ABCMdb

ABCB1 p.Ala544Asp

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Publications

PMID: 10767346 [PubMed] Dixon PH et al: "Heterozygous MDR3 missense mutation associated with intrahepatic cholestasis of pregnancy: evidence for a defect in protein trafficking."

No. Sentence Comment

71 The mutated fragment was subcloned into the plasmid pMDR1-wt to generate pMDR1-A544D, and used to transiently transfect human epithelial kidney (HEK293T) cells. Login to comment
75 In contrast, cells transiently transfected with pMDR1-A544D formed two distinct populations. Login to comment
76 The first population behaved similarly to the control cells with no UIC2-PE fluorescence and high R123 fluorescence (Fig. 3b, lower right quadrant), consistent with failure to express A544D-P-gp1 at the cell surface (and thus accumulate R123). Login to comment
103 Cells transfected with pMDR1-A544D also formed two populations (Fig. 4); the first population (M3) had a mean UIC2-PE fluorescence of 10 [barely above the mean (= 3) of the negative control cells], and the second population (M4) had a mean of 50. Login to comment
106 Western blot analysis of A544D-P-gp1 Transiently transfected cells normally express two forms of P-gp1, discernible by polyacrylamide gel electrophoresis (PAGE) and western analysis: immature, non-or core-glyco- Figure 3. Login to comment
107 FACS analysis of HEK293T cells transiently transfected with pCIneo-βgal (a), or pMDR1-A544D (b and c). Login to comment
112 This allowed the intracellular, cell surface and total P-gp of wt-P-gp1 and A544D to be calculated (calculated values are shown in italics). Login to comment
113 Mean fluorescence (relative arbitrary fluorescence units) A544D-P-gp1 Wt-P-gp1 HEK293T Intact cells 40 171 3 F&P then labelled with UIC2-PE 197 497 13 Labelled with UIC2-PE then F&P 32 144 3 Intracellular P-gp 155 343 - Cell surface P-gp 37 168 - Total P-gp 192 511 - sylated P-gp1 and mature glycosylated P-gp1. Login to comment
116 If the A544D mutation impairs the trafficking of the protein from the ER to the cell membrane then it might be expected to alter the ratio of immature to mature protein found in the cell. Login to comment
117 PAGE and western analysis (Fig. 5) showed that wild-type P-gp1 was expressed at higher levels than A544D-P-gp1 and was predominantly present as the 170 kDa mature, glycosylated form. Login to comment
118 In contrast, the A544D-P-gp1 was predominantly found in the 140 kDa immature form. Login to comment
120 The ratio of cell surface to intracellular P-gp1 is also altered in the A544D mutant Additional evidence that A544D-P-gp1 is a trafficking mutant was provided by calculating the ratio of cell surface to intracellular P-gp1 by FACS analysis (Fig. 6 and Table 2). Login to comment
124 These data, obtained for cells expressing wild-type P-gp1 and for cells expressing the A544D mutant, are summarized along with the background levels of fluorescence in Table 2. Login to comment
125 Calculation of total P-gp1 level and the ratio of intracellular to cell surface P-gp clearly showed that cells transfected with pMDR1-A544D expressed a lower level of P-gp1 than cells transfected with pMDR1-wt (total fluorescence 192 and 511, respectively), and that much less of it got to the cell surface (only 19% of the total produced, compared with 33% of wild-type protein). Login to comment
208 The mutated fragment was subcloned into pMDR1-wt to generate pMDR1-A544D for expression studies in mammalian cells. Login to comment