Department of Anatomy, Animal Physiology and Biophysics, Faculty of Biology, University of Bucharest, 91-95 Splaiul Independenței Str., 050095 Bucharest, Romania.
Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Bucharest, 91-95 Splaiul Independenței Str., 050095 Bucharest, Romania.
Molecules. 2022 May 19;27(10):3271. doi: 10.3390/molecules27103271.
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 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.
太赫兹光谱学对于研究离子通道很重要，因为它直接涉及到与其功能相关的低频集体运动。在这里，我们使用太赫兹光谱学来研究上皮钠通道（ENaC）的特异性抑制剂阿米洛利的抑制作用。实验在悬浮的 A6 细胞上进行，这些细胞是过表达 ENaC 的细胞，来源于肾脏。研究了有或没有阿米洛利的太赫兹光谱。当 ENaC 被阿米洛利抑制时，太赫兹吸收明显增加。使用分子建模方法解释了观察到的光谱差异。使用这里构建的 ENaC 和 ENaC-阿米洛利复合物的结构模型模拟了太赫兹光谱。实验与模拟之间的一致性使我们能够验证结构模型，并描述通道孔内阿米洛利的动力学。使用太赫兹光谱学验证的阿米洛利结合位点与先前的突变研究一致。总之，我们的结果表明，太赫兹光谱学可以成功地用于区分天然和抑制的 ENaC 通道，并描述在其特异性拮抗剂存在下通道的动力学。
