Institute for Behavioral Genetics, University of Colorado Boulder, USA.
Institute for Behavioral Genetics, University of Colorado Boulder, USA.
Biochem Pharmacol. 2019 Jan;159:64-73. doi: 10.1016/j.bcp.2018.11.008. Epub 2018 Nov 9.
Chronic nicotine exposure upregulates α4β2 nicotinic acetylcholine receptors (nAChRs) in the brain. The goal of this study was to examine the role of three serine residues in the large cytoplasmic loop of the α4 subunit on α4β2 upregulation in neurons.
Serine residues S336, S470 and S530 in mouse α4 were mutated to alanine and then re-expressed in primary neurons from cortex, hippocampus and subcortex of α4 KO mice. Mutant and wild type α4 expressing neurons were treated with nicotine (0.1, 1 and 10 μM) and assessed for α4β2 upregulation.
α4β2 nAChRs expressing S336A or S470A mutants were deficient at cell surface upregulation in both subcortex and hippocampal neurons. S530A α4β2 mutants exhibited aberrant surface upregulation in subcortical neurons. None of the mutants affected surface upregulation in cortical neurons or upregulation of total α4β2 binding sites in any region. Further, dense domains or clusters of α4β2 nAChRs were observed in the neuronal surface. The impact of nicotine exposure on the intensity, area, and density of these clusters was dependent upon individual mutations.
Effects of α4 nAChR mutants on surface upregulation varied among brain regions, suggesting that the cellular mechanism of α4β2 upregulation is complex and involves cellular identity. We also report for the first time that α4β2 nAChRs form clusters on the neuronal surface and that nicotine treatment alters the characteristics of the clusters in an α4 mutant-dependent manner. This finding adds a previously unknown layer of complexity to the effects of nicotine on α4β2 expression and function.
慢性尼古丁暴露会使大脑中的α4β2 烟碱型乙酰胆碱受体（nAChR）上调。本研究旨在探讨α4 亚基大胞质环中三个丝氨酸残基在神经元中α4β2 上调中的作用。
将小鼠α4 中的丝氨酸残基 S336、S470 和 S530 突变为丙氨酸，然后在α4 KO 小鼠的皮质、海马和皮质下区的原代神经元中重新表达。用尼古丁（0.1、1 和 10μM）处理突变型和野生型α4 表达神经元，并评估α4β2 的上调。
S336A 或 S470A 突变的α4β2 nAChR 表达神经元在皮质下和海马神经元中均缺乏细胞表面上调。S530Aα4β2 突变体在皮质下神经元中表现出异常的表面上调。突变体均不影响皮质神经元中的表面上调或任何区域的总α4β2 结合位点的上调。此外，还观察到神经元表面存在密集的α4β2 nAChR 致密区或簇。尼古丁暴露对这些簇的强度、面积和密度的影响取决于个体突变。
α4 nAChR 突变体对表面上调的影响在不同脑区之间存在差异，这表明α4β2 上调的细胞机制复杂，涉及细胞身份。我们还首次报道α4β2 nAChR 在神经元表面形成簇，并且尼古丁处理以依赖于α4 突变体的方式改变簇的特征。这一发现为尼古丁对α4β2 表达和功能的影响增加了一个以前未知的复杂性层次。
