Modulation and Regulation of ROMK Channels in Kidney

描述(由申请人提供)：WNK4在调节醛固酮敏感型远端肾单位(ASDN)的钠钾转运中起着重要作用。WNK4基因突变导致家族性高钾型高血压，这是一种常染色体显性遗传病，以盐敏感型高血压、高钾血症和肾小管酸中毒(假性低醛固酮血症II型)为特征。大量证据表明，WNK4抑制ROMK，调节ENaC，调节集合管中细胞旁氯的通透性。血清糖皮质激素诱导的蛋白激酶1(SGK1)介导的WNK4在“开关域”的磷酸化减弱了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，可使WNK4磷酸化，并减弱SGK1诱导的WNK4的磷酸化，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的分泌和防止容量耗竭时K的丢失中起重要作用。然而，c-Src调控SGK1与WNK4相互作用的分子机制尚不清楚。本方案的目的是验证一种假设，即SFK介导的WNK4的磷酸化激活蛋白磷酸酶1(PP1)，从而取消SGK1诱导的WNK4磷酸化的刺激，并恢复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，即WNK4的Tyr磷酸化取消了SgK1诱导的WNK4‘S开关域的磷酸化，从而将WNK4锁定在ROMK的抑制模式中。具体目的2是验证PP1与WNK4结合并参与c-Src在WNK4诱导的ROMK抑制中的作用的假设。具体目的3是验证PTP1D与WNK4结合、调节PP1活性并参与SGK1、WNK4和c-Src之间相互作用的假设，这是一种特异性调节ROMK但对ENaC没有影响的机制。该项目的意义在于扩大目前对ASDN中Na、K转运调控的认识，并提供一套完整的WNK4介导的ASDN中ROMK调控机制。 与公众健康相关：肾脏钾(K)代谢紊乱会导致高钾血症或低钾血症，从而可能导致心律失常，而肾脏钠吸收障碍则会导致高血压或低血压。虽然肾脏钠转运的增加通常伴随着钾排泄的增加，但有时肾脏的钠转运与钾的分泌是分离的。然而，其潜在的机制并不完全清楚。我们已经确定了一种新的机制，通过这种机制，肾脏的钠吸收与肾脏的钾分泌是解偶联的。这一新概念将扩展目前关于肾脏K和Na转运的知识，并揭示家族性高钾型高血压的机制，这种疾病也被称为假性低醛固酮血症2型。

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. PUBLIC HEALTH RELEVANCE: Disturbance in renal potassium (K) results in hyperkalemia or hypokalemia which could lead to cardiac arrhythmia whereas disturbance in renal sodium (Na) absorption leads to hypertension or hypotension. Although increasing renal Na transport is usually coupled with enhanced K excretion, Na transport is sometimes dissociated with K secretion in the kidney. However, the underlying mechanism is not completely clear. We have identified a novel mechanism by which renal Na absorption is decoupled with renal K secretion. The new concept will expend the current knowledge regarding renal K and Na transport and shed a light into the mechanism of familial hyperkalemic hypertension , a disease also known as pseudohypoaldosteronism type 2.