The calcium channel α2δ-2 subunit partitions with CaV2.1 into lipid rafts in cerebellum:: Implications for localization and function
The calcium channel α2δ-2 subunit partitions with CaV2.1 into lipid rafts in cerebellum:: Implications for localization and function
复制标题
DOI：
10.1523/jneurosci.2764-06.2006
复制
发表时间：
2006-08-23
影响因子：
5.3
通讯作者：
Dolphin, Annette C.
中科院分区：
文献类型：
--
作者：
Davies, Anthony;Douglas, Leon;Dolphin, Annette C.
电压门控钙通道的辅助α（2）δ亚基是高度糖基化的跨膜蛋白，其与钙通道α 1亚基相互作用以增强钙电流。我们比较了在tsA-201细胞中稳定表达的天然小脑α（2）δ-2亚基与α（2）δ-2亚基的膜定位和加工。我们发现，α（2）δ-2完全集中在小脑中富含胆固醇的微区（脂筏）中，在那里它基本上与钙通道α 1亚基Ca（V）2.1共定位，尽管Ca（V）2.1也存在于Triton X-100可溶性组分中。在tsA-201细胞中，与小脑不同，alpha（2）delta-2不完全被蛋白水解加工成alpha（2）-2和delta-2。然而，这种加工在tsA-201细胞的脂筏部分中更完全，其中α（2）δ-2也与Ca（V）2.1共定位。完整细胞的胆固醇耗竭破坏了它们的脂筏并增强了Ca（V）2.1/α（2）δ-2/β 4电流。此外，α 2 δ-2与气孔蛋白家族的脂筏相关蛋白共免疫沉淀。在小脑和稳定的α（2）δ-2细胞系中，α（2）δ-2对其配体加巴喷丁的表观亲和力在富含胆固醇的微区组分中显著增加。相比之下，含有点突变（R282 A）的α（2）δ-2对加巴喷丁具有低得多的亲和力，并且这在脂筏组分中没有增强。这种R282 A突变体α（2）δ-2在增强Ca（V）2.1/β 4钙电流方面显示出降低的功能，表明加巴喷丁结合位点的完整性对于α（2）δ-2的正常功能可能是重要的。总之，这些结果表明，α（2）δ-2和Ca（V）2.1通常与富含胆固醇的微区相关，这影响了它们的功能。
The accessory alpha(2)delta subunits of voltage-gated calcium channels are highly glycosylated transmembrane proteins that interact with calcium channel alpha 1 subunits to enhance calcium currents. We compared the membrane localization and processing of native cerebellar alpha(2)delta-2 subunits with alpha(2)delta-2 stably expressed in tsA-201 cells. We identified that alpha(2)delta-2 is completely concentrated in cholesterol-rich microdomains (lipid rafts) in cerebellum, in which it substantially colocalizes with the calcium channel alpha 1 subunit Ca(V)2.1, although Ca(V)2.1 is also present in the Triton X-100-soluble fraction. In tsA-201 cells, unlike cerebellum, alpha(2)delta-2 is not completely proteolytically processed into alpha(2)-2 and delta-2. However, this processing is more complete in the lipid raft fraction of tsA-201 cells, in which alpha(2)delta-2 also colocalizes with Ca(V)2.1. Cholesterol depletion of intact cells disrupted their lipid rafts and enhanced Ca(V)2.1/alpha(2)delta-2/beta 4 currents. Furthermore, alpha 2 delta-2 coimmunoprecipitates with lipid raft-associated proteins of the stomatin family. The apparent affinity of alpha(2)delta-2 for its ligand gabapentin is increased markedly in the cholesterol-rich microdomain fractions, in both cerebellum and the stable alpha(2)delta-2 cell line. In contrast, alpha(2)delta-2 containing a point mutation (R282A) has a much lower affinity for gabapentin, and this is not enhanced in the lipid raft fraction. This R282A mutant alpha(2)delta-2 shows reduced functionality in terms of enhancement of Ca(V)2.1/beta 4 calcium currents, suggesting that the integrity of the gabapentin binding site may be important for normal functioning of alpha(2)delta-2. Together, these results indicate that both alpha(2)delta-2 and Ca(V)2.1 are normally associated with cholesterol-rich microdomains, and this influences their functionality.