A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel

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Abstract
Pseudohypoaldosteronism type 1 (PHA-1) is an inherited disease characterized by severe neonatal salt-wasting and caused by mutations in subunits of the amiloride-sensitive epithelial sodium channel (ENaC), A missense mutation (G37S) of the human ENaC beta subunit that causes loss of ENaC function and PHA-1 replaces a glycine that is conserved in the N-terminus of all members of the ENaC gene family, We now report an investigation of the mechanism of channel inactivation by this mutation. Homologous mutations, introduced into alpha, beta or gamma subunits, all significantly reduce macroscopic sodium channel currents recorded in Xenopus laevis oocytes, Quantitative determination of the number of channel molecules present at the cell surface showed no significant differences in surface expression of mutant compared,vith wild-type channels. Single channel conductances and ion selectivities of the mutant channels were identical to that of wildtype, These results suggest that the decrease in macroscopic Na currents is due to a decrease in channel open probability (P-o), suggesting that mutations of a conserved glycine in the N-terminus of ENaC subunits change ENaC channel gating, which would explain the disease pathophysiology, Single channel recordings of channels containing the mutant or,subunit (alpha G95S) directly demonstrate a striking reduction in P-o. We propose that this mutation favors a gating mode characterized by short-open and long-closed times, We suggest that determination of the gating mode of ENaC is a key regulator of channel activity.