ABCMdb

ABCB4 p.Lys435Met

None has been submitted yet.

Publications

PMID: 21820390 [PubMed] Groen A et al: "Complementary functions of the flippase ATP8B1 and the floppase ABCB4 in maintaining canalicular membrane integrity."

No. Sentence Comment

23 Materials and Methods Plasmids Generation of the pcDNA3.1ϩ vector (Invitrogen, Carlsbad, CA) expressing human ABCB4 and its K435M derivative (replacing the conserved lysine within the Walker A motif of the first nucleotide binding domain to inhibit adenosine triphosphate binding) was described previously.11 The E558Q derivative (replacing a conserved glutamate in the Walker B motif with a glutamine to prevent adenosine triphosphate hydrolysis) was derived from the wild-type ABCB4 by site-directed mutagenesis using the mutagenic oligonucleotide 5=- GATCCTTCTGCTG- GATCAGGCGACGTCAGCATTGGAC-3= and its reverse complement to prime synthesis of the mutant vector using Quikchange II (Agilent Technologies, Santa Clara, CA). Login to comment
65 We also noted that inactive derivatives of ABCB4 carrying a point mutation in either the Walker A (K435M) or Walker B (E558Q) motifs of the first nucleotide binding domain, expressed to much higher levels than wild-type protein, suggesting a negative selective pressure against ABCB4 function (Figure 1A). Login to comment
67 In contrast, the nonfunctional K435M mutant was easily detectable on the plasma membrane and there was less evidence of nuclear breakdown (Supplementary Figure 3). Login to comment
68 Transient expression of wild-type ABCB4, but not the K435M mutant, was also associated with high levels of LDH activity in the culture media indicative of leakage of the cytoplasmic LDH from damaged cells (Figure 1D). Login to comment
88 (A) Western analysis of mutant and wild-type ABCB4 in HEK293T whole cell lysates. Lane 1, mock-transfected cells; lane 2, wild-type ABCB4 alone; lane 3, ABCB4 mutant K435M alone; lane 4, ABCB4 mutant E558Q alone. Login to comment
66 We also noted that inactive derivatives of ABCB4 carrying a point mutation in either the Walker A (K435M) or Walker B (E558Q) motifs of the first nucleotide binding domain, expressed to much higher levels than wild-type protein, suggesting a negative selective pressure against ABCB4 function (Figure 1A). Login to comment
69 Transient expression of wild-type ABCB4, but not the K435M mutant, was also associated with high levels of LDH activity in the culture media indicative of leakage of the cytoplasmic LDH from damaged cells (Figure 1D). Login to comment
89 (A) Western analysis of mutant and wild-type ABCB4 in HEK293T whole cell lysates. Lane 1, mock-transfected cells; lane 2, wild-type ABCB4 alone; lane 3, ABCB4 mutant K435M alone; lane 4, ABCB4 mutant E558Q alone. Login to comment

PMID: 17523162 [PubMed] Morita SY et al: "Bile salt-dependent efflux of cellular phospholipids mediated by ATP binding cassette protein B4."

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. Login to comment
42 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 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 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 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 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 (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-WT, -K435M and -K1075M were detected with mouse monoclonal antibody C219. Login to comment
194 (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 (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 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

PMID: 25315773 [PubMed] Takatsu H et al: "Phospholipid flippase activities and substrate specificities of human type IV P-type ATPases localized to the plasma membrane."

No. Sentence Comment

207 To address this hypothesis, we established cell lines stably expressing FLAG-tagged ABCB4(WT) or ABCB4(K435M), which is nonfunctional due to a mutation in the Walker A motif in the nucleotide-binding domain (41), with or without ATP8B1-HA. Login to comment
208 In both the singly and doubly expressing cell lines, comparable expression levels of ABCB4(WT) and ABCB4(K435M) were confirmed by immunoblotting, and the plasma membrane localization of all proteins was visualized by immunostaining with anti-FLAG antibody (Fig. 9, A and B). Login to comment
210 ABCB4(WT) and ABCB4- (K435M) colocalized with ATP8B1 at the plasma membrane (Fig. 8B). Login to comment
211 Importantly, coexpression of ATP8B1 with ABCB4- (WT), but not ABCB4(K435M), significantly suppressed PC incorporation at all time points examined relative to that in cells expressing only ATP8B1 (Fig. 9, C and D). Login to comment
214 Although we did not detect a decrease in PC incorporation in cells expressing ABCB4 alone at 5 min, we did detect a small but significant decrease at 30 min relative to parental cells or cells expressing ABCB4(K435M) (Fig. 9C). Login to comment
219 HeLa cell lines stably expressing HA-tagged ATP8B1, FLAG-tagged ABCB4(WT), FLAG-tagged ABCB4(K435M), ATP8B1-HA af9; FLAG-ABCB4(WT), or ATP8B1-HA af9; FLAG-ABCB4(K435M) were established by infection with recombinant retroviral vectors. Login to comment