ABCB4 p.Lys1075Met
| Predicted by SNAP2: | A: D (91%), C: D (91%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (91%), I: D (91%), L: D (95%), M: D (91%), N: D (95%), P: D (95%), Q: D (91%), R: D (91%), S: D (91%), T: D (91%), V: D (91%), W: D (95%), Y: D (95%), |
| 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: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Bile salt-dependent efflux of cellular phospholipi... Hepatology. 2007 Jul;46(1):188-99. Morita SY, Kobayashi A, Takanezawa Y, Kioka N, Handa T, Arai H, Matsuo M, Ueda K
Bile salt-dependent efflux of cellular phospholipids mediated by ATP binding cassette protein B4.
Hepatology. 2007 Jul;46(1):188-99., [PMID:17523162]
Abstract [show]
Human ABCB4 (multidrug resistance [MDR]3 P-glycoprotein) is expressed in the canalicular membrane of the hepatocyte. ABCB4 has been shown to be required for phosphatidylcholine (PC) secretion into the bile and to translocate PC across the plasma membrane. To further investigate the function of ABCB4, we established a cell line stably expressing ABCB4 (human embryonic kidney [HEK]/ABCB4). The efflux of phospholipids from HEK/ABCB4 cells was remarkably increased by the addition of taurocholate. In addition, the cholesterol efflux from HEK/ABCB4 cells was also enhanced in the presence of taurocholate. Light scattering measurements suggested that the taurocholate monomer plays an important role in ABCB4-mediated lipid secretion. On the other hand, the efflux of phospholipids and cholesterol was not mediated by ABCB1 (MDR1) even in the presence of taurocholate. Taurocholate promoted the efflux of phospholipids and cholesterol from HEK/ABCB4 cells more efficiently than glycocholate and cholate. ABCB4-K435M and ABCB4-K1075M, Walker A lysine mutants, did not mediate the phospholipid and cholesterol efflux in the presence of taurocholate, suggesting that ATP hydrolysis is essential for the efflux. Verapamil completely inhibited the taurocholate-dependent efflux of phospholipids and cholesterol from HEK/ABCB4 cells. Mass spectrometry revealed that, in the presence of taurocholate, HEK/ABCB4 cells preferentially secreted PC compared to sphingomyelin. PC vesicles induced cholesterol diffusion from cell membrane, but did not accept cholesterol from ABCB4. CONCLUSION: ABCB4 mediates the efflux of phospholipids into the canalicular lumen in the presence of bile salts, and plays a crucial role in bile formation and lipid homeostasis.
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No. Sentence Comment
8 ABCB4-K435M and ABCB4-K1075M, Walker A lysine mutants, did not mediate the phospholipid and cholesterol efflux in the presence of taurocholate, suggesting that ATP hydrolysis is essential for the efflux.
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ABCB4 p.Lys1075Met 17523162:8:22
status: NEW42 ABCB4 WalkerA lysine mutants (ABCB4-K435M and -K1075M) were prepared with the QuikChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) as described by the manufacturer.
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ABCB4 p.Lys1075Met 17523162:42:47
status: NEW43 The human ABCB4 gene and its mutant genes were inserted into the HindIII-XbaI site of pcDNA3.1/Hygro(ϩ) (Invitrogen, Carlsbad, CA) to make an expression vector for pcDNA3.1/Hygro(ϩ)/ABCB4, pcDNA3.1/Hygro(ϩ)/ABCB4-K435M and pcDNA3.1/ Hygro(ϩ)/ABCB4-K1075M.ThehumanABCB1genewas fused to His tag in the pCAGGSP vector (pCAGGSP/ MDR1-His).
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ABCB4 p.Lys1075Met 17523162:43:272
status: NEW47 HEK293 cells were transfected with pcDNA3.1/ Hygro(ϩ)/ABCB4, pcDNA3.1/Hygro(ϩ)/ABCB4-K435M, or pcDNA3.1/Hygro(ϩ)/ABCB4-K1075M using LipofectAMINE (Invitrogen) according to the manufacturer`s instructions.
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ABCB4 p.Lys1075Met 17523162:47:137
status: NEW188 To study the involvement of ATP hydrolysis in ABCB4-mediated secretion of phospholipids and cholesterol in the presence of NaTC, we established HEK293 cells stably expressing ABCB4-K435M (HEK/ABCB4-K435M) and ABCB4-K1075M (HEK/ABCB4-K1075M), in which the Walker A lysine in either nucleotide binding domain was substituted by methionine.
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ABCB4 p.Lys1075Met 17523162:188:215
status: NEWX
ABCB4 p.Lys1075Met 17523162:188:233
status: NEW189 NaTC-dependent efflux of phospholipids and cholesterol was not observed with HEK/ABCB4-K435M or HEK/ABCB4-K1075M cells (Fig. 6F,G), although the expression levels, glycosylation, and the surface expression of mutant ABCB4 were comparable to those of wild-type ABCB4 (Fig. 6A-E).
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ABCB4 p.Lys1075Met 17523162:189:106
status: NEW192 (A) Cell lysates (32.0 g of proteins) from HEK/ABCB4-WT cells (lane 1), HEK/ABCB4-K435M cells (lane 2) and HEK/ ABCB4-K1075M cells (lane 3) were separated by 7% polyacrylamide gel electrophoresis.
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ABCB4 p.Lys1075Met 17523162:192:126
status: NEW193 ABCB4-WT, -K435M and -K1075M were detected with mouse monoclonal antibody C219.
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ABCB4 p.Lys1075Met 17523162:193:22
status: NEW194 (B-E) HEK293 cells (B), HEK/ABCB4-WT cells (C), HEK/ABCB4-K435M cells (D), and HEK/ ABCB4-K1075M cells (E) were fixed in 70% ethanol and reacted with monoclonal antibody C219 and Alexa488-conjugated anti-mouse IgG.
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ABCB4 p.Lys1075Met 17523162:194:90
status: NEW196 (F-G) HEK/ ABCB4 cells, HEK/ABCB4-K435M cells and HEK/ABCB4-K1075M cells were incubated for 24 hours at 37°C with 0.02% BSA in the absence (control, open bars) or presence (filled bars) of 0.5 mM NaTC. Bars represent the mean Ϯ SE of 3 measurements.
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ABCB4 p.Lys1075Met 17523162:196:60
status: NEW217 Because the excretion of phospholipids and cholesterol was not observed with ABCB4-K435M or ABCB4-K1075M, in which the Walker A lysine in either nucleotide binding domain was substituted by methionine, ABCB4 mediates lipid efflux in an ATP-dependent manner, like other ABC transporters.
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ABCB4 p.Lys1075Met 17523162:217:98
status: NEW