FLAG epitope positioned in an external loop preserves normal biophysical properties of CFTR

The American Journal of Physiology
Bruce D SchultzM Howard

Abstract

We asked whether inclusion of the FLAG epitope in the fourth extracellular loop of the cystic fibrosis transmembrane conductance regulator (M2-901/CFTR), which permits detection of cell surface expression, affected CFTR's biophysical properties or channel regulation by kinases, phosphatases, and nucleotides. Channel activity of M2-901/CFTR was evaluated in numerous cell types and expression systems to characterize its gating and regulation. Our results show that M2-901/CFTR required adenosine 3',5'-cyclic monophosphate-dependent protein kinase phosphorylation to initiate channel activity. Subsequently, ATP alone was sufficient to support channel gating, and ADP inhibited channel opening. Current fluctuation analysis indicated that the nucleotide-dependent gating rates were indistinguishable from those of wild-type (wt) cystic fibrosis transmembrane conductance regulator (CFTR). Channel conductance in symmetric Cl- (11.2 pS), anion permeability ratio (1.66), and block by gluconate indicate that the anion conduction pathway is indistinguishable from wtCFTR. Sulfonylureas (glibenclamide and LY-295501) inhibited M2-901/ CFTR channel activity by an identical mechanism to that described for wtCFTR. Finally, CFTR-dependent insertion a...Continue Reading

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Citations

May 21, 2004·American Journal of Physiology. Cell Physiology·James R BroughmanBruce D Schultz
Sep 13, 2007·Journal of Cellular Physiology·Robert A Maue
Dec 20, 2011·Cellular Physiology and Biochemistry : International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology·Jiraporn OusingsawatKarl Kunzelmann
Jul 8, 2017·BioMed Research International·Elena KrachmarovaGenoveva Nacheva
Sep 26, 2000·American Journal of Physiology. Cell Physiology·G A GibsonO A Weisz
Jul 17, 1999·The American Journal of Physiology·K W PetersR A Frizzell
Aug 15, 2001·American Journal of Physiology. Renal Physiology·R L SedlacekB D Schultz
Jul 27, 2000·American Journal of Physiology. Cell Physiology·M HowardO A Weisz
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