Alberola-Die Armando, Fernández-Ballester Gregorio, González-Ros José M, Ivorra Isabel, Morales Andrés
División de Fisiología, Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante Alicante, Spain.
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández Alicante, Spain.
Front Mol Neurosci. 2016 Feb 15;9:12. doi: 10.3389/fnmol.2016.00012. eCollection 2016.
Lidocaine bears in its structure both an aromatic ring and a terminal amine, which can be protonated at physiological pH, linked by an amide group. Since lidocaine causes multiple inhibitory actions on nicotinic acetylcholine receptors (nAChRs), this work was aimed to determine the inhibitory effects of diethylamine (DEA), a small molecule resembling the hydrophilic moiety of lidocaine, on Torpedo marmorata nAChRs microtransplanted to Xenopus oocytes. Similarly to lidocaine, DEA reversibly blocked acetylcholine-elicited currents (I ACh ) in a dose-dependent manner (IC 50 close to 70 μM), but unlike lidocaine, DEA did not affect I ACh desensitization. I ACh inhibition by DEA was more pronounced at negative potentials, suggesting an open-channel blockade of nAChRs, although roughly 30% inhibition persisted at positive potentials, indicating additional binding sites outside the pore. DEA block of nAChRs in the resting state (closed channel) was confirmed by the enhanced I ACh inhibition when pre-applying DEA before its co-application with ACh, as compared with solely DEA and ACh co-application. Virtual docking assays provide a plausible explanation to the experimental observations in terms of the involvement of different sets of drug binding sites. So, at the nAChR transmembrane (TM) domain, DEA and lidocaine shared binding sites within the channel pore, giving support to their open-channel blockade; besides, lidocaine, but not DEA, interacted with residues at cavities among the M1, M2, M3, and M4 segments of each subunit and also at intersubunit crevices. At the extracellular (EC) domain, DEA and lidocaine binding sites were broadly distributed, which aids to explain the closed channel blockade observed. Interestingly, some DEA clusters were located at the α-γ interphase of the EC domain, in a cavity near the orthosteric binding site pocket; by contrast, lidocaine contacted with all α-subunit loops conforming the ACh binding site, both in α-γ and α-δ and interphases, likely because of its larger size. Together, these results indicate that DEA mimics some, but not all, inhibitory actions of lidocaine on nAChRs and that even this small polar molecule acts by different mechanisms on this receptor. The presented results contribute to a better understanding of the structural determinants of nAChR modulation.
利多卡因的结构中既有芳香环又有末端胺基，在生理pH值下可被质子化，二者通过酰胺基相连。由于利多卡因对烟碱型乙酰胆碱受体（nAChRs）具有多种抑制作用，本研究旨在确定二乙胺（DEA）这种类似于利多卡因亲水部分的小分子对微移植到非洲爪蟾卵母细胞中的斑纹电鳐nAChRs的抑制作用。与利多卡因类似，DEA以剂量依赖性方式可逆地阻断乙酰胆碱引发的电流（I ACh）（IC50接近70 μM），但与利多卡因不同的是，DEA不影响I ACh脱敏。DEA对I ACh的抑制在负电位时更为明显，提示其对nAChRs的开放通道阻滞作用，尽管在正电位时仍有大约30%的抑制作用，表明孔外存在其他结合位点。与单独将DEA和乙酰胆碱共同应用相比，在将DEA与乙酰胆碱共同应用之前预先施加DEA时I ACh抑制作用增强，这证实了DEA对静息状态（关闭通道）下nAChRs的阻滞作用。虚拟对接分析从不同药物结合位点的参与角度为实验观察结果提供了合理的解释。因此，在nAChR跨膜（TM）结构域，DEA和利多卡因在通道孔内共享结合位点，支持了它们的开放通道阻滞作用；此外，利多卡因而非DEA与每个亚基的M1、M2、M3和M4片段之间的腔以及亚基间缝隙处的残基相互作用。在细胞外（EC）结构域，DEA和利多卡因的结合位点广泛分布，这有助于解释所观察到的关闭通道阻滞作用。有趣的是，一些DEA簇位于EC结构域的α-γ界面，在正构结合位点口袋附近的一个腔内；相比之下，利多卡因与构成ACh结合位点的所有α亚基环接触，包括α-γ和α-δ界面以及相间区域，这可能是由于其分子尺寸较大。总之，这些结果表明DEA模拟了利多卡因对nAChRs的部分而非全部抑制作用，并且即使是这种小的极性分子对该受体的作用机制也不同。所呈现的结果有助于更好地理解nAChR调节的结构决定因素。
