Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry

Cystic fibrosis (CF) is a frequent autosomal recessive disease caused by mutations that impair the CF transmembrane conductance regulator (CFTR) protein function. CFTR is a chloride channel activated by cyclic AMP (CAMP) via protein kinase A (PKA) and ATP hydrolysis. We describe here a method to measure CFTR activity in a monolayer of cultured cells using a fluorescence spectrophotometer and the chloride-sensitive probe 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Modifying a slice holder, the spectrophotometer quartz cuvette was converted in a perfusion chamber, allowing measurement of CFTR activity in real time, in a monolayer of T84 colon carcinoma cells. The SPQ Stern-Volmer constant (K(Cl-)) for chloride in water solution was 115.0 +/- 2.8 M(-1), whereas the intracellular K(Cl-) was 17.8 +/- 0.8 M(-1) for T84 cells. A functional analysis was performed by measuring CFTR activity in T84 cells. The CFTR transport inhibitors CFT'R(inh)-172 (5 mu M) and glibenclamide (100 mu M) showed a significant reduction (P < 0.05) in CFTR activity. This simple method allows measuring CFTR activity in a very simple, reproducible, and sensitive way. (C) 2011 Elsevier Inc. All rights reserved.