REGULATION OF NA+, K+ -ATPASE TOPOGENESIS IN MDCK CELLS

MDCK细胞NA,K-ATP酶拓扑发生的调控

• 批准号: 3288431
• 负责人: W. James Nelson
• 金额: $ 14.1万
• 依托单位: INSTITUTE FOR CANCER RESEARCH
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-04-01 至 1990-03-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3288431
• 关键词: adenosinetriphosphatase; affinity chromatography; apical membrane; basolateral membrane; cell membrane; cytoskeleton; densitometry; density gradient ultracentrifugation; enzyme complex; fluorescence microscopy; gel electrophoresis; immunoprecipitation; potassium; proteolysis; radiotracer; sodium; spectrometry; stoichiometry; tissue /cell culture

Cells of transporting epithelia in the kidney and intestine form highly specialized monolayers which function as selective permeability barriers between the external medium and the circulatory system. This process depends upon a high degree of structural and functional polarity within the cells, which is reflected in the asymmetric distribution of enzymes and transport activities between the apical and basolateral domains of the plasma membrane. An essential enzyme in the function of these cells is the Na+,K+-ATPase. The Na+K+-ATPase is localized to the basolateral plasma membrane from where it generates significant transcellular fluxes of Na+ required for the active uptake of metabolites, the transport of water and the control of cell volume. The biological importance of the Na+K+-ATPase is reflected in the fact that altered levels of enzyme activity have profound effects at the cellular and organismal level, and are associated with hypertension, renal tubular transport defects, and uremia. The studies proposed here seek to elucidate the regulatory mechanisms involved in the assembly of the active AlphaBeta-unit complex, and the mechanism(s) involved in establishing and maintaining the polarized distribution of this important protein in differentiated epithelial cell layers in culture. Experiments are planned to analyze quantitatively the post-translational spatial and temporal regulation of assembly and turnover of newly synthesized subunits and their appearance at the plasma membrane. Experiments are planned also to selectively perturb the intracellular transport of the subunits to determine the subcellular site of assembly of the AlphaBeta-complex. The molecular mechanisms involved in establishing and maintaining the polarized distribution of the Na+,K+-ATPase will be investigated during transition of the enzyme from a nonpolarized to a polarized distribution, and by observing the fate of native Na+,K+-ATPase implanted directly into the apical plasma membrane of polarized epithelial cells. In addition, experiments are planned to identify proteins that bind specifically to the cytoplasmic domain of the Na+,K+-ATPase which may function to maintain the polarized distribution of the protein in the cell. Together, the results of this study will provide, for the first time, a comprehensive understanding of the regulatory mechanisms involved in the assembly and topogenesis of the Na+,K+-ATPase and, thereby, provide also a new insight into the assembly of nonviral multisubunit complexes and the formation of distinct protein domains on the plasma membrane.

肾和肠中的运输上皮细胞高度形成 充当选择性渗透屏障的特殊单分子层 外部介质和循环系统之间。 这个过程 取决于内部高度的结构和功能极性 细胞内酶的分布不对称， 顶端域和基底外侧域之间的运输活动 质膜。 这些细胞功能中必需的酶是 Na+,K+-ATP 酶。 Na+K+-ATP酶位于基底外侧血浆 膜从中产生显着的 Na+ 跨细胞通量 代谢物的主动吸收、水的运输和 细胞体积的控制。 Na+K+-ATP酶的生物学重要性 反映在酶活性水平改变的事实 在细胞和有机体水平上产生深远的影响，并与之相关 患有高血压、肾小管运输缺陷和尿毒症。 这 这里提出的研究旨在阐明所涉及的监管机制 活性 AlphaBeta 单元复合物的组装以及机制 参与建立和维持这种极化分布 培养物中分化的上皮细胞层中的重要蛋白质。 计划进行实验来定量分析翻译后 新事物的集结和周转的时空调节 合成的亚基及其在质膜上的出现。 还计划进行实验来选择性地干扰细胞内 亚基的运输以确定亚细胞组装位点 AlphaBeta复合体。 参与建立的分子机制 并维持 Na+,K+-ATPase 的极化分布 在酶从非极化到极化的转变过程中进行了研究 极化分布，并通过观察天然 Na+,K+-ATP 酶的命运 直接植入极化上皮细胞的顶端质膜 细胞。 此外，计划进行实验来鉴定结合的蛋白质 特异性针对 Na+,K+-ATPase 的胞质结构域，这可能 维持蛋白质极化分布的功能 细胞。 总之，这项研究的结果将首次提供 时间，全面了解所涉及的监管机制 参与 Na+,K+-ATP 酶的组装和拓扑发生，从而提供 也是对非病毒多亚基复合物组装的新见解 质膜上不同蛋白质结构域的形成。