Regulation of ENaC by Casein Kinase 2

酪蛋白激酶 2 对 ENaC 的调节

• 批准号: 10241447
• 负责人: James D Stockand
• 金额: $ 34.31万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-05 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10241447
• 关键词: ANK3 gene; Address; Affect; Aldosterone; Amino Acid Motifs; Amino Acid Sequence; Animals; Ankyrins; Binding; Binding Sites; Biopsy; Blood Pressure; Cell Line; Cell membrane; Cells; Clinic; Clinical; Consensus; Consensus Sequence; Cytoskeleton; Diagnosis; Duct (organ) structure; Ductal Epithelial Cell; Electrophysiology (science); Evolution; Excretory function; Family; Functional disorder; Harvest; Human; Hypertension; Investigation; Ion Channel; Kidney; Knock-out; Knockout Mice; Knowledge; Literature; Locales; Location; MAPK1 gene; Measurement; Mediating; Membrane; Molecular; Molecular Genetics; Myocardial Infarction; Phosphorylation; Phosphorylation Site; Physiological; Potassium Channel; Preparation; Protein Kinase; Regulation; Research Design; Risk Factors; Shapes; Site; Sodium; Sodium Chloride; Stroke; Testing; Thinking; Transmembrane Domain; Treatment Efficacy; United States; Urine; Water; blood pressure regulation; casein kinase II; epithelial Na+ channel; extracellular; gain of function mutation; gamma ENaC; improved; in vivo; interdisciplinary approach; loss of function; novel; patch clamp; tool; trafficking; urinary; voltage; wasting

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.

高血压会影响美国和全球数百万人。提高了对 高血压的分子和细胞起源将提高治疗和诊断的效率。 ENAC上皮Na+通道是Kidneys中Na+排泄物的最后仲裁者。因此，酌处控制 ENAC微型肾脏排泄。适当的肾脏排泄是正常调节的关键因素 动脉血压。因此，ENAC及其上游调节器的功能障碍引起失调 由于排泄异常而引起的血压。酪蛋白激酶2（CK2）已知磷酸化ENAC。这 但是，生理上的重要性是晦涩的。我们的初步结果表明磷酸化 CK2对于正常的ENAC活性和肾脏Na+排泄是必需的。 CK2磷酸化位点 在规范的“锚固” ANKYRIN结合基序中的ENAC住宅。 ENAC中的此网站与CK2有相似之处 无关的NAV和KCNQ通道中的磷酸化位点，它们也位于“锚”基序内。 CK2对NAV和KCNQ通道的磷酸化充当分子“开关”，有利于结合 Ankyrin-3（ANK-3）。 ANK-3的结合促进了这些通道的适当膜定位 增加活动。主细胞（PC）中ANK-3的靶向缺失显着降低了ENAC 在我们的PC-AK-3 KO鼠标中的活动。考虑到我们强大的初步结果以及可能的 CK2通过CK2的ENAC，NAV和KCNQ的融合演变调节，我们提出了测试前提。 CK2在“锚定”基序中对ENAC的磷酸化是必要的，足以与ANK-3结合到与 通道，这是正常通道位置和功能所需的，以及肾脏NA+的适当调节 排泄。我们将使用包括新颖思维和工具的多学科方法来测试这些想法 包括PC特异性CK2和ANK-3 KO小鼠，以及与研究的高度严格程度 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 动物生理后果。以下特定目标将用于测试我们的想法：1）确定 CK2调节ENAC活性的细胞和分子机制； 2）量化 CK2调节ENAC的身体功能； 3）确定CK2的ENAC调节是否为 在人类中保守。如果CK2的ENAC调节是临床和身体重要性的 必须在整个门中保守调节，尤其是在人类中。我们量化ENAC的开创性努力 健康供体人类肾脏中的小管中的活动使我们能够在最相关的环境中测试我们的想法 可能：天然收集管中的人类主要细胞。完成这些目标后，我们将 知道ENAC的CK2磷酸化是否，如何以及何时作为“开关”起作用以增加ANK-3的结合 渠道活动，包括对介导该调节的机制有详细的了解，并 对这种调节的身体后果的丰富欣赏。