Abstract
An atomic absorption spectroscopy-based detection system was employed to develop a new non-radioactive flux assay for chloride (Cl–) channels in a high throughput format. Cl– flux is assayed by measuring the extent to which Cl– precipitates an excess amount of silver ions (Ag+). A linear correlation was observed between theoretical and determined Cl– concentration with an r 2 value of 0.996. The assay was found to be free from interference from various ions and proteins. The assay was used to study the physiology of endogenously expressed Cl– channels in a Chinese hamster ovary-K1 cell line. Cl– efflux was activated in response to an increased concentration of K+ (100 mM), Ca2+ (4 mM), and ionomycin (10 µM) as calcium ionophore. The efflux was also sensitive to pH as slightly higher efflux of Cl– was observed at an acidic pH of 3.2 in comparison to the neutral pH of 7.4. The Cl– efflux was inhibited by 100 µM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 500 µM 5-nitro-2-(3-phenylpropylamino) benzoate (NPPB) but not by tolbutamide, niflumic acid, or glybenclamide, indicating that the channel current is not sensitive to other cystic fibrosis transmembrane conductance regulator inhibitors. The 50% inhibitory concentration (IC50) values of DIDS at pH 7.4 and pH 3.2 were 17 µM and 19 µM, respectively. An IC50 of 26 µM was observed for NPPB. The assay had a Z' factor of 0.678.
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