ABCMdb

PMID: 17523162

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., [PubMed]

Sentences

No. Mutations Sentence Comment

8 ABCB4 p.Lys435Met ABCB4 p.Lys1075Met 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. Login to comment 42 ABCB4 p.Lys435Met ABCB4 p.Lys1075Met 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. Login to comment 43 ABCB4 p.Lys435Met ABCB4 p.Lys1075Met 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). Login to comment 47 ABCB4 p.Lys435Met ABCB4 p.Lys1075Met 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. Login to comment 188 ABCB4 p.Lys435Met ABCB4 p.Lys435Met ABCB4 p.Lys1075Met ABCB4 p.Lys1075Met 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. Login to comment 189 ABCB4 p.Lys435Met ABCB4 p.Lys1075Met 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). Login to comment 192 ABCB4 p.Lys435Met ABCB4 p.Lys1075Met (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. Login to comment 193 ABCB4 p.Lys435Met ABCB4 p.Lys1075Met ABCB4-WT, -K435M and -K1075M were detected with mouse monoclonal antibody C219. Login to comment 194 ABCB4 p.Lys435Met ABCB4 p.Lys1075Met (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. Login to comment 196 ABCB4 p.Lys435Met ABCB4 p.Lys1075Met (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. Login to comment 217 ABCB4 p.Lys435Met ABCB4 p.Lys1075Met 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. Login to comment