Modulation and Regulation of ROMK Channels in Kidney

描述（由申请人提供）：无赖氨酸激酶4（WNK 4）在调节醛固酮敏感性远端肾单位（ASDN）中的Na和K转运中起重要作用。WNK 4突变导致家族性高钾血症性高血压，这是一种常染色体显性遗传疾病，其特征为盐敏感性高血压、高钾血症和肾小管酸中毒（假性醛固酮减少症II型）。大量证据表明，WNK 4抑制ROMK，调节ENaC并调节集合管中的细胞旁Cl渗透性。WNK 4对ROMK的抑制作用被血清糖皮质激素诱导的激酶1（SGK 1）介导的WNK 4在“开关结构域”中的磷酸化减弱。最近，我们进一步证明Src家族蛋白酪氨酸激酶（SFK），如c-Src，磷酸化WNK 4并减少SGK 1诱导的WNK 4磷酸化，从而恢复WNK 4对ROMK通道的抑制作用。WNK 4、SFK和SGK 1之间的相互作用在增加钾摄入时刺激钾分泌和防止容量耗竭时钾流失方面起着重要作用。然而，c-Src调节SGK 1与WNK 4相互作用的分子机制尚不清楚。本提案的目的是检验以下假设：SFK介导的WNK 4磷酸化激活蛋白磷酸酶1（PP 1），从而消除SGK 1诱导的WNK 4磷酸化刺激并恢复WNK 4对ROMK 1的抑制作用。具体目的1是检验c-Src结合并磷酸化WNK 4以及WNK 4的Tyr磷酸化消除SGK 1诱导的WNK 4开关结构域磷酸化从而将WNK 4锁定在ROMK抑制模式的假设。具体目的2是检验PP 1与WNK 4结合并参与介导c-Src对WNK 4诱导的ROMK抑制作用的假设。具体目的3是检验以下假设：PTP 1D结合WNK 4，调节PP 1活性，并参与SGK 1、WNK 4和c-Src之间的相互作用，这是一种特异性调节ROMK但对ENaC无影响的机制。该项目的意义在于扩展目前对ASDN中Na和K转运调控的理解，并提供ASDN中WNK 4介导的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.