Modulation and Regulation of ROMK Channels in Kidney

描述（由申请人提供）：withno - lysine Kinase 4 （WNK4）在醛固酮敏感远端肾元（ASDN）中调节Na和K转运中起重要作用。WNK4突变导致家族性高钾血症高血压，这是一种常染色体显性疾病，以盐敏感性高血压、高钾血症和肾小管酸中毒为特征（假性低醛固酮症II型）。大量证据表明，WNK4抑制ROMK，调节ENaC并调节集管中细胞旁Cl的通透性。血清糖皮质激素诱导的激酶1 （SGK1）介导的WNK4“开关结构域”磷酸化可减弱WNK4对ROMK的抑制作用。最近，我们进一步证明了src家族蛋白酪氨酸激酶（SFK），如c-Src，磷酸化WNK4，并减少sgk1诱导的WNK4磷酸化，从而恢复WNK4对ROMK通道的抑制作用。WNK4、SFK和SGK1之间的相互作用在增加K摄入量时刺激K分泌和在体积消耗时防止K损失中起重要作用。然而，c-Src调节SGK1与WNK4相互作用的分子机制尚不清楚。本研究的目的是验证sfk介导的WNK4磷酸化激活蛋白磷酸酶1 （PP1）的假设，从而消除sgk1诱导的WNK4磷酸化刺激，恢复WNK4对ROMK1的抑制作用。具体目的1是验证c-Src结合并磷酸化WNK4的假设，以及WNK4的Tyr磷酸化消除了sgk1诱导的WNK4开关结构域的磷酸化，从而将WNK4锁定在ROMK的抑制模式。特异性目的2是验证PP1与WNK4结合并参与介导c-Src对WNK4诱导的ROMK抑制作用的假设。特异性目的3是验证PTP1D结合WNK4，调节PP1活性，参与SGK1、WNK4和c- Src之间相互作用的假设，该机制特异性调节ROMK，但对ENaC没有影响。本项目的意义在于扩大目前对ASDN中Na和K转运调控的认识，并提供wnk4介导的ASDN中ROMK调控的综合机制。

DESCRIPTION (provided by applicant): With-No-Lysine Kinase 4 (WNK4) plays an important role in regulating Na and K transport in the aldosterone-sensitive distal nephron (ASDN). Mutation of WNK4 causes familial hyperkalemic hypertension, an autosomal dominant disease characterized by salt-sensitive hypertension, hyperkalemia and renal tubule acidosis (Pseudohypoaldosteronism type II). A large body of evidence shows that WNK4 inhibits ROMK, regulates ENaC and modulates the paracellular Cl permeability in the collecting duct. The inhibitory effect of WNK4 on ROMK is attenuated by serum-glucocorticoid- induced kinase1 (SGK1)-mediated phosphorylation of WNK4 in the "switch-domain". Recently, we have further demonstrated that Src-family protein tyrosine kinases (SFK), such as c-Src, phosphorylates WNK4 and diminishes SGK1-induced phosphorylation of WNK4 thereby restoring the inhibitory effect of WNK4 on ROMK channels. The interaction among WNK4, SFK and SGK1 plays an important role in stimulating K secretion during increasing K intake and in preventing K loss during volume depletion. However, the molecular mechanism by which c-Src modulates the interaction of SGK1 with WNK4 is not understood. The goal of the present proposal is to test the hypothesis that SFK-mediated phosphorylation of WNK4 activates protein phosphatase 1 (PP1) thereby abolishing the SGK1-induced stimulation of WNK4 phosphorylation and restoring the inhibitory effect of WNK4 on ROMK1. Specific Aim 1 is to test the hypothesis that c-Src binds and phosphorylates WNK4 and that the Tyr phosphorylation of WNK4 abolishes SGK1-induced phosphorylation at WNK4's switch domain thereby locking WNK4 in inhibitory mode for ROMK. Specific Aim 2 is to test the hypothesis that PP1 binds to WNK4 and is involved in mediating the effect of c-Src on WNK4-induced inhibition of ROMK. Specific Aim 3 is to test the hypothesis that PTP1D binds to WNK4, modulates PP1 activity and participates in the interaction among SGK1, WNK4 and c- Src, a mechanism which specifically regulates ROMK but has no effect on ENaC. The significance of the project is to expand the current understanding regarding the regulation of Na and K transport in the ASDN and to provide an integrated mechanism of WNK4-mediated regulation of ROMK in the ASDN.