Binding of 3H-phenamil, an irreversible amiloride analog, to toad urinary bladder: effects of aldosterone and vasopressin.

Phenamil, an analog of amiloride, has previously been shown to bind specifically to sodium channels in toad bladder (J.L. Garvin et al., J. Membrane Biol. 87:45-54, 1985). In this paper, 3H-phenamil was used to measure sodium channel density in both isolated epithelial cells and intact bladders. From the specific binding to intact bladders, a channel density of 455 +/- 102 channels/micron2 was calculated. No correlation between specific binding and the magnitude of irreversible inhibition of short-circuit current was found. Pretreatment of intact bladders with 1 mg/ml trypsin reduced specific binding to isolated cells by 82 +/- 5%. In isolated cells, neither aldosterone nor vasopressin had any significant effect on specific phenamil binding. It is inferred that phenamil binds to both open and closed channels which may be either in the mucosal membrane or in the submembrane space. Finally, and rather surprisingly, we found that 3H-phenamil binds irreversibly to the basolateral membrane at concentrations as low as 4 X 10(-7) M. Therefore, care must be used in interpreting binding studies with amiloride or its analog at such concentrations.