Stimulatory Role of SPAK Signaling in the Regulation of Large Conductance Ca2+-Activated Potassium (BK) Channel Protein Expression in Kidney
Stimulatory Role of SPAK Signaling in the Regulation of Large Conductance Ca2+-Activated Potassium (BK) Channel Protein Expression in Kidney
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DOI：
10.3389/fphys.2020.00638
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发表时间：
2020-07-02
影响因子：
4
通讯作者：
Cai, Hui
中科院分区：
文献类型：
--
作者：
Bi, Ye;Li, Chunmei;Cai, Hui
SPS 1相关的脯氨酸/富含丙氨酸激酶（SPAK）在调节多种离子通道和转运蛋白的功能中起重要作用。无赖氨酸（WNK）激酶磷酸化SPAK激酶以激活SPAK信号通路。我们的前期研究表明WNK激酶通过ERK 1/2信号通路调节大电导Ca ~（2+）激活的K ~+（BK）通道的活性及其蛋白表达。SPAK激酶是否直接调节肾脏BK蛋白的表达尚不清楚。在这项研究中，我们研究了SPAK对HEK 293细胞和小鼠肾脏BK蛋白表达的影响。在HEK 293细胞中，siRNA介导的SPAK表达的敲低显著降低BK蛋白表达并增加ERK 1/2磷酸化，而SPAK的过表达以剂量依赖性方式显著增强BK表达并降低ERK 1/2磷酸化。ERK 1/2的敲低阻止了SPAK siRNA介导的BK表达抑制。同样，用溶酶体抑制剂巴弗洛霉素A1或蛋白酶体抑制剂MG 132预处理HEK 293细胞，逆转了SPAK敲低对BK表达的抑制作用。我们还发现，在SPAK KO小鼠的CCD的PC中没有BK通道活性，使用分离的分裂开放小管单细胞贴敷。此外，我们发现SPAK基因敲除小鼠肾脏中BK蛋白丰度显著降低，ERK 1/2磷酸化显著增强。高钾饮食显著增加BK蛋白丰度和SPAK磷酸化水平，同时降低ERK 1/2磷酸化水平。这些发现表明，SPAK通过减少ERK 1/2信号介导的BK通道的溶酶体和蛋白酶体降解来增强BK蛋白表达。
SPS1-related proline/alanine-rich kinase (SPAK) plays important roles in regulating the function of numerous ion channels and transporters. With-no-lysine (WNK) kinase phosphorylates SPAK kinase to active the SPAK signaling pathway. Our previous studies indicated that WNK kinases regulate the activity of the large-conductance Ca2+-activated K+(BK) channel and its protein expression via the ERK1/2 signaling pathway. It remains largely unknown whether SPAK kinase directly modulates the BK protein expression in kidney. In this study, we investigated the effect of SPAK on renal BK protein expression in both HEK293 cells and mouse kidney. In HEK293 cells, siRNA-mediated knockdown of SPAK expression significantly reduced BK protein expression and increased ERK1/2 phosphorylation, whereas overexpression of SPAK significantly enhanced BK expression and decreased ERK1/2 phosphorylation in a dose-dependent manner. Knockdown of ERK1/2 prevented SPAK siRNA-mediated inhibition of BK expression. Similarly, pretreatment of HEK293 cells with either the lysosomal inhibitor bafilomycin A1 or the proteasomal inhibitor MG132 reversed the inhibitory effects of SPAK knockdown on BK expression. We also found that there is no BK channel activity in PCs of CCD in SPAK KO mice using the isolated split-open tubule single-cell patching. In addition, we found that BK protein abundance in the kidney of SPAK knockout mice was significantly decreased and ERK1/2 phosphorylation was significantly enhanced. A high-potassium diet significantly increased BK protein abundance and SPAK phosphorylation levels, while reducing ERK1/2 phosphorylation levels. These findings suggest that SPAK enhances BK protein expression by reducing ERK1/2 signaling-mediated lysosomal and proteasomal degradations of the BK channel.