Epithelial Sodium Channel Inhibition by Amiloride Addressed with THz Spectroscopy and Molecular Modeling
Maria Mernea,
Roxana Ștefania Ulăreanu,
Dana Cucu,
Jasim Hafedh Al-Saedi,
Cristian-Emilian Pop,
Sergiu Fendrihan,
Giorgiana Diana Carmen Anghelescu,
Dan Florin Mihăilescu
Affiliations
Maria Mernea
Department of Anatomy, Animal Physiology and Biophysics, Faculty of Biology, University of Bucharest, 91–95 Splaiul Independenței Str., 050095 Bucharest, Romania
Roxana Ștefania Ulăreanu
Department of Anatomy, Animal Physiology and Biophysics, Faculty of Biology, University of Bucharest, 91–95 Splaiul Independenței Str., 050095 Bucharest, Romania
Dana Cucu
Department of Anatomy, Animal Physiology and Biophysics, Faculty of Biology, University of Bucharest, 91–95 Splaiul Independenței Str., 050095 Bucharest, Romania
Jasim Hafedh Al-Saedi
Department of Anatomy, Animal Physiology and Biophysics, Faculty of Biology, University of Bucharest, 91–95 Splaiul Independenței Str., 050095 Bucharest, Romania
Cristian-Emilian Pop
Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Bucharest, 91–95 Splaiul Independenței Str., 050095 Bucharest, Romania
Sergiu Fendrihan
Non-Governmental Research Organization Biologic, 14 Schitului Str., 032044 Bucharest, Romania
Giorgiana Diana Carmen Anghelescu
Department of Anatomy, Animal Physiology and Biophysics, Faculty of Biology, University of Bucharest, 91–95 Splaiul Independenței Str., 050095 Bucharest, Romania
Dan Florin Mihăilescu
Department of Anatomy, Animal Physiology and Biophysics, Faculty of Biology, University of Bucharest, 91–95 Splaiul Independenței Str., 050095 Bucharest, Romania
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.
