ABCMdb

PMID: 21820390

Groen A, Romero MR, Kunne C, Hoosdally SJ, Dixon PH, Wooding C, Williamson C, Seppen J, Van den Oever K, Mok KS, Paulusma CC, Linton KJ, Oude Elferink RP
Complementary functions of the flippase ATP8B1 and the floppase ABCB4 in maintaining canalicular membrane integrity.
Gastroenterology. 2011 Nov;141(5):1927-37.e1-4. Epub 2011 Aug 4., [PubMed]

Sentences

No. Mutations Sentence Comment

23 ABCB4 p.Lys435Met ABCB4 p.Glu558Gln 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 ABCB4 p.Lys435Met ABCB4 p.Glu558Gln 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 66 ABCB4 p.Lys435Met ABCB4 p.Glu558Gln 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 ABCB4 p.Lys435Met 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 ABCB4 p.Lys435Met ABCB4 p.Lys435Met 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 69 ABCB4 p.Lys435Met 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 ABCB4 p.Lys435Met ABCB4 p.Glu558Gln (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 89 ABCB4 p.Lys435Met ABCB4 p.Glu558Gln (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