On the interaction between amiloride and its putative alpha-subunit epithelial Na+ channel binding site

Area of Science:

• Ion channel physiology
• Molecular biology
• Biophysics

Background:

• The epithelial sodium channel (ENaC) is crucial for sodium transport and belongs to the ENaC/Degenerin superfamily.
• Amiloride and its analogs are known blockers of ENaC, with specific amino acid residues implicated in binding.
• Previous studies identified alphaSer-583, betaGly-525, and gammaGly-542 as key residues for amiloride binding.

Purpose of the Study:

• To investigate the precise role of alphaSer-583 in amiloride binding to ENaC.
• To understand how mutations at alphaSer-583 affect amiloride's inhibitory effects and channel gating.
• To elucidate the structural and functional relationship between alphaSer-583 and amiloride interaction.

Main Methods:

• Systematic mutagenesis of the alphaSer-583 residue in ENaC.
• Two-electrode voltage clamp electrophysiology to analyze mutant channel function.
• Assessment of amiloride binding affinities (K(i)) and voltage dependence of channel gating.

Main Results:

• Most mutations at alphaSer-583 resulted in moderate alterations in amiloride block.
• Mutations introducing ring structures at alpha583 dramatically weakened amiloride block.
• Introduction of a beta-methyl group at alpha583 altered ENaC's electric field, impacting amiloride binding and gating.
• The His mutation at alpha583 significantly weakened amiloride binding and introduced pH-dependent voltage-sensitive gating.

Conclusions:

• The wild-type serine side chain at alpha583 is likely not critical for amiloride binding affinity.
• Amiloride binds at or near the alphaSer-583 site.
• alphaSer-583 plays a role in ion permeation through ENaC, with specific mutations influencing channel gating and electrical properties.