Subcellular trafficking, pentameric assembly, and subunit stoichiometry of neuronal nicotinic acetylcholine receptors containing fluorescently labeled α6 and β3 Subunits
Subcellular trafficking, pentameric assembly, and subunit stoichiometry of neuronal nicotinic acetylcholine receptors containing fluorescently labeled α6 and β3 Subunits
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DOI：
10.1124/mol.107.039180
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发表时间：
2008-01-01
影响因子：
3.6
通讯作者：
Lester, Henry A.
中科院分区：
文献类型：
--
作者：
Drenan, Ryan M.;Nashmi, Raad;Lester, Henry A.
神经元烟碱乙酰胆碱（ACh）受体是配体门控的阳离子选择性离子通道。含有α 4、α 6、β 2和β 3亚基的尼古丁受体在中脑多巴胺能神经元中表达，并且它们与对吸烟尼古丁的反应有关。在这里，我们研究了细胞的生物学和生物物理特性的受体含有α 6和β 3亚基，通过使用荧光蛋白融合在M3-M4细胞内环。与未标记β 3亚基的受体相比，含有荧光标记β 3亚基的受体功能完全。我们发现β 3和α 6受体在神经元中高度表达，并且它们与共表达的荧光α 4和β 2亚基在神经元索马和树突中共定位。福斯特共振能量转移（FRET）揭示了β 3和α 6有效、特异地组装成各种亚基组成的烟碱受体五聚体。使用FRET，我们直接证明，只有一个单一的β 3亚基被纳入烟碱乙酰胆碱受体（nAChRs）含有这个亚基，而多个亚基的化学计量存在的α 4-和α 6-含有受体。最后，我们证明，烟碱乙酰胆碱受体定位在不同的微域或附近的质膜，使用全内反射荧光（TIRF）显微镜。我们认为，神经元含有大的，组装的，功能性烟碱受体，这可能为他们提供了快速上调内源性配体，如乙酰胆碱，或外源性试剂，如尼古丁的烟碱反应的能力，细胞内池。此外，这份报告是第一个直接测量nAChR亚基化学计量使用FRET和质膜定位的α 6-和β 3-含受体使用TIRF。
Neuronal nicotinic acetylcholine (ACh) receptors are ligand-gated, cation-selective ion channels. Nicotinic receptors containing alpha 4, alpha 6, beta 2, and beta 3 subunits are expressed in midbrain dopaminergic neurons, and they are implicated in the response to smoked nicotine. Here, we have studied the cell biological and biophysical properties of receptors containing alpha 6 and beta 3 subunits by using fluorescent proteins fused within the M3-M4 intracellular loop. Receptors containing fluorescently tagged beta 3 subunits were fully functional compared with receptors with untagged beta 3 subunits. We find that beta 3- and alpha 6- containing receptors are highly expressed in neurons and that they colocalize with coexpressed, fluorescent alpha 4 and beta 2 subunits in neuronal soma and dendrites. Forster resonance energy transfer (FRET) reveals efficient, specific assembly of beta 3 and alpha 6 into nicotinic receptor pentamers of various subunit compositions. Using FRET, we demonstrate directly that only a single beta 3 subunit is incorporated into nicotinic acetylcholine receptors (nAChRs) containing this subunit, whereas multiple subunit stoichiometries exist for alpha 4- and alpha 6- containing receptors. Finally, we demonstrate that nicotinic ACh receptors are localized in distinct microdomains at or near the plasma membrane using total internal reflection fluorescence (TIRF) microscopy. We suggest that neurons contain large, intracellular pools of assembled, functional nicotinic receptors, which may provide them with the ability to rapidly up-regulate nicotinic responses to endogenous ligands such as ACh, or to exogenous agents such as nicotine. Furthermore, this report is the first to directly measure nAChR subunit stoichiometry using FRET and plasma membrane localization of alpha 6- and beta 3- containing receptors using TIRF.