Kudryavtsev Denis, Isaeva Anastasia, Barkova Daria, Spirova Ekaterina, Mukhutdinova Renata, Kasheverov Igor, Tsetlin Victor
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.
Moscow Institute of Physics and Technology, Dolgoprudny, 141700 Moscow, Russia.
Molecules. 2021 Feb 26;26(5):1278. doi: 10.3390/molecules26051278.
Slow-channel congenital myasthenic syndromes (SCCMSs) are rare genetic diseases caused by mutations in muscle nicotinic acetylcholine receptor (nAChR) subunits. Most of the known SCCMS-associated mutations localize at the transmembrane region near the ion pore. Only two SCCMS point mutations are at the extracellular domains near the acetylcholine binding site, α1(G153S) being one of them. In this work, a combination of molecular dynamics, targeted mutagenesis, fluorescent Ca imaging and patch-clamp electrophysiology has been applied to G153S mutant muscle nAChR to investigate the role of hydrogen bonds formed by Ser 153 with C-loop residues near the acetylcholine-binding site. Introduction of L199T mutation to the C-loop in the vicinity of Ser 153 changed hydrogen bonds distribution, decreased acetylcholine potency (EC 2607 vs. 146 nM) of the double mutant and decay kinetics of acetylcholine-evoked cytoplasmic Ca rise (τ 14.2 ± 0.3 vs. 34.0 ± 0.4 s). These results shed light on molecular mechanisms of nAChR activation-desensitization and on the involvement of such mechanisms in channelopathy genesis.
慢通道先天性肌无力综合征（SCCMSs）是由肌肉烟碱型乙酰胆碱受体（nAChR）亚基突变引起的罕见遗传病。大多数已知的与SCCMS相关的突变位于离子孔附近的跨膜区域。只有两个SCCMS点突变位于乙酰胆碱结合位点附近的细胞外结构域，α1（G153S）就是其中之一。在这项研究中，分子动力学、定点诱变、荧光钙成像和膜片钳电生理学相结合的方法被应用于G153S突变型肌肉nAChR，以研究由丝氨酸153与乙酰胆碱结合位点附近的C环残基形成的氢键的作用。在丝氨酸153附近的C环引入L199T突变改变了氢键分布，降低了双突变体的乙酰胆碱效力（EC 2607对146 nM）以及乙酰胆碱诱发的细胞质钙升高的衰减动力学（τ 14.2±0.3对34.0±0.4秒）。这些结果揭示了nAChR激活-脱敏的分子机制以及这些机制在通道病发生中的作用。
