title: Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry.
abstract: 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 ± 2.8M(-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 CFTR(inh)-172 (5 μM) and glibenclamide (100 μM) 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.
authors.affiliation: Program for Biomedical Research, Laboratory of Cellular and Molecular Biology, School of Medicine, Pontifical Catholic University of Argentina and National Research Council, Buenos Aires, Argentina.
authors.forename: Ángel G
authors.initials: ÁG
authors.lastname: Valdivieso
authors.forename: María C
authors.initials: MC
authors.lastname: Marín
authors.forename: Mariángeles
authors.initials: M
authors.lastname: Clauzure
authors.forename: Tomás A
authors.initials: TA
authors.lastname: Santa-Coloma
mesh_terms: D002478:Cells, Cultured; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000378:metabolism; D003550:Cystic Fibrosis / Q000378:metabolism / Q000473:pathology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000032:analysis* / Q000378:metabolism; D005905:Glyburide / Q000494:pharmacology; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology; D011791:Quartz; D011806:Quinolinium Compounds / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D018118:Chloride Channels; D007004:Hypoglycemic Agents; D011806:Quinolinium Compounds; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D011791:Quartz; C052335:6-methoxy-N-(3-sulfopropyl)quinolinium; D005905:Glyburide