Regulation of Renal BK Channel by WNK Kinase and its Interacting Proteins

 描述（由申请人提供）： WNK激酶是丝氨酸/苏氨酸激酶的一个家族（1）。WNK 1和WNK 4激酶的突变导致II型假性醛固酮减少症（PHA II），以高血压、高钾血症和代谢性酸中毒为特征（4）。WNK激酶在维持电解质稳态方面发挥重要作用（5）。WNK不仅调节氯化钠协同转运蛋白（NCC）（6）、氯化钠钾协同转运蛋白（NKCC）（7; 8）、氯化钾协同转运蛋白（KCC）（7）和上皮钠通道（ENaC）（9; 10），而且影响肾外髓钾通道（ROMK）（11; 12）。在之前的资助期间，我们发现WNK 4抑制BK通道（另一种主要的肾脏钾通道）的活性和蛋白表达（13），而WNK 1通过抑制ERK 1/2信号通路增加其活性和蛋白表达（14）。在肾脏中，BK通道主要在远端肾单位中表达（15; 16）。先前的研究已经表明，WNK 1的肾脏特异性同种型（KS-WNK 1）在ROMK的调节中也起重要作用（17; 18）。高钾（K）饮食影响ROMK和BK通道（16; 19; 20）。膳食钾的变化也影响WNK激酶的表达（17; 21）。我们的初步数据表明，饮食中钾的变化影响BK蛋白的丰度通过WNK 1介导的ERK 1/2信号通路，涉及醛固酮。我们发现KS-WNK 1抑制BK蛋白的表达。我们使用WNK 4的部分作为诱饵的酵母双杂交筛选揭示了来自MAPK信号通路的相互作用蛋白，即，RanBPM是ERK 1/2的潜在调节剂。此外，我们发现14-3-3 γ降低BK蛋白表达，同时增强ERK 1/2磷酸化。因此，我们的总体假设是1）WNK通过ERK 1/2信号通路通过其与RanBPM和14-3-3的相互作用调节BK通道蛋白表达，和2）醛固酮调节WNK介导的BK调节。为了验证这一假设，我们提出了以下具体目标，如图1所示：具体目标1：研究WNK 4、WNK 1和KS-WNK 1之间的相互作用在细胞和小鼠中对BK通道活性和蛋白表达的调节中的作用。具体目的2：研究RanBPM在细胞和ERK 1/2基因敲除小鼠中对BK蛋白表达的调节作用。具体目标3：通过检查肾上皮细胞中ERK 1/2信号通路引起的BK泛素化和降解，表征14-3-3介导的BK蛋白表达减少。我们将应用膜片钳技术、细胞生物学、分子生物学、蛋白质生物化学和动物体内研究等方法进行实验。进一步探讨WNK及其相互作用蛋白对BK通道功能的影响，阐明WNK激酶突变引起的PHA II的病理生理机制，将为WNK激酶信号通路调控钾通道活性和钾分泌提供新的视角。

 DESCRIPTION (provided by applicant): Project Summary 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 γ 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.