Interactions between β-catenin and the HSlo potassium channel regulates HSlo surface expression.
Interactions between β-catenin and the HSlo potassium channel regulates HSlo surface expression.
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DOI：
10.1371/journal.pone.0028264
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Navaratnam DS
中科院分区：
文献类型：
--
作者：
Bian S;Bai JP;Chapin H;Le Moellic C;Dong H;Caplan M;Sigworth FJ;Navaratnam DS
大电导钙激活钾通道α亚基（Slo）广泛分布于全身，在多种疾病中发挥重要作用。先前的工作已经表明，Slo通过其S10区域与β-连环蛋白相互作用，β-连环蛋白是细胞骨架框架和Wnt信号通路的关键组分。然而，这种相互作用的生理意义尚不清楚。使用蛋白质组学和细胞生物学工具的组合，我们显示在Slo中存在额外的多个结合位点，并详细探索β-连环蛋白与S10区域的相互作用。我们证明，该区域的缺失降低了HEK细胞中的Slo表面表达，这表明与β-连环蛋白的相互作用对于Slo表面表达是重要的。这通过用针对β-连环蛋白的siRNA处理的HEK细胞和鸡（Gallus gallus recticus leghorn白色）毛细胞中Slo的表达减少来证实。HSlo缺失突变体与β-连环蛋白免疫共沉淀，表明这两种蛋白质之间的稳定结合，S10缺失突变体与β-连环蛋白的结合减少。我们还观察到S10区域内两个推定的GSK磷酸化位点的突变影响Slo的表面表达和通道的电压和钙敏感性。有趣的是，外源性Slo在HEK细胞中的表达抑制β-连环蛋白依赖性经典Wnt信号传导。这些研究首次确定了β-连环蛋白在介导Slo表面表达中的核心作用。此外，我们发现Slo过表达可导致Wnt信号转导的下调。
The large conductance calcium-activated potassium channel alpha-subunit (Slo) is widely distributed throughout the body and plays an important role in a number of diseases. Prior work has shown that Slo, through its S10 region, interacts with β-catenin, a key component of the cytoskeleton framework and the Wnt signaling pathway. However, the physiological significance of this interaction was not clear. Using a combination of proteomic and cell biology tools we show the existence of additional multiple binding sites in Slo, and explore in detail β-catenin interactions with the S10 region. We demonstrate that deletion of this region reduces Slo surface expression in HEK cells, which indicates that interaction with beta-catenin is important for Slo surface expression. This is confirmed by reduced expression of Slo in HEK cells and chicken (Gallus gallus domesticus leghorn white) hair cells treated with siRNA to β-catenin. HSlo reciprocally co-immunoprecipitates with β-catenin, indicating a stable binding between these two proteins, with the S10 deletion mutant having reduced binding with β-catenin. We also observed that mutations of the two putative GSK phosphorylation sites within the S10 region affect both the surface expression of Slo and the channel's voltage and calcium sensitivities. Interestingly, expression of exogenous Slo in HEK cells inhibits β-catenin-dependent canonical Wnt signaling. These studies identify for the first time a central role for β-catenin in mediating Slo surface expression. Additionally we show that Slo overexpression can lead to downregulation of Wnt signaling.
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