Lipid raft localization of GABAA receptor and Na+, K+-ATPase in discrete microdomain clusters in rat cerebellar granule cells
Lipid raft localization of GABAA receptor and Na+, K+-ATPase in discrete microdomain clusters in rat cerebellar granule cells
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DOI：
10.1016/j.neuint.2004.11.010
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发表时间：
2005-05-01
影响因子：
4.2
通讯作者：
Danielsen, EM
中科院分区：
文献类型：
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作者：
Dalskov, SM;Immerdal, L;Danielsen, EM
用亚细胞分离、荧光和免疫金电镜技术研究了大鼠小脑颗粒细胞GABA（A）受体的微区定位。受体存在于脂筏中，脂筏在37 ℃下用非离子去污剂Brij 98提取制备，但密度梯度离心后漂浮部分中的标记物神经节苷脂GM（1）所定义的脂筏部分在密度和蛋白质组成上是不均匀的。因此，另一个主要的筏相关的膜蛋白，Na+，K+-ATP酶，被发现在离散的筏密度较低，反映集群的两种蛋白质在单独的膜微区。这两种蛋白质在细胞表面以及孤立的脂筏中观察到斑片状的“热点”。它们在Brij 98中的不溶性仅受甲基-β-环糊精的轻微影响。相反，GABA（A）受体和Na+，K+-ATP酶在冰冷的Triton X-100中大部分可溶。这表明Brij 98提取物定义了一种不寻常类型的胆固醇非依赖性脂筏，其含有也与基础支架/细胞骨架蛋白如桥蛋白（GABA（A）受体）和锚蛋白G（Na+，K+-ATP酶）相关的膜蛋白。通过提供有序的膜微环境，脂筏可能有助于GABA（A）受体和Na+，K+-ATP酶在细胞表面不同功能位置的聚集。(c)2005爱思唯尔有限公司保留所有权利。
The microdomain localization of the GABA(A) receptor in rat cerebellar granule cells was studied by subcellular fractionation and fluorescence- and immunogold electron microscopy. The receptor resided in lipid rafts, prepared at 37 degrees C by extraction with the nonionic detergent Brij 98, but the raft fraction, defined by the marker ganglioside GM(1) in the floating fractions following density gradient centrifugation, was heterogeneous in density and protein composition. Thus, another major raft-associated membrane protein, the Na+, K+-ATPase, was found in discrete rafts of lower density, reflecting clustering of the two proteins in separate membrane microdomains. Both proteins were observed in patchy 'hot spots' at the cell surface as well as in isolated lipid rafts. Their insolubility in Brij 98 was only marginally affected by methyl-p-cyclodextrin. In contrast, both the GABA(A) receptor and Na+, K+-ATPase were largely soluble in ice cold Triton X-100. This indicates that Brij 98 extraction defines an unusual type of cholesterol-independent lipid rafts that harbour membrane proteins also associated with underlying scaffolding/cytoskeletal proteins such as gephyrin (GABA(A) receptor) and ankyrin G (Na+, K+-ATPase). By providing an ordered membrane microenvironment, lipid rafts may contribute to the clustering of the GABA(A) receptor and the Na+, K+-ATPase at distinct functional locations on the cell surface. (c) 2005 Elsevier Ltd. All rights reserved.