SPAK Signaling Stimulates the Activity and Protein Expression of Large Conductance Ca<sup>2+</sup> Activated Potassium (BK) Channels
October 22, 2020 | 10:00 AM - 12:00 PM
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SPAK Signaling Stimulates the Activity and Protein Expression of Large Conductance Ca2+ Activated Potassium (BK) Channels
- Fluid, Electrolyte, and Acid-Base Disorders: Basic
October 22, 2020 | Location: On-Demand
Abstract Time: 10:00 AM - 12:00 PM
Category: Fluid, Electrolyte, and Acid-Base Disorders
- 901 Fluid, Electrolyte, and Acid-Base Disorders: Basic
- Bi, Ye, Emory University School of Medicine, Atlanta, Georgia, United States
- Li, Chunmei, Emory University School of Medicine, Atlanta, Georgia, United States
- Wang, Xiaonan H., Emory University School of Medicine, Atlanta, Georgia, United States
- Delpire, Eric J., Vanderbilt University Medical Center, Nashville, Tennessee, United States
- Hoover, Robert S., Emory University School of Medicine, Atlanta, Georgia, United States
- Eaton, Douglas C., Emory University School of Medicine, Atlanta, Georgia, United States
- Cai, Hui, Emory University School of Medicine, Atlanta, Georgia, United States
Xiaonan H. Wang,
Eric J. Delpire,
Robert S. Hoover,
Douglas C. Eaton,
Ste20-like 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 activity and protein expression in kidney.
Electrophysiology, cell culture, western blot, siRNA knockdown, and SPAK knockout (KO) mice were used in the study.
We first determined the effects of SPAK gene depletion using SPAK KO mice on BK channel activity in the isolated, split-opened renal collecting ducts (CD) from WT and SPAK KO mice. We found that there is no BK channel activity in principal cells (PCs) of cortical CD (CCD) in SPAK KO mice, whereas there is BK channel activity in PCs from WT mice. We further investigated the effects of overexpression and siRNA knockdown of SPAK expression on BK in HEK293 cells. Overexpression of SPAK significantly increased BK protein expression with decreasing ERK 1/2 phosphorylation, whereas knockdown of SPAK expression using siRNA significantly reduced BK protein expression associated with increased ERK1/2 phosphorylation, both in a dose-dependent manner. Knockdown of ERK1/2 prevented SPAK siRNA-mediated inhibition of BK protein expression. Similarly, pretreatment of HEK293 cells with either the lysosomal inhibitor bafilomycin A1 or proteasomal inhibitor MG132, reversed the inhibitory effects of SPAK knockdown on BK protein expressions. In addition, we found that BK protein abundance in the renal cortex of SPAK KO mice was significantly decreased and ERK1/2 phosphorylation was significantly enhanced. A high potassium diet significantly increased BK protein abundance and SPAK phosphorylation levels in WT mice, while reducing ERK1/2 phosphorylation levels.
These findings suggest that SPAK stimulates BK channel activity and protein expression by reducing ERK1/2 signaling-mediated lysosomal and proteasomal degradations of the BK channel.
- NIDDK Support