Abstract
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.
Highlights
Pseudohypoaldosteronism type II (PHAII), characterized by hypertension, hyperkalemia, and metabolic acidosis (Hadchouel et al, 2016), results from mutations in WNK kinase family members WNK1 and WNK4 (Wilson et al, 2001)
Our previous studies have shown that a HK diet increased BK channel expression in the kidney and that this was associated with a decrease in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation status (Liu et al, 2015)
To determine whether SPAK signaling is involved in the regulation of BK protein, we investigated the effects of an HK (10%) diet (10 days) on the levels of BK protein, and phosphorylation levels of SPAK and ERK signaling in WT mice
Summary
Pseudohypoaldosteronism type II (PHAII), characterized by hypertension, hyperkalemia, and metabolic acidosis (Hadchouel et al, 2016), results from mutations in WNK kinase family members WNK1 and WNK4 (Wilson et al, 2001). The renal distal nephron contains two major potassium channels, ROMK ( known as Kir1.1 or KCNJ1) and BK (big potassium, Maxi-K, or Slo) channels (Bailey et al, 2006). The BK channels, which are expressed in the apical membrane of intercalated cells and principal cells (Welling, 2016), are large-conductance, voltageand calcium-activated potassium channels responsible for “flowinduced potassium secretion” (Woda et al, 2001; Grimm and Sansom, 2007). The alpha subunit functions as an independent channel. Several studies have shown that members of the WNK family are involved in the regulation of BK channels (Zhuang et al, 2011; Liu et al, 2015)
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