Abstract

The amiloride-sensitive epithelial Na⁺ channel (ENaC) regulates Na⁺ homeostasis in cells and across epithelia. Four homologous ENaC subunits (α, β, γ, and δ) have been isolated in mammals. The chemical activators acting on ENaC, however, are largely unknown. More recently, we have found that capsazepine activates human ENaCδ (hENaCδ), which is mainly expressed in the brain. In addition, here we show that icilin, which is a tetrahydropyrimidine-2-one derivative unrelated structurally to capsazepine, markedly enhanced the activity of hENaCδβγ heteromultimer expressed in Xenopus laevis oocytes. The inward currents at a holding potential of -60 mV in hENaCδβγ-expressing oocytes were increased by the application of icilin in a concentration-dependent manner with an EC₅₀ value of 33 μM. The icilin-elicited current was mostly abolished by the addition of 100 μM amiloride or by the removal of external Na⁺. Homomeric hENaCδ was also significantly activated by icilin, whereas hENaCα activity was not affected by icilin, and icilin caused a slight inhibition of the hENaCαβγ current. Furthermore, icilin acted together with protons or capsazepine on hENaCδβγ. These findings identify icilin as a novel chemical activator of ENaCδ, providing us with a lead compound for drug development in the degenerin/ENaC superfamily.