Development of a colorimetric method for functional chloride channel assay.
Anion channels play significant physiological roles in humans and animals. However, the effort of screening for anion channel modulators was limited by the available assay technologies. This report discusses the development of a cell-based functional chloride channel assay using iodine as the chloride channel functional indicator. Iodine concentrations were measured with modified Sandell-Kolthoff reaction using colorimetric detection. The assay was rapid and quantitative. When WSS-1 cells were activated by gamma-aminobutyric acid (GABA) in the condition that gamma-aminobutyric acid type A receptor (GABAA receptor) conducted outwardly rectifying chloride channel function, the EC50 of GABA was 7.69 microM. IC50s were 0.53 microM for bicuculline and 3.1 microM for picrotoxin, respectively, in the presence of 10 microM GABA. When Capan-1 cells were activated by forskolin, the EC50 was 0.14 microM. The assay can also be applied to inwardly rectifying anion channels as exemplified by GABAA channel with an EC50 of 294 microM. Thus, the assay is universal and reliable and can be used for anion channel high-throughput screening.
Pharmaceutical Research and Development, Johnson & Johnson, Raritan, NJ 08869, USA. wtang1@prdus.jnj.com
Weimin
W
Tang
Mary Jo
MJ
Wildey
D001681:Biological Assay / Q000379:methods*; D018118:Chloride Channels / Q000378:metabolism*; D003124:Colorimetry / Q000379:methods*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism; D018756:GABA Antagonists / Q000494:pharmacology; D018757:GABA Modulators / Q000494:pharmacology; D058787:GABA-A Receptor Antagonists; D006801:Humans; D007455:Iodine / Q000378:metabolism; D011963:Receptors, GABA-A / Q000378:metabolism; D014407:Tumor Cells, Cultured; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
C505032:CFTR protein, human; D018118:Chloride Channels; D018756:GABA Antagonists; D018757:GABA Modulators; D058787:GABA-A Receptor Antagonists; D011963:Receptors, GABA-A; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005680:gamma-Aminobutyric Acid; D007455:Iodine