Paradoxical stimulation of a DEG ENaC channel by amiloride

Extracellular amiloride inhibits all known DEG/ENaC ion channels, including BNC1, a proton-activated human neuronal cation channel. Earlier studies sh owed that protons cause a conformational change that activates BNC1 and exp oses residue 430 to the extracellular solution, Here we demonstrate that, i n addition to blocking BNC1, amiloride also exposes residue 430, This resul t suggested that, like protons, amiloride might be capable of activating th e channel. To test this hypothesis, we introduced a mutation in the BNC1 po re that reduces amiloride block, and found that amiloride stimulated these channels. Amiloride inhibition was voltage-dependent, suggesting block with in the pore, whereas stimulation was not, suggesting binding to an extracel lular site. These data show that amiloride can have two distinct effects on BNC1, and they suggest two different interaction sites. The results sugges t that extracellular amiloride binding may have a stimulatory effect simila r to that of protons in BNC1 or extracellular ligands in other DEG/ENaC cha nnels.