Ren Yanyan, Pan Lanying, Zhao Wei, Xu Lei, Xu Jianwei, Lu Haiyin, Chen Yuan
Chinese Herb Medicine Division, Zhejiang Agriculture and Forestry University, 666 Wusu street, Lin'an 311300, PR China; The State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, 88 North Circle Road, Lin'an 311300, PR China.
Institute of Bioinformatics and Medical Engineering, School of Electrical and Information Engineering, Jiangsu University of Technology, Changzhou 213001, PR China.
Brain Res. 2018 Mar 15;1683:17-26. doi: 10.1016/j.brainres.2018.01.013. Epub 2018 Jan 31.
Stellera chamaejasme L. (Thymelaeaceae) is a toxic perennial herb and widespread in Mongolia and the northern parts of China. Previous studies have revealed that Neochamaejasmin A (NCA), one of the main active ingredients in the plant roots, has many bioactivities such as inhibiting the P-gp-mediated efflux. But whether NCA affects ion channels is unknown. Here the whole cell patch clamp technique was used to investigate whether NCA affects ion channels, especially how it inhibits K1.4. Mutagenesis and structure-based molecular simulation were used for analysis of inhibition mechanism and identification of binding site. Among all the channels assayed, K1.4 stood out as the one on which NCA showed strongest inhibition activity with IC of 7.55 µM. Compared with NCA's isomerides, neochamaejasmin B (NCB) and chamaechromone (CMC), NCA also exhibited superior inhibition ability on K1.4. Three mutations, V549A, A553V and V560A, occurred inside the pore, were found to significantly alleviate the NCA blocking effects, suggesting that they are the important binding sites of NCA. Structure-based modelling showed that the phenolic hydroxyl group of NCA can form hydrogen bonds with main chains of Val549 and Ala553 in IS6 and IVS6 segment respectively, which support our in vitro results. In conclusion, data suggest that NCA might inhibit K1.4 channels via direct binding to the pore domain.
狼毒（瑞香科）是一种有毒的多年生草本植物，广泛分布于蒙古和中国北方。先前的研究表明，该植物根部的主要活性成分之一新狼毒素A（NCA）具有多种生物活性，如抑制P-糖蛋白介导的外排。但NCA是否影响离子通道尚不清楚。在此，采用全细胞膜片钳技术研究NCA是否影响离子通道，特别是其如何抑制K1.4。利用诱变和基于结构的分子模拟分析抑制机制并确定结合位点。在所有检测的通道中，K1.4是NCA表现出最强抑制活性的通道，IC为7.55 µM。与NCA的异构体新狼毒素B（NCB）和狼毒素色原酮（CMC）相比，NCA对K1.4也表现出更强的抑制能力。发现在孔内发生的三个突变V549A、A553V和V560A可显著减轻NCA的阻断作用，表明它们是NCA的重要结合位点。基于结构的建模显示，NCA的酚羟基可分别与IS6和IVS6段中Val549和Ala553的主链形成氢键，这支持了我们的体外实验结果。总之，数据表明NCA可能通过直接结合孔道结构域来抑制K1.4通道。
