ABCMdb

PMID: 18989824

Hughes LK, Ju M, Sheppard DN
Potentiation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents by the chemical solvent tetrahydrofuran.
Mol Membr Biol. 2008 Sep;25(6-7):528-38., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Gly551Asp This THF-induced increase in Isc was enhanced by forskolin (10 mM), inhibited by the PKA inhibitor H-89 (10 mM) and the thiazolidinone CFTRinh-172 (10 mM) and attenuated greatly in FRT epithelia expressing the cystic fibrosis mutants F508del- and G551D-CFTR. Login to comment 7 ABCC7 p.Ser660Ala Similar results were obtained with the DR-S660A-CFTR Cl( channel that is not regulated by PKA-dependent phosphorylation and using 2?deoxy-ATP, which gates wild-type CFTR more effectively than ATP. Login to comment 28 ABCC7 p.Gly551Asp ABCC7 p.Ser660Ala Materials and methods Cells and cell culture For this study, we used Fischer rat thyroid (FRT) epithelial cells expressing wild-type, F508del- and G551D- human CFTR [8] and mouse mammary epithelial (C127) cells stably expressing either wild-type human CFTR or the variant DR-S660A-CFTR Figure 1. Login to comment 102 ABCC7 p.Gly551Asp To provide further evidence that THF increases Isc by acting on CFTR, we tested its effects on the cystic fibrosis (CF) mutants F508del and G551D, both of which are located in NBD1 of CFTR and associated with a severe disease phenotype. Login to comment 104 ABCC7 p.Gly551Asp However, like G551D [11], F508del also perturbs severely CFTR channel gating [14]. Login to comment 106 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Figure 2H demonstrates that THF (100 mM), itself, had little or no effect on Isc in FRT epithelia expressing F508del- and G551D-CFTR, whereas forskolin (10 mM) elicited a modest increase in Isc in F508del- and G551D-expressing FRT epithelia. Login to comment 107 ABCC7 p.Gly551Asp Moreover, THF (100 mM) failed to potentiate Isc following treatment of F508del- and G551D-expressing FRT epithelia with forskolin (10 mM; Figure 2H). Login to comment 108 ABCC7 p.Gly551Asp By contrast, genistein (50 mM) enhanced robustly cAMP-stimulated Isc in FRT epithelia expressing F508del- and G551D-CFTR (Figure 2H). Login to comment 115 ABCC7 p.Gly551Asp (G, H) The change in Isc evoked in wild-type, F508del- and G551D-CFTR FRT epithelia by different agents. Login to comment 179 ABCC7 p.Ser660Ala THF potentiates the DR-S660A-CFTR Cl( channel and wild-type CFTR gated by 2?-deoxy-ATP The lack of effect of THF on the CFTR Cl( channel after phosphorylation by PKA, suggests that the RD might be a target for the actions of THF. Login to comment 180 ABCC7 p.Ser660Ala To test this hypothesis, we used the CFTR construct DR-S660A [9], performing the same experiments on this CFTR variant as those with wild-type CFTR. Login to comment 181 ABCC7 p.Ser660Ala The CFTR mutant DR-S660A lacks much of the RD (residues 708Á835) and disables the dibasic phosphorylation site at serine 660. Login to comment 183 ABCC7 p.Ser660Ala Supplementary Information (SI) Figure 1 (online version only) demonstrates that THF (100 mM) augments the activity of DR-S660A-CFTR Cl( channels by increasing the frequency of channel openings. Login to comment 227 ABCC7 p.Ser660Ala Our observation that THF potentiates the DR-S660A-CFTR Cl( channel argues that the solvent interacts with a site distinct from the RD. Login to comment