Abstract

The epithelial Na⁺ channel ENaC mediates transepithelial Na⁺ transport in the distal kidney, the colon, and the lung and is a key element for the maintenance of Na⁺ balance and the regulation of blood pressure. Mutagenesis studies have identified residues αS583 and the homologous βG525 and γG537 in the outer pore entrance that are critical for ENaC block by the K⁺-sparing diuretic amiloride. The aim of the present study was to determine first, whether these residues are part of the amiloride binding site, and second, whether they are general determinants of ENaC block by amiloride and its derivatives. Kinetic analysis of the association and dissociation rates of amiloride and benzamil to ENaC showed that mutation of residue αS583C and the homologous βG525C increased the dissociation rate of the drugs from the binding site, with little changes in their association rate. Thus, these mutations destabilize the binding interaction between the blockers and the receptor on the channel, favoring the unbinding of the ligand. This strongly suggests that they are part of the binding site. Because mutations of αS583, βG525, and γG537 have similar effects on amiloride, benzamil, and triamterene block, we conclude that these three ENaC blockers share a common receptor within the ion channel pore.