AJP - Cell Physiology, Vol 266, Issue 4 C1105-C1111, Copyright © 1994 by American Physiological Society
Characterization and localization of epithelial Na+ channels in toad urinary bladder
T. R. Kleyman, P. R. Smith and D. J. Benos
Department of Medicine, University of Pennsylvania, Philadelphia.
The toad urinary bladder and epithelial cell lines derived from the urinary
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 amiloride
analogue [3H]benzamil bound to a single class of high-affinity binding
sites in membrane vesicles from toad urinary bladder with a dissociation
constant (Kd) 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 recognized a 135,000-Da polypeptide
in TBM cells. Polyclonal anti-Na+ channel antibodies generated against
purified bovine epithelial Na+ channel specifically recognized a 235,000-Da
polypeptide in toad urinary bladder and localized Na+ channels to the
apical plasma membrane of urinary bladder epithelial cells. The biochemical
characteristics and the cellular localization of epithelial Na+ channels in
toad urinary bladder are similar to those previously described in mammalian
kidney and in the A6 cell line.
