HEK ‐293 cells expressing the cystic fibrosis transmembrane conductance regulator ( CFTR ): a model for studying regulation of Cl − transport

The Human Embryonic Kidney 293 cell line ( HEK ‐293) readily lends itself to genetic manipulation and is a common tool for biologists to overexpress proteins of interest and study their function and molecular regulation. Although these cells have some limitations, such as an inability to form resistive monolayers necessary for studying transepithelial ion transport, they are nevertheless valuable in studying individual epithelial ion transporters. We report the use of HEK ‐293 cells to study the cystic fibrosis transmembrane conductance regulator ( CFTR ) Cl − channel. While HEK ‐293 cells endogenously express mRNA for the Cl − channels, ClC‐2 and TMEM 16 A , they neither express CFTR mRNA nor protein. Therefore, we stably transfected HEK ‐293 cells with EGFP‐CFTR ( HEK‐CFTR ) and demonstrated CFTR function by measuring forskolin‐stimulated iodide efflux. This efflux was inhibited by CFTR inh 172, and the protein kinase A inhibitor H89, but not by Ca 2+ chelation. In contrast to intestinal epithelia, forskolin stimulation does not increase surface CFTR expression and does not require intact microtubules in HEK‐CFTR . To investigate the role of an endogenous G α S ‐coupled receptor, we examined the bile acid receptor, TGR 5. Although HEK‐CFTR cells express TGR 5, the potent TGR 5 agonist lithocholic acid ( LCA ; 5–500  μ mol/L) did not activate CFTR . Furthermore, forskolin, but not LCA , increased [ cAMP ] i in HEK ‐ CFTR suggesting that endogenous TGR 5 may not be functionally linked to Gα S . However, LCA did increase [Ca 2+ ] i and interestingly, abolished forskolin‐stimulated iodide efflux. Thus, we propose that the stable HEK‐CFTR cell line is a useful model to study the multiple signaling pathways that regulate CFTR .