Up-regulation of cell-surface α4β2 neuronal nicotinic receptors by lower temperature and expression of chimeric subunits
Up-regulation of cell-surface α4β2 neuronal nicotinic receptors by lower temperature and expression of chimeric subunits
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DOI：
10.1074/jbc.274.38.27145
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发表时间：
1999-09-17
影响因子：
4.8
通讯作者：
Millar, NS
中科院分区：
文献类型：
--
作者：
Cooper, ST;Harkness, PC;Millar, NS
在脊椎动物脑中表达的主要烟碱乙酰胆碱受体（nAChR）是含有α 4和β 2亚基的五聚体。在这项研究中，我们已经研究了温度和亚基嵌合体的表达如何影响大鼠α 4 β 2 nAChR的细胞表面表达效率，功能性重组α 4 β 2 nAChR，显示出烟碱放射性配体的高亲和力结合（[（3）H]地棘蛙素的K（d）= 41 +/-22 pM），在稳定和瞬时转染的哺乳动物细胞系中表达，尽管如此，在保持在37 ℃的转染的哺乳动物细胞的细胞表面上只能检测到非常低水平的α 4 β 2 nAChR。然而，在30 ℃下，表达α 4 β 2 nAChR的细胞显示放射性配体结合增加12倍（亲和力没有变化），细胞表面受体上调5倍，总亚基蛋白没有增加。与“野生型”α 4和β 2亚基相反，嵌合烟碱/烟碱能亚基（“α 4 χ”和“β 2 χ”）在细胞表面上非常有效地表达（在30 ° C或37 ° C下），作为异源寡聚复合物（例如α 4 χ + β 2或α 4 χ + β 2 χ）或当单独表达时。与α 4 β 2 nAChR相比，含有嵌合亚基的复合物的表达通常导致烟碱放射性配体结合位点增加高达20倍（亲和力无变化），细胞表面受体增加相似，尽管总嵌合蛋白和野生型蛋白水平相似。
The predominant nicotinic acetylcholine receptor (nAChR) expressed in vertebrate brain is a pentamer containing alpha 4 and beta 2 subunits, In this study we have examined how temperature and the expression of subunit chimeras can influence the efficiency of cell-surface expression of the rat alpha 4 beta 2 nAChR, Functional recombinant alpha 4 beta 2 nAChRs, showing high affinity binding of nicotinic radioligands (K(d) = 41 +/- 22 pM for [(3)H]epibatidine), are expressed in both stably and transiently transfected mammalian cell lines, Despite this, only very low levels of alpha 4 beta 2 nAChRs can be detected on the cell surface of transfected mammalian cells maintained at 37 degrees C. At 30 degrees C, however, cells expressing alpha 4 beta 2 nAChRs show a 12-fold increase in radioligand binding (with no change in affinity), and a 5-fold up-regulation in cell-surface receptors with no increase in total subunit protein. In contrast to "wild-type" alpha 4 and beta 2 subunits, chimeric nicotinic/serotonergic subunits ("alpha 4 chi" and "beta 2 chi") are expressed very efficiently on the cell surface (at 30 degrees C or 37 degrees C), either as hetero-oligomeric complexes (e.g. alpha 4 chi+beta 2 or alpha 4 chi+beta 2 chi) or when expressed alone. Compared with alpha 4 beta 2 nAChRs, expression of complexes containing: chimeric subunits typically results in up to 20-fold increase in nicotinic radioligand binding sites (with no change in affinity) and a similar increase in cell-surface receptor, despite a similar level of total chimeric and wild-type protein.