Dopamine inhibition of proximal tubule Na, K-ATPase

描述（由申请人提供）：肾钠稳态是血压的主要决定因素，受多种内分泌、自分泌和神经因素调节。这些因素起作用的分子机制尚不清楚，但涉及管状钠重吸收速率的调节。一些证据表明，Na+，K+- atp酶活性的调节至少在一定程度上是多巴胺在肾近端小管诱导的钠重吸收减少的原因。在细胞水平上，多巴胺触发一个特定的信号级联，最终导致细胞膜Na+，K+- atp酶的内吞，我们已经证明这一过程是导致Na+，K+- atp酶活性降低的原因。本研究的目的是：1)确定Na+，K+- atp酶α -亚基Ser-18磷酸化产生的信号如何传递到a-亚基的其他结构域，如脯氨酸位点（81-TPPPTTP-87）和“内噬序列”（537-YLEL）。2)验证PI3K结合Na+，K+- atp酶α -亚基脯氨酸位点（81-TPPPTTP-87）并形成支架，募集参与Na+，K+- atp酶分子内吞作用的其他蛋白的假设。3)确定多巴胺诱导Na+，K+- atp酶内吞过程中与质膜交换Na+，K+- atp酶分子的胞内池的位置和大小。4)确定多巴胺信号级联中对细胞内钠浓度变化敏感的阶段。5)通过产生Na+，K+- atp酶耐多巴胺调节的转基因小鼠，验证多巴胺在体内的调节作用。这些研究将深入了解多巴胺促进肾近端小管钠排泄的分子机制。在充血性心力衰竭、肝硬化、肾功能衰竭和高血压患者中经常发现肾钠重吸收调节的改变。更好地了解参与肾钠转运的蛋白质的调节机制将有助于开发治疗这些疾病的新方法。

DESCRIPTION (provided by applicant): Renal sodium homeostasis, a major determinant of blood pressure, is regulated by a variety of endocrine, autocrine, and neural factors. The molecular mechanism by which these factors act is not well understood but involves regulation of the rate of tubular sodium reabsorption. Several lines of evidence indicate that regulation of Na+,K+-ATPase activity is, at least in part, responsible for the reduced sodium reabsorption induced by dopamine in renal proximal tubules. At the cellular level, dopamine triggers a specific signaling cascade that ultimately leads to endocytosis of membrane Na+,K+-ATPase, a process that we have demonstrated is responsible for the decreased Na+,K+-ATPase activity. The aims of this proposal are: 1) To determine how the signal originated by phosphorylation of Na+,K+-ATPase alpha-subunit Ser-18 is transmitted to other domains of the a-subunit, such as the polyproline site (81-TPPPTTP-87) and the "endocytic sequence" (537-YLEL). 2) To test the hypothesis that PI3K binds to the Na+,K+-ATPase alpha-subunit polyproline site (81-TPPPTTP-87) and forms a scaffold for recruitment of other proteins that participate in the endocytosis of Na+,K+-ATPase molecules. 3) To identify the location and size of the intracellular pool that exchanges Na+,K+-ATPase molecules with the plasma membrane during dopamine induced Na+,K+-ATPase endocytosis. 4) To identify the stage(s) within the dopamine signaling cascade that is/are sensitive to changes of intracellular sodium concentration. 5) To test the in vivo regulatory role of dopamine by producing transgenic mice that express Na+,K+-ATPase molecules that are resistant to regulation by dopamine. These studies will provide insight into the molecular mechanism by which dopamine promotes renal sodium excretion in proximal tubules. Alterations in the regulation of kidney sodium reabsorption are often found on patients with congestive heart failure, cirrhosis, renal failure, and hypertension. Better knowledge of the mechanism of regulation of proteins involved in renal sodium transport will facilitate the development of new approaches in the treatment of patients with these disorders.