Identification of a highly conserved sequence at the N-terminus of the epithelial Na+ channel α subunit involved in gating

The epithelial Na+ channel (ENaC) is responsible for Na+ reabsorption in aldosterone target tissues such as distal nephron and colon. ENaC is a heterotetramer composed of three homologous subunits, alpha, beta, and gamma ENaC. Mutations leading to loss of function or reduced channel activity have been identified in all three subunits in patients with pseudohypoaldosteronism type-1. One missense mutation substituting a glycine (G95S) which is completely conserved throughout the gene family reduced ENaC open probability, P-o. In this study we have performed systematic alanine substitutions of 28 residues of alpha ENaC encompassing the glycine (G95). This screen identified a stretch of ten consecutive amino acids (alpha T92-alpha C101) which, when mutated, lead to a decrease in Na+ current (I-Na) expressed with no significant changes in channel surface expression. This inhibitory effect was strongest for G95 and for two additional highly conserved amino acids - H94 and R98. The R98A mutant led to an important reduction in channel P-o with no change in single-channel conductance, indicating that the segment encompassing H94, G95 and R98 is involved in modulation of channel gating kinetics.