ABCMdb

PMID: 24381502

Kim TH, Park HJ, Choi JH
Functional Characterization of ABCB4 Mutations Found in Low Phospholipid-Associated Cholelithiasis (LPAC).
Korean J Physiol Pharmacol. 2013 Dec;17(6):525-30. doi: 10.4196/kjpp.2013.17.6.525. Epub 2013 Dec 16., [PubMed]

Sentences

No. Mutations Sentence Comment

8 ABCB4 p.Ser320Phe ABCB4 p.Phe165Ile Through a membrane vesicular transport assay, we observed that the uptake of paclitaxel was significantly reduced in membrane vesicles expressing 2 ABCB4 mutations, F165I and S320F. Login to comment 20 ABCB4 p.Ile541Phe For example, a ABCB4 mutation found in PFIC3 patients, I541F, was shown to decrease transport activity through reduction of membrane MDR3 expression [10,11]. Login to comment 26 ABCB4 p.Ser320Phe ABCB4 p.Phe165Ile ABCB4 p.Met301Thr In this study, we selected 3 novel missense mutations of ABCB4 that were first reported by Rosmorduc et al. [3] and F165I 5`-AGG AAA TAG GAT GGA TTG ACA TCA ATG ACA-3` M301T 5`-GCA AAC ATT TCC ACG GGT ATT GCC TTS CTG-3` S320F 5`-AGG ACA CAA ATC AGA CAG CAT CAA AGG GAA-3` The SNP sites were marked by bold-faced letters with underlines. Login to comment 56 ABCB4 p.Ser320Phe ABCB4 p.Phe165Ile ABCB4 p.Met301Thr RESULTS Mutations of ABCB4 examined in this study To perform a molecular characterization of ABCB4 muta- cDNA position Amino acid substitution Predicted domain a c.495T&#ff1e;A F165I ICD1 c.902T&#ff1e;C M301T TM5 c.959C&#ff1e;T S320F TM5 Position of each mutant was based upon the translational start site. Login to comment 80 ABCB4 p.Ser320Phe ABCB4 p.Phe165Ile ABCB4 p.Met301Thr The ABCB4 mutant, F165I might be located in the ICD1, while other two mutants, M301T and S320F are located in the TM5 [3]. Login to comment 84 ABCB4 p.Ser320Phe ABCB4 p.Phe165Ile ABCB4 p.Met301Thr To exclude ATPase activity by other endogenous ABC transporters including MDR1, values for transport activity were obtained by subtracting the uptake in empty vector-transfected cells from that in cells transfected with ABCB4 reference or mutant-bearing vectors, at each corresponding paclitaxel concentration. The uptake of paclitaxel Vmax (nmol mg &#ff0d;1 per min) Km (mM) Vmax/Km ratio (nmol mg&#ff0d;1 min &#ff0d;1 per mM) Reference 28.98&#b1;1.565 1.114&#b1;0.1391 27.13&#b1;5.232 F165I 16.82&#b1;1.565* 1.028&#b1;0.1189 15.56&#b1;4.658* M301T 23.23&#b1;0.8641 1.206&#b1;0.2875 20.60&#b1;5.628 S320F 18.55&#b1;2.726* 1.185&#b1;0.1064 15.99&#b1;3.736* Data (mean&#b1;SD) are from 5 separate experiments. Login to comment 92 ABCB4 p.Ser320Phe ABCB4 p.Phe165Ile was significantly reduced in membrane vesicles expressing 2 ABCB4 mutations, F165I and S320F. Login to comment 93 ABCB4 p.Met301Thr M301T mutant also showed a reduction in the transport activity compared to that of the reference, although statistical significance was not observed. Login to comment 95 ABCB4 p.Ser320Phe ABCB4 p.Phe165Ile We observed that the average values of Vmax/Km for F165I and S320F were significantly reduced compared to that of the ABCB4 reference. Login to comment 100 ABCB4 p.Ser320Phe ABCB4 p.Phe165Ile The 2 mutations, F165I and S320F that showed decreased transport activities as compared to the reference, had significantly decreased MDR3 expression as compared to reference. Login to comment 101 ABCB4 p.Met301Thr The MDR3 expression level of M301T was comparable with that of the reference. Login to comment 102 ABCB4 p.Ser320Phe ABCB4 p.Phe165Ile Then, we investigated MDR3 expression levels of these mutants on the plasma membrane by cell surface biotinylation and observed that those of F165I and S320F were significantly decreased by 31%, compared to that of the reference (Fig. 2B). Login to comment 103 ABCB4 p.Ser320Phe ABCB4 p.Phe165Ile The results from immunoblotting or cell surface biotinylation experiments could explain the reason of altered transport activities of ABCB4 mutants, F165I and S320F; the decreased transport activities of these mutants were due to the reduced expression of functional MDR3 on the plasma membrane. Login to comment 106 ABCB4 p.Ser320Phe ABCB4 p.Phe165Ile ABCB4 p.Met301Thr The subcellular expression of M301T was comparable with that of the reference while the co-localization of F165I and S320F with plasma membrane was decreased. Login to comment 115 ABCB4 p.Ser320Phe ABCB4 p.Phe165Ile Through a membrane vesicular transport assay using paclitaxel, we found that 2 mutants, F165I and S320F, showed significantly decreased transport activity compared to the reference (Fig. 1). Login to comment 116 ABCB4 p.Ser320Phe ABCB4 p.Phe165Ile F165I and S320F are located in the ICD1 and TM5 of MDR3, respectively, that might be involved in coupling the energy from ATP hydrolysis to substrate transport and the conformational change involved in substrate extrusion, respectively [3,15]. Login to comment 117 ABCB4 p.Met301Thr M301T, showing a comparable transport activity with that of the reference is also located in TM5. Login to comment 120 ABCB4 p.Ser320Phe ABCB4 p.Phe165Ile The reduced transport function of F165I and S320F mutants also might be due to the decreased expression of functional MDR3 on the plasma membrane (Fig. 2). Login to comment 123 ABCB4 p.Ile541Phe One of the ABCB4 mutations, I541F, was found in PFIC3 patients and was shown to be a trafficking-defective mutation [10,11]. Login to comment 125 ABCB4 p.Ser320Phe ABCB4 p.Ile541Phe ABCB4 p.Phe165Ile 2 mutants, F165I and S320F, examined in this study also showed decreased transport activity and protein expression, although the extent of reduction was less than that of I541F. Login to comment