Characterization and localization of epithelial Na+ channels in toad urinary bladder.
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.
Kleyman
T R
TR
Department of Medicine, University of Pennsylvania, Philadelphia.
Smith
P R
PR
Benos
D J
DJ
D000584:Amiloride / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002024:Bufo marinus; D004848:Epithelium / Q000378:metabolism; D015222:Sodium Channels / Q000378:metabolism*; D014018:Tissue Distribution; D001743:Urinary Bladder / Q000378:metabolism*
D015222:Sodium Channels; C013407:benzamil; C076676:2'-methoxy-5'-nitrobenzamil; D000584:Amiloride; C064150:6-bromobenzamil