External Cu2+ inhibits human epithelial Na+ channels by binding at a subunit interface of extracellular domains

Epithelial Na(+) channels (ENaCs) play an essential role in the regulation of body fluid homeostasis. Certain transition metals activate or inhibit the activity of ENaCs. In this study, we examined the effect of extracellular Cu(2+) on human ENaC expressed in Xenopus oocytes and investigated the structural basis for its effects. External Cu(2+) inhibited human αβγ ENaC with an estimated IC(50) of 0.3 μM. The slow time course and a lack of change in the current-voltage relationship were consistent with an allosteric (non pore-plugging) inhibition of human ENaC by Cu(2+). Experiments with mixed human and mouse ENaC subunits suggested that both the α and β subunits were primarily responsible for the inhibitory effect of Cu(2+) on human ENaC. Lowering bath solution pH diminished the inhibition by Cu(2+). Mutations of two α, two β, and two γ His residues within extracellular domains significantly reduced the inhibition of human ENaC by Cu(2+). We identified a pair of residues as potential Cu(2+)-binding sites at the subunit interface between thumb subdomain of αhENaC and palm subdomain of βhENaC, suggesting a counterclockwise arrangement of α, β, and γ ENaC subunits in a trimeric channel complex when viewed from above. We conclude that extracellular Cu(2+) is a potent inhibitor of human ENaC and binds to multiple sites within the extracellular domains including a subunit interface.