Photoaffinity labeling of the epithelial sodium channel.
Sodium enters tight epithelia across the apical plasma membrane through a sodium channel, a process inhibited by submicromolar concentrations of amiloride and benzamil. Using membrane vesicles from bovine kidney cortex, we found that sodium transport through the sodium channel was inhibited by benzamil with an IC50 of 4 nM. Amiloride (IC50 = 400 nM) was a weaker inhibitor of sodium transport. [3H]Benzamil bound to the vesicles at a single class of high affinity binding sites with a Kd of 5 nM, the similarity of which to the IC50 suggests that these binding sites are associated with the sodium channel. Amiloride displaced bound [3H]benzamil with a Ki of 2,500 nM. Bromobenzamil is a photoactive amiloride analog with potency similar to benzamil in inhibiting sodium transport (IC50 = 5 nM) and binding to the sodium channel (Kd = 6 nM). [3H]Bromobenzamil was specifically photoincorporated into three molecular weight classes of polypeptides with apparent Mr values of 176,000, 77,000, and 47,000. The photoincorporation of [3H]bromobenzamil into these three classes of polypeptides was blocked by addition of excess benzamil and by amiloride in a dose-dependent manner. These data suggest that these polypeptides are components of the epithelial sodium channel.
T R
TR
Kleyman
T
T
Yulo
C
C
Ashbaugh
D
D
Landry
E
E
Cragoe
A
A
Karlin
Q
Q
Al-Awqati
D000345:Affinity Labels / Q000378:metabolism*; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism*; D007672:Kidney Cortex / Q000378:metabolism; D007700:Kinetics; D010777:Photochemistry; D012964:Sodium / Q000378:metabolism*
D000345:Affinity Labels; D007473:Ion Channels; C013407:benzamil; D000584:Amiloride; D012964:Sodium