Engineering α4β2 nAChRs with reduced or increased nicotine sensitivity via selective disruption of consensus sites in the M3-M4 cytoplasmic loop of the α4 subunit.
Engineering α4β2 nAChRs with reduced or increased nicotine sensitivity via selective disruption of consensus sites in the M3-M4 cytoplasmic loop of the α4 subunit.
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DOI：
10.1016/j.neuropharm.2015.04.022
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发表时间：
2015-12
影响因子：
4.7
通讯作者：
Lasalde-Dominicci JA
中科院分区：
文献类型：
--
作者：
Biaggi-Labiosa NM;Avilés-Pagán E;Caballero-Rivera D;Báez-Pagán CA;Lasalde-Dominicci JA
α4β2神经元烟碱乙酰胆碱受体（nAChR）在尼古丁成瘾中起着关键作用。已知这些受体在慢性尼古丁暴露后脱敏和上调，但其机制尚不清楚。目前，nAChR的胞内结构域的结构和功能作用尚不清楚。为了研究亚基磷酸化对α4β2 nAChR功能和表达的影响，将位于M3-M4胞质环中的11个残基突变为丙氨酸和天冬氨酸。双电极电压钳和125碘标记的epibatidine结合试验进行爪蟾卵母细胞，以评估激动剂激活和受体表达。当乙酰胆碱被用作激动剂，受体激活的减少，观察到大多数的突变。当尼古丁用作激动剂时，四种突变对尼古丁表现出统计学显著的超敏反应（S438 D、S469 A、Y 576 A和S589 A）。此外，两个突变（S516 D和T536 A）显示正常激活ACh显示对尼古丁的敏感性显着降低。结合试验显示，这两个尼古丁突变的组成性上调与尼古丁敏感性降低。这些结果表明，α4亚基M3-M4胞质环中的共有磷酸化残基在调节α4β2 nAChR激动剂选择性和功能表达方面发挥着至关重要的作用。此外，这些结果表明，破坏PKC推定共识位点的特异性相互作用可以使α4β2 nAChR对尼古丁几乎不敏感，而对正常AChR功能没有实质性影响。因此，α4 nAChR亚基的M3-M4胞质环中的这些PKC共有位点可能是戒烟药物的靶点。
The α4β2 neuronal nicotinic acetylcholine receptor (nAChR) plays a crucial role in nicotine addiction. These receptors are known to desensitize and up-regulate after chronic nicotine exposure, but the mechanism remains unknown. Currently, the structure and functional role of the intracellular domains of the nAChR are obscure. To study the effect of subunit phosphorylation on α4β2 nAChR function and expression, eleven residues located in the M3-M4 cytoplasmic loop were mutated to alanine and aspartic acid. Two-electrode voltage clamp and 125I-labeled epibatidine binding assays were performed on Xenopus oocytes to assess agonist activation and receptor expression. When ACh was used as an agonist, a decrease in receptor activation was observed for the majority of the mutations. When nicotine was used as an agonist, four mutations exhibited a statistically significant hypersensitivity to nicotine (S438D, S469A, Y576A, and S589A). Additionally, two mutations (S516D and T536A) that displayed normal activation with ACh displayed remarkable reductions in sensitivity to nicotine. Binding assays revealed a constitutive up-regulation in these two nicotine mutations with reduced nicotine sensitivity. These results suggest that consensus phosphorylation residues in the M3-M4 cytoplasmic loop of the α4 subunit play a crucial role in regulating α4β2 nAChR agonist selectivity and functional expression. Furthermore, these results suggest that disruption of specific interactions at PKC putative consensus sites can render α4β2 nAChRs almost insensitive to nicotine without substantial effects on normal AChR function. Therefore, these PKC consensus sites in the M3-M4 cytoplasmic loop of the α4 nAChR subunit could be a target for smoking cessation drugs.