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 Cu2+ on human ENaC expressed in Xenopus oocytes and investigated the structural basis for its effects. External Cu2+ inhibited human alpha beta gamma ENaC with an estimated IC50 of 0.3 mu M. 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 Cu2+. Experiments with mixed human and mouse ENaC subunits suggested that both the alpha and beta subunits were primarily responsible for the inhibitory effect of Cu2+ on human ENaC. Lowering bath solution pH diminished the inhibition by Cu2+. Mutations of two alpha, two beta, and two gamma His residues within extracellular domains significantly reduced the inhibition of human ENaC by Cu2+. We identified a pair of residues as potential Cu2+-binding sites at the subunit interface between thumb subdomain of alpha hENaC and palm subdomain of beta hENaC, suggesting a counterclockwise arrangement of alpha, beta, and gamma ENaC subunits in a trimeric channel complex when viewed from above. We conclude that extracellular Cu2+ is a potent inhibitor of human ENaC and binds to multiple sites within the extracellular domains including a subunit interface.