WNK kinases are a family of serine/threonine kinases (1). Mutations of WNK1 and WNK4 kinases result in pseudohypoaldosteronism type II (PHA II), featuring hypertension, hyperkalemia and metabolic acidosis (4). WNK kinases play an important role in maintaining electrolyte homeostasis (5). WNKs not only modulate sodium chloride cotransporter (NCC) (6), sodium potassium chloride cotransporter (NKCC) (7; 8), potassium chloride cotransporter (KCC) (7), and epithelial sodium channel (ENaC) (9; 10), but also affect renal outer medullary potassium channel (ROMK) (11; 12). During the previous funding period, we showed that WNK4 inhibits the activity and protein expression of the BK channel, another major renal potassium channel (13), whereas WNK1 increases its activity and protein expression by inhibiting the ERK 1/2 signaling pathway (14). In kidney, BK channels are mainly expressed in the distal nephron (15; 16). Previous studies have shown that the kidney specific isoform of WNK1 (KS-WNK1) also plays an important role in the regulation of ROMK (17; 18). High potassium (K) diets affect ROMK and BK channels (16; 19; 20). Changes in dietary K also affect WNK kinase expression (17; 21). Our preliminary data showed that changes in dietary potassium affect BK protein abundance through a WNK1-mediated ERK 1/2 signaling pathway involving aldosterone. We found that KS-WNK1 inhibits BK protein expression. Our yeast-two hybrid screen using parts of WNK4 as baits reveals a interacting protein from the MAPK signaling pathway, i.e., RanBPM, a potential modulator of ERK 1/2. In addition, we found that 14-3-3 ? decreases BK protein expression while enhancing ERK 1/2 phosphorylation. Therefore, our overall hypothesis is that 1) WNK modulates BK channel protein expression via ERK 1/2 signaling pathway through its interaction with RanBPM and 14-3-3, and 2) aldosterone modulates WNK-mediated regulation of BK. To test this hypothesis we have proposed the following specific aims as seen in Figure 1:
Specific Aim 1 : Investigate the role of interactions among WNK4, WNK1 and KS-WNK1 in the regulation of BK channel activity and protein expression in both cells and mice.
Specific Aim 2 : Investigate the role of RanBPM in the regulation of BK protein expression in cells and ERK 1/2 knockout mice.
Specific Aim 3 : Characterize the 14-3-3-mediated reduction of BK protein expression by examining BK ubiquitination and degradation through an ERK 1/2 signaling pathway in renal epithelial cells. We will apply the methodologies of patch-clamp technique, cell biology, molecular biology, protein biochemistry and in vivo animal study to perform the proposed experiments. Further exploration of how WNK and its interacting proteins affect BK channel function as well as the elucidation of the underlying pathophysiologic mechanism of PHA II caused by mutations of WNK kinases will provide a novel view on the regulation of the potassium channel activity and K secretion involving WNK kinase signaling in this research field.
WNK kinase plays an important role in the regulation of blood pressure and potassium homeostasis. Big potassium (BK) channel is one of the two major potassium (K) channels responsible for K excretion. BK becomes crucial in renal K excretion in response to high K dietary challenges. Hypertension and hyperkalemia are common problems, especially in those with kidney diseases. The aim of the present project is to investigate the role of WNK and its interacting proteins in the regulation of BK function and elucidate the mechanism of how high K diet and aldosterone affect WNK-mediated regulation of BK channel and its underlying signaling pathway. Our studies will form the basis for future translational studies of BK regulation and for providing an insight into developing therapeutic agents for treating hypertension and hyperkalemia.
