Regulation of ENaC by Casein Kinase 2

高血压影响着美国和全世界数百万人。更好地了解 高血压的分子和细胞起源将提高治疗和诊断的功效。的 上皮Na+通道（ENaC）是肾脏Na+排泄的最终仲裁者。因此，酌情控制权 的ENaC微调肾脏排泄。适当的肾脏排泄是正常调节的关键因素， 动脉血压因此，ENaC及其上游调节剂的功能障碍导致调节异常 由于排泄异常而引起的高血压。已知酪蛋白激酶2（CK 2）使ENaC磷酸化。的 然而，这在生理上的重要性还不清楚。我们的初步结果表明， CK 2是正常ENaC活性和肾脏Na+排泄所必需的。CK 2磷酸化位点 ENaC位于典型的“锚”锚蛋白结合基序内。ENaC中的该网站与CK 2有相似之处 在不相关的NaV和KCNQ通道中的磷酸化位点，其也位于“锚”基序内。 CK 2对NaV和KCNQ通道的磷酸化作用是一种分子“开关”，有利于与 锚蛋白-3（Ank-3）。Ank-3的结合促进了这些通道的适当膜定位 增加他们的活动。在主细胞（PC）中靶向缺失Ank-3显著降低ENaC 在我们的PC-Ank-3 KO小鼠中的活性。考虑到我们强有力的初步结果和可能的 CK 2对ENaC、NaV和KCNQ的收敛演化整形规则，我们提出测试的前提是， 在“锚”基序内，CK 2对ENaC的磷酸化是Ank-3结合到ENaC上所必需和充分的。 通道，这是正常的通道位置和功能所需的，以及肾脏Na+的适当调节 排泄我们将使用多学科的方法来测试这些想法，包括新颖的思维和工具， 包括PC特异性CK 2和Ank-3 KO小鼠，以及与研究相关的高度严谨性。 一个范围广泛的设计，询问有关分子和细胞机制以及整体的问题， 动物的生理后果。以下具体目标将用于测试我们的想法：1）确定 CK 2调节ENaC活性的细胞和分子机制; 2）定量分析ENaC活性的变化， ENaC的CK 2调节的生理功能;和3）确定ENaC的CK 2调节是否是 在人类中保存。如果CK 2对ENaC的调节具有临床和生理重要性， 调节必须在整个门中保持，特别是在人类中。我们量化ENaC的开创性努力 来自健康捐赠者肾脏的小管活性使我们能够在最相关的环境中测试我们的想法 可能的：天然集合管内的人类主细胞。在实现这些目标后，我们将 了解ENaC的CK 2磷酸化是否、如何以及何时起“开关”作用，以促进Ank-3结合增加 通道活动，包括对调节这种调节的机制的详细了解，以及 对这种调节的生理后果的丰富认识。

Hypertension affects millions of people in the United States and worldwide. Improved understanding of the molecular and cellular origins of hypertension will improve the efficacy of treatment and diagnosis. The Epithelial Na+ Channel, ENaC, is the final arbiter of Na+ excretion in the kidneys. As such, discretionary control of ENaC fine-tunes renal excretion. Appropriate renal excretion is a key factor in the normal regulation of arterial blood pressure. Consequently, dysfunction of ENaC and its upstream modulators cause dysregulation of blood pressure due to abnormal excretion. Casein kinase 2 (CK2) is known to phosphorylate ENaC. The physiological importance of this, though, is obscure. Our preliminary results demonstrate that phosphorylation by CK2 is necessary for normal ENaC activity and renal Na+ excretion. The CK2 phosphorylation site within ENaC resides within a canonical “anchor” ankyrin binding motif. This site in ENaC shares similarity to CK2 phosphorylation sites in the unrelated NaV and KCNQ channels, which also lie within “anchor” motifs. Phosphorylation of NaV and KCNQ channels by CK2 acts as a molecular “switch” favoring the binding of ankyrin-3 (Ank-3). The binding of Ank-3 facilitates the proper membrane localization of these channels increasing their activity. The targeted deletion of Ank-3 in principal cells (PC) significantly decreased ENaC activity in our PC-Ank-3 KO mouse. In consideration of our strong preliminary results and the possible convergent evolution shaping regulation of ENaC, NaV and KCNQ by CK2, we propose testing the premise that phosphorylation of ENaC by CK2 within “anchor” motifs is necessary and sufficient for Ank-3 binding to the channel, which is required for normal channel locale and function, and the proper regulation of renal Na+ excretion. We will test these ideas using a multidisciplinary approach that includes novel thinking and tools, including PC-specific CK2 and Ank-3 KO mice, and a high degree of rigor in conjunction with a research design that is broad in scope asking questions about molecular and cellular mechanisms as well as whole animal physiological consequences. The following specific aims will be used to test our ideas: 1) To determine the cellular and molecular mechanisms of CK2 regulation of ENaC activity; 2) To quantify the physiological function of CK2 regulation of ENaC; and 3) To determine if CK2 regulation of ENaC is conserved in humans. If CK2 regulation of ENaC is to be of clinic and physiological importance such regulation must be conserved across phyla particularly in humans. Our pioneering efforts to quantify ENaC activity in tubules from healthy donor human kidneys allows us to test our ideas in the most relevant setting possible: the human principal cell within the native collecting duct. After accomplishing these aims, we will know if, how and when CK2 phosphorylation of ENaC functions as a “switch” to favor Ank-3 binding to increase channel activity to include having a detailed understanding of the mechanisms mediating this regulation, and a rich appreciation of the physiological consequences of such regulation.