. Login to comment
123 The decrease in transport activity of K578A was mainly due to the decrease in the expression level. Login to comment
147 Except for a mutant K578A, the protein expression level of these mutants in COS-7 cell was comparable with that of wild type (Figs. 5 and 6). Login to comment
148 Among these mutants, K578A, R1210A, and R1257A were the most influenced by mutation concerning substrate translocating activity FIG. 6. Login to comment
161 As to K578A, the excretion of GS-MF and protein expression was coincidentally lowered (Figs. 5, 6, and 8). Login to comment