Extracellular disulfide bridges stabilize TRPC5 dimerization, trafficking, and activity
Extracellular disulfide bridges stabilize TRPC5 dimerization, trafficking, and activity
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DOI：
10.1007/s00424-014-1540-0
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发表时间：
2015-04-01
影响因子：
4.5
通讯作者：
So, Insuk
中科院分区：
文献类型：
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作者：
Hong, Chansik;Kwak, Misun;So, Insuk
关键的半胱氨酸残基可以通过巯基（-SH）基团的修饰参与蛋白质活性的调节。在这些反应中，二硫键（S-S）在膜蛋白的折叠、稳定性和活性中起着关键作用。然而，胞外半胱氨酸在经典瞬时受体电位（TRPC）通道中的调控仍存在争议。在这里，我们研究了TRPC5细胞外二硫键在调节通道门控和运输中的功能重要性。具体来说，我们研究了瞬时转染的HEK293细胞中TRPC5的活性，这些细胞具有野生型（WT）或半胱氨酸（C553和C558）突变。使用还原剂，我们确定了二硫键介导TRPC5通道的四聚体形成。通过测量TRPC5电流，我们观察到C553S或C558S突变体在镧系元素或受体刺激下完全失去通道活性。TRPC5 （WT）与突变体共表达在突变体中表现为显性负表达，抑制了TRPC5 （WT）的活性。我们生成了TRPC5-TRPC5二聚体，并观察到与WT-WT二聚体相比，wt -突变体（C553S或C558S）二聚体的活性降低。当还原剂预处理12 h时，由于膜上TRPC5分布减少，TRPC5电流减小。此外，我们发现C553S突变体在质膜中的表达减少。我们使用共免疫沉淀和FRET方法分析了野生型和突变型TRPC5的二聚体相互作用，表明二聚体伴侣之间存在弱相互作用。这些结果表明，保守的细胞外半胱氨酸之间的二硫键，特别是C553，是通过通道多化和运输功能TRPC5活性所必需的。
Crucial cysteine residues can be involved in the modulation of protein activity via the modification of thiol (-SH) groups. Among these reactions, disulfide bonds (S-S) play a key role in the folding, stability, and activity of membrane proteins. However, the regulation of extracellular cysteines in classical transient receptor potential (TRPC) channels remains controversial. Here, we examine the functional importance of the extracellular disulfide bond in TRPC5 in modulating channel gating and trafficking. Specifically, we investigated TRPC5 activity in transiently transfected HEK293 cells with wild-type (WT) or cysteine (C553 and C558) mutants in the pore loop. Using reducing agents, we determined that a disulfide linkage mediates the tetrameric formation of the TRPC5 channel. By measuring the TRPC5 current, we observed that C553S or C558S mutants completely lose channel activity induced by lanthanides or receptor stimulation. Co-expression of TRPC5 (WT) with mutants demonstrated a dominant-negative function in mutants, which inhibited the activity of TRPC5 (WT). We generated TRPC5-TRPC5 dimers and observed reduced activity of WT-mutant (C553S or C558S) dimers compared to WT-WT dimers. When pretreated with reducing agents for 12 h, the TRPC5 current decreased due to a reduction in membrane TRPC5 distribution. In addition, we identified a reduced expression of C553S mutant in plasma membrane. We analyzed a dimeric interaction of wild-type and mutant TRPC5 using co-immunoprecipitation and FRET method, indicating a weak interaction between dimeric partners. These results indicated that the disulfide bond between conserved extracellular cysteines, especially C553, is essential for functional TRPC5 activity by channel multimerization and trafficking.