Role of the C-terminal part of the extracellular domain of the alpha-ENaC in activation by sulfonylurea glibenclamide.

The epithelial sodium channel (ENaC) is regulated by hormones and by other intracellular or extracellular factors. It is activated by the sulfonylurea drug glibenclamide. The activator effect of glibenclamide is fast and reversible and was observed in Xenopus oocytes coexpressing the alpha subunit from human, Xenopus, or guinea pig (but not rat) with betagamma-rat ENaC subunits. The mechanism of this effect is not yet well understood. We hypothesize that the extracellular loop of ENaC plays a major role in this activation. Mutants and chimeras of alpha subunits harboring different parts of the rat and guinea pig alpha-subunit extracellular loops were generated and coexpressed with betagamma-rat subunits in Xenopus oocytes. The effect of glibenclamide on ENaC activity was measured using two-electrode voltage-clamp technique. The alpha-rat ENaC chimera containing the C-terminal part of the extracellular loop of the alpha-guinea pig ENaC was significantly stimulated by glibenclamide (1.26-fold), whereas the rat-alpha combination was not activated by this sulfonylurea. Mutagenesis of specific residues on the rat alpha subunit did not generate channels sensitive to glibenclamide, suggesting that the overall structure of the extracellular loop is required for activation of the channel by this drug. These results support the hypothesis of the existence of a role played by the last 100 amino acids of the extracellular loop and confirm that the ENaC behaves as a ligand-gated channel similar to several other members of the ENaC/degenerin family.