Development and validation of HTS flux assay for endogenously expressed chloride channels in a CHO-K1 cell line.
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 r2 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 microM) 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 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 500 microM 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 microM and 19 microM, respectively. An IC50 of 26 microM was observed for NPPB. The assay had a Z' factor of 0.678.
Gill
Sikander
S
Aurora Biomed Inc., Vancouver, BC, Canada. gill@aurorabiomed.com
Gill
Rajwant
R
Xie
Yong
Y
Wicks
David
D
Liang
Dong
D
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D018118:Chloride Channels / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D002712:Chlorides / Q000494:pharmacology; D006224:Cricetinae; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008954:Models, Biological; D011188:Potassium / Q000494:pharmacology; D015203:Reproducibility of Results; D012834:Silver / Q000494:pharmacology
D018118:Chloride Channels; D002712:Chlorides; D012834:Silver; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D011188:Potassium