CHARACTERIZATION AND LOCALIZATION OF EPITHELIAL NA-BLADDER( CHANNELS IN TOAD URINARY)

The toad urinary bladder and epithelial cell lines derived from the ur inary bladder, including TBM, serve as model systems for the study of transepithelial Na+ transport. We examined biochemical characteristics of epithelial Na+ channels in toad urinary bladder and TBM cells and their cellular localization in the urinary bladder. The radiolabeled a miloride analogue [H-3]benzamil bound to a single class of high-affini ty binding sites in membrane vesicles from toad urinary bladder with a dissociation constant (K-d) Of 10 nM. Photoactive benzamil analogues specifically labeled a 135,000-Da polypeptide in toad urinary bladder and TBM cells. A monoclonal anti-Na+ channel antibody directed against the amiloride-binding component of the channel specifically recognize d a 135,000-Da polypeptide in TBM cells. Polyclonal anti-Na+ channel a ntibodies generated against purified bovine epithelial Na+ channel spe cifically recognized a 235,000-Da polypeptide in toad urinary bladder and localized Na+ channels to the apical plasma membrane of urinary bl adder epithelial cells. The biochemical characteristics and the cellul ar localization of epithelial Na+ channels in toad urinary bladder are similar to those previously described in mammalian kidney and in the A6 cell line.