DISCRIMINATION BETWEEN CYSTIC FIBROSIS AND CFTR-CORRECTED EPITHELIAL CELLS BY A MEMBRANE POTENTIAL-SENSITIVE PROBE

Abstract

Methods to detect functional cystic fibrosis transmembrane conductance regulator (CFTR) are needed for the assessment of new therapies in cystic fibrosis (CF). We have combined patch-clamp and fluorimetric techniques to investigate whether the fluorescent voltage-sensitive probe bis-(1,3-diethylthiobarbituric acid) trimethine oxonol (DiSBAC 2 (3)) discriminates between changes of membrane potential (V m) evoked by cAMP in CF and CFTR-corrected epithelial cells. About 60% of the CFTR-corrected cells increased their membrane conductance and depolarized in response to cAMP, as compared to about 20% of CF cells. CFTR was found to contribute only to a fraction of the cAMP-induced responses, as indicated by the differential effects of Cl - channel blockers. Simultaneous recording of fluorescence (Δ F) and membrane potential revealed that Δ F detected V m changes as small as 10 mV. The relationship between Δ F and Δ V m, however, was not proportional. When a large number of cells were analyzed by digital imaging, an increase in Δ F in response to cAMP was detected in the majority of CFTR-corrected cells, but only in a small proportion of CF cells. The results indicate that the DiSBAC 2 (3) approach is a valid tool to compare cell populations with different proportions of cells responding to CFTR activation by cAMP. It cannot be used, however, for quantitative assessment of functional CFTR in individual CF cells.