ABCMdb

PMID: 21520952

Loo TW, Bartlett MC, Clarke DM
Benzbromarone stabilizes DeltaF508 CFTR at the cell surface.
Biochemistry. 2011 May 31;50(21):4393-5. Epub 2011 May 3., 2011-05-31 [PubMed]

Sentences

No. Mutations Sentence Comment

14 ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp It was recently reported that the stability of ΔF508 CFTR at the cell surface approached that of wild-type CFTR when NBDÀTMD2 interactions were restored.21 It was shown that introduction of a V510D mutation into NBD1 promoted the maturation and stability of ΔF508 CFTR by forming a a salt bridge with Arg1070 of TMD2.21 Similarly, maturation of ΔF508 CFTR was promoted by a R1070W suppressor mutation in TMD2.5 These suppressor mutation results suggested that direct binding of a compound to the TMDs may promote the maturation and stability of ΔF508 CFTR. Login to comment 22 ABCC7 p.His1085Arg ABCC7 p.His1085Arg The H1085R mutation is located in TMD2 within the intracellular loop (ICL) connecting TM10 and TM11.24 Mature protein was observed when H1085R CFTR was expressed in the presence of 38À100 μM benzbromarone (Figure 1A). Login to comment 26 ABCC7 p.His1085Arg To test if the CFTR mutants were active after benzbromarone rescue, we performed iodide efflux assays on BHK cells stably expressing ΔF508, H1085R, or wild-type CFTR proteins. Login to comment 27 ABCC7 p.His1085Arg It was found that both ΔF508 and H1085R exhibited forskolin-activated iodide efflux after rescue with benzbromarone (Figure 1B). Login to comment 28 ABCC7 p.Thr1142Cys ABCC7 p.Met348Cys It was shown that benzbromarone appeared to interact with the CFTR TMDs because 200 μM benzbromarone blocked cross-linking between cysteines introduced into TM segments 6 and 12 (M348C/T1142C).23 This concentration of benzbromarone is now shown to inhibit maturation of CFTR (Figure 1A). Login to comment 30 ABCC7 p.Val510Cys Figure 1C shows that 50 μM benzbromarone inhibited cross-linking between TMD1 and TMD2 [M348C(TM6)/ T1142C(TM12)] but not between NBD1 and TMD2 [V510C- (NBD1)/A1067C(ICL4)]. Login to comment 38 ABCC7 p.Val510Asp ABCC7 p.Val510Asp The slow maturation was similar to what was previously observed with the V510D/ΔF508 CFTR suppressor mutant.8 Maturation of mutant V510D/ΔF508 CFTR required ~4À8 h (Figure 2A) compared to 1À2 h for the wild-type enzyme.21 The half-life of the mature ΔF508 CFTR in the pulseÀchase assays was ~16 h after rescue with benzbromarone (data not shown). Login to comment 47 ABCC7 p.His1085Arg (A) Immunoblot analysis of cells expressing CFTR mutant ΔF508 or H1085R or the P-gp G251V processing mutant after treatment with the indicated concentrations of benzbromarone (Benz) for 40 h. Login to comment 48 ABCC7 p.His1085Arg (B) Iodide efflux assays performed on BHK cells stably expressing wild-type, ΔF508, or H1085R CFTR. Login to comment 50 ABCC7 p.Thr1142Cys ABCC7 p.Met348Cys ABCC7 p.Val510Cys ABCC7 p.Ala1067Cys (C) Effect of benzbromarone on cross-linking (X-link) between cysteines in TMD1 and TMD2 (M348C/T1142C) or NBD1 and TMD2 (V510C/A1067C).7 (D) Immunoblot of cells expressing CFTR TMD1þ2 in the absence (À) or presence (þ) of 0.05 mM benzbromarone. Login to comment