Biophysical Approach to Determine the Subunit Stoichiometry of the Epithelial Sodium Channel Using t

The amiloride-sensitive epithelial Na+ channel (ENaC) is typically composed of three structurally related subunits termed α, β, and γ. We describe methods to determine the functional subunit stoichiometry of ENaC based on a biophysical approach that was first introduced in 1991 to determine the subunit stoichiometry of a voltage-gated K+ channel. The strategy is to analyze channel sensitivity to a specific blocker when various mixtures of block-sensitive and blocker-insensitive subunits are coexpressed in a heterologous expression system. Details related to the expression of wild type and mutant ENaCs in Xenopus oocytes and the examination of blocker sensitivity by two-electrode voltage clamp, as well as analysis of data are provided.