scholarly journals Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

10.3791/55700 ◽  
2017 ◽  
Author(s):  
Bao Li Zhang ◽  
Da Yuan Gao ◽  
Xiao Xu Zhang ◽  
Shuo Shi ◽  
Winnie Shum
Keyword(s):  
Epithelial Cells ◽  
Patch Clamp ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp

scholarly journals Generation and functional characterization of epithelial cells with stable expression of SLC26A9 Cl− channels

2016 ◽  
Vol 310 (7) ◽  
pp. L593-L602 ◽  
Author(s):  
Johanna J. Salomon ◽  
Stephan Spahn ◽  
Xiaohui Wang ◽  
Joachim Füllekrug ◽  
Carol A. Bertrand ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Patch Clamp ◽  
Western Blotting ◽  
Therapeutic Target ◽  
Stable Expression ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Rat Thyroid

Recent studies identified the SLC26A9 Cl− channel as a modifier and potential therapeutic target in cystic fibrosis (CF). However, understanding of the regulation of SLC26A9 in epithelia remains limited and cellular models with stable expression for biochemical and functional studies are missing. We, therefore, generated Fisher rat thyroid (FRT) epithelial cells with stable expression of HA-tagged SLC26A9 via retroviral transfection and characterized SLC26A9 expression and function using Western blotting, immunolocalization, whole cell patch-clamp, and transepithelial bioelectric studies in Ussing chambers. We demonstrate stable expression of SLC26A9 in transfected FRT (SLC26A9-FRT) cells on the mRNA and protein level. Immunolocalization and Western blotting detected SLC26A9 in different intracellular compartments and to a lesser extent at the cell surface. Whole cell patch-clamp recordings demonstrated significantly increased constitutive Cl− currents in SLC26A9-FRT compared with control-transduced FRT (Control-FRT) cells ( P < 0.01). Similar, transepithelial measurements showed that the basal short circuit current was significantly increased in SLC26A9-FRT vs. Control-FRT cell monolayers ( P < 0.01). SLC26A9-mediated Cl− currents were increased by cAMP-dependent stimulation (IBMX and forskolin) and inhibited by GlyH-101, niflumic acid, DIDS, and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), as well as RNAi knockdown of WNK1 implicated in epithelial osmoregulation. Our results support that these novel epithelial cells with stable expression of SLC26A9 will be a useful model for studies of pharmacological regulation including the identification of activators of SLC26A9 Cl− channels that may compensate deficient cystic fibrosis transmembrane regulator (CFTR)-mediated Cl− secretion and serve as an alternative therapeutic target in patients with CF and potentially other muco-obstructive lung diseases.

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