Complexes of the α1C and β subunits generate the necessary signal for membrane targeting of class C L-type calcium channels
Complexes of the α1C and β subunits generate the necessary signal for membrane targeting of class C L-type calcium channels
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DOI：
10.1074/jbc.274.4.2137
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发表时间：
1999-01-22
影响因子：
4.8
通讯作者：
Hosey, MM
中科院分区：
文献类型：
--
作者：
Gao, TY;Chien, AJ;Hosey, MM
在本研究中，我们研究了通道亚基在电压依赖性L-型钙通道复合物膜靶向中的作用。我们在HEK-tsA 201细胞中共表达钙通道成孔α（1C）亚基和不同的辅助β亚基，并使用共聚焦显微镜和全细胞放射性配体结合研究通过免疫组织化学检查通道亚基的亚细胞定位。虽然成孔α（1C）亚基在单独表达时表现出核周染色，并且几种野生型和突变体β亚基也表现出细胞内染色，但α（1C）亚基与野生型β（2a）亚基、棕榈酰化缺陷的β（2a）（C3 S/C4 S）突变体或三种其它非棕榈酰化β同种型（β（1b），β（3），和β（4）亚基）导致α（1C）和β亚基沿细胞表面沿着重新分布成簇。此外，当α（1C）与仅含有中心保守区的N-和C-末端截短突变体β（2a）共表达时，观察到钙通道复合物向质膜的再分布。然而，当α（1C）亚基与α（1）β相互作用缺陷突变体β（2a）BID（-）共表达时，我们没有观察到质膜上通道的形成。此外，不能与α（1C）亚基相互作用的β（2a）的Src同源性3基序突变体也未能将通道复合物靶向质膜。有趣的是，成孔α（1C）亚基与主要是外周辅助α（2）δ亚基的共表达在将α（1C）募集到质膜方面是无效的，而当β（2a）与α（1C）和α（2）δ亚基共表达时，观察到所有三种亚基的共分布。我们的结果表明，C类L型钙通道复合物的正确质膜靶向所必需的信号是由于α（1）和β亚基之间的功能性相互作用而产生的。
In the present study, we investigated the role of channel subunits in the membrane targeting of voltage-dependent L-type calcium channel complexes. We co-expressed the calcium channel pore-forming alpha(1C) Subunit with different accessory beta subunits in HEK-tsA201 cells and examined the subcellular localization of the channel subunits by immunohistochemistry using confocal microscopy and whole-cell radioligand binding studies. While the pore-forming alpha(1C) subunit exhibited perinuclear staining when expressed alone, and several of the wild-type and mutant beta subunits also exhibited intracellular staining, co-expression of the alpha(1C) subunit with either the wild-type beta(2a) subunit, a palmitoylation-deficient beta(2a)(C3S/C4S) mutant or three other nonpalmitoylated beta isoforms (beta(1b), beta(3), and beta(4) subunits) resulted in the redistribution of both the alpha(1C) and beta subunits into clusters along the cell surface. Furthermore, the redistribution of calcium channel complexes to the plasma membrane was observed when alpha(1C) was co-expressed with an N- and C-terminal truncated mutant beta(2a) containing only the central conserved regions. However, when the alpha(1C) subunit was co-expressed with an alpha(1)beta interaction-deficient mutant, beta(2a)BID(-), we did not observe formation of the channels at the plasma membrane. In addition, an Src homology 3 motif mutant of beta(2a) that was unable to interact with the alpha(1C) subunit also failed to target channel complexes to the plasma membrane. Interestingly, co-expression of the pore-forming alpha(1C) subunit with the largely peripheral accessory alpha(2)delta subunit was ineffective in recruiting alpha(1C) to the plasma membrane, while codistribution of all three subunits was observed when beta(2a) was co-expressed with the alpha(1C) and alpha(2)delta subunits, Taken together, our results suggested that the signal necessary for correct plasma membrane targeting of the class C L-type calcium channel complexes is generated as a result of a functional interaction between the alpha(1) and beta subunits.