Chloride transport is involved in a wide variety of cellular functions, including regulation of the membrane potential, cell volume, acid base balance, and epithelial fluid absorption and secretion. Alterations in chloride transport mechanisms are related to a number of physio-pathological conditions. The aim of this study was to investigate chloride transport across the plasma membrane of cervical cancer cell lines (HeLa), relying on the halide binding properties of a green (GFP) fluorescent protein variant (YFP) that, if halide complexed, decreases the basal fluorescence intensity. The fluorescence changes of HeLa cells expressing YFP was analyzed by confocal and spectrofluorometric analysis. Our results highlights the presence of a transmembrane halide transport through fluorescence quenching when the transfected cells were exposed to NaI, used as a surrogate ion, under isotonic conditions. This quenching was inhibited by bumetanide, a specific inhibitor of Na+-K+-2Cl- cotransporter, demonstrating the role of such transporter in intracellular halide absorption and allowing cellular fluorescence assay for a functional study of Na+-K+-2Cl-. The kinetic parameters of the transporter were quantified. The present work provides an alternative approach for the measurement and characterization of Cl- and halide transport in living cells expressing YFP-H148Q/I152L based on a non-invasive analysis method.
Cloride transport in HeLa cells: a study by a recombinant cell-based assay in living cells
GIORDANO, Maria Elena;LIONETTO, Maria Giulia;CARICATO, Roberto;SCHETTINO, Trifone
2017-01-01
Abstract
Chloride transport is involved in a wide variety of cellular functions, including regulation of the membrane potential, cell volume, acid base balance, and epithelial fluid absorption and secretion. Alterations in chloride transport mechanisms are related to a number of physio-pathological conditions. The aim of this study was to investigate chloride transport across the plasma membrane of cervical cancer cell lines (HeLa), relying on the halide binding properties of a green (GFP) fluorescent protein variant (YFP) that, if halide complexed, decreases the basal fluorescence intensity. The fluorescence changes of HeLa cells expressing YFP was analyzed by confocal and spectrofluorometric analysis. Our results highlights the presence of a transmembrane halide transport through fluorescence quenching when the transfected cells were exposed to NaI, used as a surrogate ion, under isotonic conditions. This quenching was inhibited by bumetanide, a specific inhibitor of Na+-K+-2Cl- cotransporter, demonstrating the role of such transporter in intracellular halide absorption and allowing cellular fluorescence assay for a functional study of Na+-K+-2Cl-. The kinetic parameters of the transporter were quantified. The present work provides an alternative approach for the measurement and characterization of Cl- and halide transport in living cells expressing YFP-H148Q/I152L based on a non-invasive analysis method.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
