Stereoselective blockade of amphibian epithelial sodium channels by amiloride analogs

The blockade of amphibian epithelial sodium channels by amiloride has been shown to not be a simple diffusion-limited encounter but rather a selective binding process with the rate of approach of the molecule for the channel site determined by the side chain at position 2 of the pyrazine ring and the stability of the blocking complex provided largely by the bond of the ligand at position 6. The presence of such a putative channel-associated receptor suggests that the sodium channel might have chiral recognition for its blockers. Using the short-circuit technique to measure the amiloride-sensitive sodium current through the apical membrane of the toad urinary bladder and frog skins, we evaluated the sodium channel-blocking potency of three enantiomeric pairs of amiloride analogs with chiral centers on the side chain. We demonstrated the stereoselectivity in sodium channel blockade of those analogs and confirmed the existence of a second attachment site on the channel for interacting with the lipophilic ligand on the side chain of the analog molecule to enhance its blocking activity. Depending on the separation between the chiral carbon and the pyrazine ring and the chemical groups on the chiral carbon, the eudismic ratio for the 50% inhibition constant of the enantiomers, i.e., the ratio of the sodium channel-blocking potency of the more active enantiomer to that of the less active enantiomer, was found to range from slightly more than 1 to more than 100.