Discrimination between cystic fibrosis and CFTR-corrected epithelial cells by a membrane potential-sensitive probe.
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-diethylthiobarbituic anid) trimethine oxonol (Di5BAC2(3)) discriminates between changes of membrane potential (Vm evoked by cAMP in CF and CYFT-corrected epithelial cells. About 60% of the (FTR-correrced 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 reocording of fluorescence (AF) and membrane potential revealed that AF detected Vm changes as small as 10 mV . The relationship between deltaF and deltaVm however, was not proportional. When a large number of cells were analyzed by digital imaging, an increase in deltaF 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 DiSBAC2(3) approach is a valid tool to compare cell populations with different proportions of cells responding to CFTR arcivation by cAMP. It cannot be used, however, for quantitative assessment of functional CFTR in individual CF cells.
Coclet-Ninin
Joelle
J
Pulmonary Division, University Hospital of Geneva, Switzerland.
Rochat
Thierry
T
Poitry
Serge
S
Chanson
Marc
M
D000242:Cyclic AMP / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000473:pathology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology*; D004847:Epithelial Cells / Q000502:physiology*; D005456:Fluorescent Dyes; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000592:standards; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques; D012680:Sensitivity and Specificity; D013858:Thiobarbiturates; D014407:Tumor Cells, Cultured
C505032:CFTR protein, human; D005456:Fluorescent Dyes; D013858:Thiobarbiturates; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C025284:bis(1,3-diethylthiobarbiturate)trimethineoxonol; D000242:Cyclic AMP