A Microplate Assay Measuring Chloride Ion Channel Activity

Publisher： Elsevier

ISSN： 0003-2697

Source： Analytical Biochemistry, Vol.241, Iss.1, 1996-10, pp. : 51-58

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Abstract

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 EC 50 of 0.27 ± 0.05 mu m , illustrating the reproducibility of the assay. Forskolin exposure of CFPAC-1 cells, containing DeltaF508 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 Ca 2+ dependent. Removal of Na + ions severely inhibited basal and stimulated Cl - efflux from T84 cells. However, furosemide did not affect forskolin-stimulated J Cl , 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 m m . 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.