Structure-activity relationship of amiloride analogs as blockers of epithelial Na channels: II. Side-chain modifications.

The overall on- and off-rate constants for blockage of epithelial Na channels by amiloride analogs were estimated by noise analysis of the stationary Na current traversing frog skin epithelium. The (2-position) side chain structure of amiloride was varied in order to obtain structure/rate constant relationships. Hydrophobic chain elongations (benzamil and related compounds of high blocking potency) increase the stability of the blocking complex (lowered off-rate), explained by attachment of the added phenyl moiety to a hydrophobic area near the site of side chain interaction with the channel protein. Some other chain modifications show that the on-rate, which is smaller than a diffusion-limited rate, varies with side chain structure. In several cases this effect is not attributable to steric hindrance on encounter, and implies that the side chain interacts briefly with the channel protein (encounter complex) before the main blocking position of the molecule is attained. The encounter complex must be labile since the overall rate constants of blockage are not concentration-dependent. In two cases, changes at the 2-position side chain and at other ring ligands, with known effects on the blocking rate constants, could be combined in one analog. The rate constants of blocking by the resulting compounds indicate that the structural changes have additive effects in terms of activation energies. Along with other observations (voltage dependence of the rate constants and competition with the transported Na ion), these results suggest a blocking process of at least two steps. It appears that initially the 2-position side chain invades the outward-facing channel entrance, establishing a labile complex. Then the molecule is either released completely (no block) or the 6-ligand of the pyrazine ring gains access to its receptor counterpart, thus establishing the blocking complex, the lifetime of which is strongly determined by the electronegativity of the 6-ligand.