IDENTIFICATION OF AMINO-ACID-RESIDUES IN THE ALPHA-SUBUNIT, BETA-SUBUNIT, AND GAMMA-SUBUNIT OF THE EPITHELIAL SODIUM-CHANNEL (ENAC) INVOLVED IN AMILORIDE BLOCK AND ION PERMEATION

The amiloride-sensitive epithelial Na channel (ENaC) is a heteromultim eric channel made of three alpha beta gamma subunits. The structures i nvolved in the ion permeation pathway have only been partially identif ied, and the respective contributions of each subunit in the formation of the conduction pore has not pet been established. Using a site-dir ected mutagenesis approach, we have identified in a short segment prec eding the second membrane-spanning domain (the pre-M2 segment) amino a cid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in beta and gamma sub units at position beta G525 and gamma G537 increased the apparent inhi bitory constant (K-i) for amiloride by >1,000-fold and decreased chann el unitary current without affecting ion selectivity. The correspondin g mutation S583 to C in the alpha subunit increased amiloride K-i by 2 0-fold, without changing channel conducting properties. Coexpression o f these mutated alpha beta gamma subunits resulted in a nonconducting channel expressed at the cell surface. Finally, these Cys substitution s increased channel affinity for block by external Zn2+ ions, in parti cular the alpha S583C mutant showing a K-i for Zn2+ of 29 mu M. Mutati ons of residues alpha W582L or beta G522D also increased amiloride K-i , the later mutation generating a Ca2+ blocking site located 15% withi n the membrane electric field. These experiments provide strong eviden ce that alpha beta gamma ENaCs are pore-forming subunits involved in i on permeation through the channel. The pre-M2 segment of alpha beta ga mma subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We pro pose that amiloride interacts with Na+ ions at an external Na+ binding site preventing ion permeation through the channel pore.