A microplate assay measuring chloride ion channel activity.
A microplate chloride ion channel assay, using N-(6-methoxyquinolyl) acetoethyl ester (MQAE) fluorescence changes has been developed. Forskolin stimulation of T84 cells caused cAMP-dependent, increased Cl- loss. Forskolin responses after 6 min gave an EC50 of 0.27 +/- 0.05 microM, illustrating the reproducibility of the assay. Forskolin exposure of CFPAC-1 cells, containing delta F508 CFTR, and CFPAC-1 PLJ4.7 cells, transfected with WT-CFTR stimulated Cl- secretion only from the latter, showing that the assay can be used to measure CFTR function. Stimulation of CFPAC-1 cells with ionomycin increased Cl- efflux, demonstrating functional Ca(2+)-mediated Cl- secretion in these cells. Ionomycin also induced a dose-responsive Cl- efflux from T84 cells that, unlike the forskolin response, was Ca2+ dependent. Removal of Na+ ions severely inhibited basal and stimulated Cl- efflux from T84 cells. However, furosemide did not affect forskolin-stimulated JCl, although the magnitude of the Cl- loss was reduced. The Stern-Volmer constant for MQAE fluorescence in T84 cells was calculated as 28.3 +/- 0.9 M-1 and the [Cl-]i in untreated T84 cells was determined as 52.4 +/- 0.6 mM. Stimulation of T84 cells with ionomycin and forskolin before inducing Cl- efflux allowed calculation of initial efflux rates without interference by second messenger generation and ion channel activation kinetics.
West
M R
MR
Respiratory Diseases Unit, Glaxo Wellcome Medicines Research Centre, Hertfordshire, United Kingdom.
Molloy
C R
CR
D002460:Cell Line; D018118:Chloride Channels / Q000032:analysis*; D005576:Colforsin / Q000494:pharmacology; D005456:Fluorescent Dyes; D006801:Humans; D017136:Ion Transport / Q000187:drug effects; D011804:Quinolines
D018118:Chloride Channels; D005456:Fluorescent Dyes; D011804:Quinolines; C078229:N-(6-methoxyquinolyl)acetoethyl ester; D005576:Colforsin