Epithelial Sodium Channel Inhibition by Amiloride Addressed with THz Spectroscopy and Molecular Modeling

Molecules. 2022 May 19;27(10):3271. doi: 10.3390/molecules27103271.

Abstract

THz spectroscopy is important for the study of ion channels because it directly addresses the low frequency collective motions relevant for their function. Here we used THz spectroscopy to investigate the inhibition of the epithelial sodium channel (ENaC) by its specific blocker, amiloride. Experiments were performed on A6 cells' suspensions, which are cells overexpressing ENaC derived from Xenopus laevis kidney. THz spectra were investigated with or without amiloride. When ENaC was inhibited by amiloride, a substantial increase in THz absorption was noticed. Molecular modeling methods were used to explain the observed spectroscopic differences. THz spectra were simulated using the structural models of ENaC and ENaC-amiloride complexes built here. The agreement between the experiment and the simulations allowed us to validate the structural models and to describe the amiloride dynamics inside the channel pore. The amiloride binding site validated using THz spectroscopy agrees with previous mutagenesis studies. Altogether, our results show that THz spectroscopy can be successfully used to discriminate between native and inhibited ENaC channels and to characterize the dynamics of channels in the presence of their specific antagonist.

Keywords: THz spectroscopy; amiloride binding; epithelial sodium channel; ion channel; spectra simulation.

MeSH terms

  • Amiloride* / metabolism
  • Amiloride* / pharmacology
  • Animals
  • Epithelial Sodium Channels* / genetics
  • Epithelial Sodium Channels* / metabolism
  • Oocytes / metabolism
  • Spectrum Analysis
  • Xenopus laevis / metabolism

Substances

  • Epithelial Sodium Channels
  • Amiloride