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, 
, and 
Subunits of the
Epithelial Sodium Channel (ENaC) Involved in Amiloride Block and
Ion Permeation
From the Institut de Pharmacologie et de Toxicologie, Université de Lausanne, 1005 Lausanne, Switzerland
The amiloride-sensitive epithelial Nachannel (ENaC) is a heteromultimeric channel made of three
subunits. The structures involved in the ion permeation pathway have only been partially identified, and the
respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel
block by amiloride. Cys substitutions of Gly residues in and subunits at position G525 and G537 increased
the apparent inhibitory constant (Ki) for amiloride by >1,000-fold and decreased channel unitary current without
affecting ion selectivity. The corresponding mutation S583 to C in the subunit increased amiloride Ki by 20-fold,
without changing channel conducting properties. Coexpression of these mutated subunits resulted in a nonconducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by externalZn2+ ions, in particular the S583C mutant showing a Ki for Zn2+of 29 µM. Mutations of residues W582L or G522D also increased amiloride Ki, the later mutation generating a Ca2+blocking site located
15% within the membrane electric field. These experiments provide strong evidence that ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of subunits may
form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na+ions at an external Na+binding site preventing ion permeation through the channel pore.
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