Chen Dejie, Gao Fenfei, Ma Xiaokuang, Eaton Jason Brek, Huang Yuanbing, Gao Ming, Chang Yongchang, Ma Zegang, Der-Ghazarian Taleen, Neisewander Janet, Whiteaker Paul, Wu Jie, Su Quanxi
Department of Neurology, Yunfu People's Hospital, Yunfu, China.
Division of Neurobiology, Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, AZ, United States.
Front Pharmacol. 2019 Feb 14;10:72. doi: 10.3389/fphar.2019.00072. eCollection 2019.
Alpha6-containing nicotinic acetylcholine receptors are primarily found in neurons of the midbrain dopaminergic (DA) system, suggesting these receptors are potentially involved in drug reward and dependence. Here, we report a novel effect that cocaine directly inhibits α6N/α3Cβ2β3-nAChR (α6*-nAChRs) function. Human α6*-nAChRs were heterologously expressed within cells of the SH-EP1 cell line for functional characterization. Mechanically dissociated DA neurons from mouse ventral tegmental area (VTA) were used as a model of presynaptic α6*-nAChR activation since this method preserves terminal boutons. Patch-clamp recordings in whole-cell configuration were used to measure α6*-nAChR function as well as evaluate the effects of cocaine. In SH-EP1 cells containing heterologously expressed human α6*-nAChRs, cocaine inhibits nicotine-induced inward currents in a concentration-dependent manner with an IC value of 30 μM. Interestingly, in the presence of 30 μM cocaine, the maximal current response of the nicotine concentration-response curve is reduced without changing nicotine's EC value, suggesting a noncompetitive mechanism. Furthermore, analysis of whole-cell current kinetics demonstrated that cocaine slows nAChR channel activation but accelerates whole-cell current decay time. Our findings demonstrate that cocaine-induced inhibition occurs solely with bath application, but not during intracellular administration, and this inhibition is not use-dependent. Additionally, in oocytes, cocaine inhibits both α6N/α3Cβ2β3-nAChRs and α6M211L/α3ICβ2β3-nCAhRs similarly, suggesting that cocaine may not act on the α3 transmembrane domain of chimeric α6N/α3Cβ2β3-nAChR. In mechanically isolated VTA DA neurons, cocaine abolishes α6*-nAChR-mediated enhancement of spontaneous inhibitory postsynaptic currents (sIPSCs). Collectively, these studies provide the first evidence that cocaine directly inhibits the function of both heterologously and naturally expressed α6*-nAChRs. These findings suggest that α6*-nAChRs may provide a novel pharmacological target mediating the effects of cocaine and may underlie a novel mechanism of cocaine reward and dependence.
含α6的烟碱型乙酰胆碱受体主要存在于中脑多巴胺能（DA）系统的神经元中，这表明这些受体可能参与药物奖赏和成瘾。在此，我们报告了一种新的效应，即可卡因直接抑制α6N/α3Cβ2β3 - nAChR（α6* - nAChRs）的功能。人α6* - nAChRs在SH - EP1细胞系的细胞中进行异源表达以进行功能表征。从小鼠腹侧被盖区（VTA）机械分离的DA神经元被用作突触前α6* - nAChR激活的模型，因为这种方法保留了终末小体。采用全细胞模式的膜片钳记录来测量α6* - nAChR的功能以及评估可卡因的作用。在含有异源表达人α6* - nAChRs的SH - EP1细胞中，可卡因以浓度依赖的方式抑制尼古丁诱导的内向电流，IC值为30μM。有趣的是，在30μM可卡因存在的情况下，尼古丁浓度 - 反应曲线的最大电流反应降低，而不改变尼古丁的EC值，提示一种非竞争性机制。此外，对全细胞电流动力学的分析表明，可卡因减慢了nAChR通道的激活，但加速了全细胞电流的衰减时间。我们的研究结果表明，可卡因诱导的抑制仅在浴槽给药时发生，而在细胞内给药时不发生，并且这种抑制不依赖于使用情况。此外，在卵母细胞中，可卡因同样抑制α6N/α3Cβ2β3 - nAChRs和α6M211L/α3ICβ2β3 - nCAhRs，这表明可卡因可能不作用于嵌合α6N/α3Cβ2β3 - nAChR的α3跨膜结构域。在机械分离的VTA DA神经元中，可卡因消除了α6* - nAChR介导的自发性抑制性突触后电流（sIPSCs）的增强。总的来说，这些研究提供了首个证据，即可卡因直接抑制异源表达和天然表达的α6* - nAChRs的功能。这些发现表明，α6* - nAChRs可能提供了一个介导可卡因作用的新的药理学靶点，并且可能是可卡因奖赏和成瘾新机制的基础。
