Uspenska Kateryna, Lykhmus Olena, Gergalova Galyna, Chernyshov Volodymyr, Arias Hugo R, Komisarenko Sergiy, Skok Maryna
Department of Molecular Immunology, Palladin Institute of Biochemistry NAS of Ukraine, 9, Leontovycha str., 01030 Kyiv, Ukraine.
Department of Basic Sciences, California Northstate University College of Medicine, 9700 W. Taron Dr., Elk Grove, CA 95757, USA.
Neurosci Lett. 2017 Aug 24;656:43-50. doi: 10.1016/j.neulet.2017.07.009. Epub 2017 Jul 9.
Several nicotinic acetylcholine receptor (nAChR) subtypes are expressed in mitochondria to regulate the internal pathway of apoptosis in ion channel-independent manner. However, the mechanisms of nAChR activation in mitochondria and targeting to mitochondria are still unknown. Nicotine has been shown to favor nAChR pentamer assembly, folding, and maturation on the way of biosynthesis. The idea of the present work was to determine whether nicotine affects the content, glycosylation, and function of mitochondrial nAChRs. Experiments were performed in isolated liver mitochondria from mice, that either consumed or not nicotine with the drinking water (200μL/L) for 7days. Mitochondria detergent lysates were studied by sandwich or lectin ELISA for the presence and carbohydrate composition of different nAChR subunits. Intact mitochondria were examined by flow cytometry for the binding of fluorescently labeled α-cobratoxin and were tested in functional assay of cytochrome c release under the effect of either Ca or wortmannin in the presence or absence of nAChR-selective ligands, including PNU-282987 (1nM), dihydro-β-erythroidine (DhβE, 1μM), PNU-120596 (0.3, 3, or 10μM) and desformylflustrabromine hydrochloride (dFBr, 0.001, 0.3, or 1μM). It was found that nicotine consumption increased the ratio of mitochondrial vs non-mitochondrial nAChRs in the liver, enhanced fucosylation of mitochondrial nAChRs, but prevented the binding of α-cobratoxin and the cytochrome c release-attenuating effects of nAChR-specific agonists, antagonists, or positive allosteric modulators. It is concluded that nicotine consumption in vivo favors nAChR glycosylation and trafficking to mitochondria but makes them less susceptible to the effects of specific ligands.
几种烟碱型乙酰胆碱受体（nAChR）亚型在线粒体中表达，以不依赖离子通道的方式调节细胞凋亡的内在途径。然而，nAChR在线粒体中的激活机制以及靶向线粒体的机制仍不清楚。已表明尼古丁在生物合成过程中有利于nAChR五聚体的组装、折叠和成熟。本研究的目的是确定尼古丁是否影响线粒体nAChR的含量、糖基化和功能。实验在从小鼠分离的肝线粒体中进行，这些小鼠饮用含或不含尼古丁（200μL/L）的水7天。通过夹心ELISA或凝集素ELISA研究线粒体去污剂裂解物中不同nAChR亚基的存在和碳水化合物组成。通过流式细胞术检测完整线粒体与荧光标记的α-银环蛇毒素的结合，并在存在或不存在nAChR选择性配体（包括PNU-282987（1nM）、二氢-β-刺桐啶（DhβE，1μM）、PNU-120596（0.3、3或10μM）和盐酸去甲氟溴马钱子碱（dFBr，0.001、0.3或1μM））的情况下，在钙或渥曼青霉素作用下进行细胞色素c释放的功能测定。发现摄入尼古丁会增加肝脏中线粒体与非线粒体nAChR的比例，增强线粒体nAChR的岩藻糖基化，但会阻止α-银环蛇毒素的结合以及nAChR特异性激动剂、拮抗剂或正变构调节剂对细胞色素c释放的减弱作用。得出的结论是，体内摄入尼古丁有利于nAChR的糖基化和向线粒体的转运，但使它们对特异性配体的作用不太敏感。
