EFFECT OF CELL SWELLING ON MEMBRANE AND CYTOPLASMIC DISTRIBUTION OF PI(CLN)

pI(Cln) is found ubiquitously in mammalian cells and is postulated to play a critical role in cell volume regulation. Mutagenesis studies le d to the proposal that pI(Cln) is a swelling-activated anion channel. However, recent studies in Madin-Darby canine kidney cells and endothe lial cells have shown that the protein is localized primarily to the c ytoplasm. It has therefore been postulated that activation involves re versible translocation of pI(Cln) from the cytoplasm and insertion int o the plasma membrane. We tested this hypothesis using several differe nt approaches. Fractionation of C-6 glioma cells into plasma membrane- and cytoplasm-containing fractions demonstrated that similar to 90% of the recovered pI(Cln), was confined to the cytosol. Swelling had no e ffect on the relative amount of protein present in the plasma membrane fraction. Immunofluorescence microscopy revealed that pI(Cln) is loca lized primarily, if not exclusively, to the cytoplasm of swollen and n onswollen cells. Similarly, transfection of cells with a green fluores cent protein-labeled pI(Cln) construct failed to reveal any membrane l ocalization of the protein. These findings do not support the hypothes is that pI(Cln) is a volume regulatory anion channel activated by swel ling-induced membrane insertion.