Membrane lipid rafts are necessary for the maintenance of the α7 nicotinic acetylcholine receptor in somatic spines of ciliary neurons
Membrane lipid rafts are necessary for the maintenance of the α7 nicotinic acetylcholine receptor in somatic spines of ciliary neurons
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DOI：
10.1523/jneurosci.21-02-00504.2001
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发表时间：
2001-01-15
影响因子：
5.3
通讯作者：
Rutishauser, U
中科院分区：
文献类型：
--
作者：
Brusés, JL;Chauvet, N;Rutishauser, U
钙渗透性神经递质受体被集中到结构和生化隔离的细胞隔室中，以定位神经传递期间的钙介导事件。细胞质膜含有称为脂筏的脂质微区，其可以聚集成显微镜可见的簇，因此特定蛋白质与脂筏的缔合可以导致其在细胞表面上的重新分布。本研究询问脂筏是否参与形成和维持的钙渗透性α 7亚基烟碱乙酰胆碱受体（α 7 nAChR）集群中发现的体细胞的纤毛神经元的棘。脂筏和α 7 nAChR在突触发生过程中逐渐在体细胞棘内共定位。为了确定这些筏是否需要α 7 nAChR聚集体的维护，胆固醇提取与甲基-β-环糊精治疗从解离的睫状神经元。这种处理导致脂筏的分散和α 7 nAChR在细胞表面重新分布成小簇，表明脂筏的完整性是维持受体聚集所必需的。然而，脂筏分散也引起F-肌动蛋白细胞骨架的解聚，这也可以在特定的网站拴受体。为了评估筏和α 7 nAChR之间的相互作用是否独立于F-肌动蛋白丝，脂质筏补丁稳定与霍乱毒素B亚基（CTX），它特异性地结合到筏组件神经节苷脂GM 1，和针对CTX的抗体的组合。然后用latrunculin-A处理稳定化的筏以使F-肌动蛋白脱辅基。在这些条件下，大补丁的CTX持续存在，并与α 7 nAChR共定位，表明受体的聚集体可以独立于底层的F-肌动蛋白细胞骨架而维持。此外，发现α 7 nAChR在4 ℃下对去污剂提取具有抗性，并且在密度梯度离心期间与含有小窝蛋白的富含脂质的级分一起漂浮，这些性质与受体和膜微区之间的直接关联一致。
Calcium-permeable neurotransmitter receptors are concentrated into structurally and biochemically isolated cellular compartments to localize calcium-mediated events during neurotransmission. The cytoplasmic membrane contains lipid microdomains called lipid rafts, which can gather into microscopically visible clusters, and thus the association of a particular protein with lipid rafts can result in its redistribution on the cell surface. The present study asks whether lipid rafts participate in the formation and maintenance of the calcium-permeable alpha7-subunit nicotinic acetylcholine receptor (alpha 7nAChR) clusters found in somatic spines of ciliary neurons. Lipid rafts and alpha 7nAChR become progressively colocalized within somatic spines during synaptogenesis. To determine whether these rafts are required for the maintenance of alpha 7nAChR aggregates, cholesterol was extracted from dissociated ciliary neurons by treatment with methyl-beta -cyclodextrin. This treatment caused the dispersion of lipid rafts and the redistribution of alpha 7nAChR into small clusters over the cell surface, suggesting that the integrity of lipid rafts is required to maintain the receptor clustering. However, lipid raft dispersion also caused the depolymerization of the F-actin cytoskeleton, which can also tether the receptor at specific sites. To assess whether interaction between rafts and alpha 7nAChR is independent of F-actin filaments, the lipid raft patches were stabilized with a combination of the cholera toxin B subunit (CTX), which specifically binds to the raft component ganglioside GM1, and an antibody against CTX. The stabilized rafts were then treated with latrunculin-A to depolymerize F-actin. Under these conditions, large patches of CTX persisted and were colocalized with alpha 7nAChR, indicating that the aggregates of receptors can be maintained independently of the underlying F-actin cytoskeleton. Moreover, it was found that the alpha 7nAChR is resistant to detergent extraction at 4 degrees C and floats with the caveolin-containing lipid-rich fraction during density gradient centrifugation, properties that are consistent with a direct association between the receptor and the membrane microdomains.