PHYSIOLOGY OF ION TRANSPORT MECHANISM OF THE NA PUMP

NA 泵离子传输机制的生理学

• 批准号: 3280094
• 负责人: BLISS FORBUSH
• 金额: $ 28.82万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-04-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3280094
• 关键词: Xenopus; adenosinetriphosphatase; affinity labeling; chemical binding; complementary DNA; dogs; enzyme biosynthesis; enzyme mechanism; enzyme structure; fluorescent dye /probe; gel electrophoresis; genetic manipulation; genetic transcription; high performance liquid chromatography; immunofluorescence technique; ion transport; kidney; laboratory rabbit; membrane potentials; membrane proteins; messenger RNA; molecular cloning; phosphorylation; protein sequence; radiotracer; sodium potassium exchanging ATPase

Coupling the energy of ATP hydrolysis to the transport of sodium and potassium, the Na, K-ATPase of animal cell plasma membranes maintains the non-equilibrium ion distributions that are necessary for cellular function. The maintenance of high K and low Na levels inside the cell is of fundamental importance in control of cell volume, in the movement of other solutes and water across cell membranes, in the net movement of salt and water across epithelial cells, and in the electrical excitability of nerve and muscle. Thus an understanding of this membrane transport process is vital to progress in all areas of human physiology, metabolism, and disease. While studies in the last twenty-five years have led to detailed knowledge of the steady-state characteristics of ion transport and of rapid changes in enzyme conformation we have only recently begun to assign the movements of Na and K to particular steps in the enzymatic cycle. 1) Our recent investigations have led to an understanding of the K-transporting phase of the cycle, involving an "occluded" state of Na, K-ATPase in which K ions are hidden within the structure of the protein during their transport across the membrane. In the proposed work we will continue to use novel rapid kinetic techniques developed in this laboratory to study the events involved in Na and K transport, concentrating on events in the Na-transporting phase of the cycle. We will study the time course of Na release from Na K-ATPase during transport, and the formation and breakdown of a state in which Na is occluded. The Na translocation events will be temporally correlated with formation and breakdown of phosphorylated intermediates, with changes in conformational state of the protein, and with transmembrane charge movement. 2) We will also attempt to discover the identify of amino acids involved in cation transport, by labeling the Na, K-ATPase with photosensitive organic cations that become occluded at the Na or K sites. 3) Previous investigations have shown that in addition to the alpha and beta subunits of Na, K-ATPase, a low molecular weight membrane protein, a putative gamma subunit, is photolabeled by ouabain derivatives that are specifically bound to the enzyme. We propose to further characterize this protein with immunological and biochemical techniques, as well as through the isolation of cDNA clones encoding its peptide sequences. These molecular probes will be used to examine the level of expression in various tissues, to deduce the amino acid sequence of the peptide and to determine its functional role through in vitro expression. 4) Finally, we plan to use molecular biologic techniques to study the biosynthetic assembly, processing and sorting of Na, K-ATPase. We will transcribe mRNA from cDNA clones encoding the subunits of Na, K- ATPase, and inject this mRNA into Xenopus laevis oocytes. Immunofluorescence and immunoprecipitation will be used to determine the requirements for transport of individual or assembled subunits to the cell surface, as well as to determine the involvement of endogenous Xenopus components.

将 ATP 水解的能量与钠的运输耦合 和钾，动物细胞质膜的Na，K-ATP酶 维持必要的非平衡离子分布 用于细胞功能。 维持高钾和低钠水平 细胞内部对于细胞的控制至关重要 体积，其他溶质和水穿过细胞的运动 膜，盐和水穿过上皮的净运动 细胞，以及神经和肌肉的电兴奋性。 因此，了解这种膜运输过程至关重要 人类生理学、新陈代谢和各个领域的进步 疾病。 虽然过去二十五年的研究已经导致 离子稳态特性的详细知识 运输和酶构象的快速变化我们只有 最近开始将 Na 和 K 的运动分配给特定的 酶促循环中的步骤。 1) 我们最近的调查 导致了对循环中 K 运输阶段的理解， 涉及 Na、K-ATP 酶的“封闭”状态，其中 K 离子 在运输过程中隐藏在蛋白质结构内 穿过膜。 在拟议的工作中，我们将继续使用 本实验室开发的新型快速动力学技术 研究涉及 Na 和 K 运输的事件，重点关注 循环中钠运输阶段的事件。 我们将学习 运输过程中 Na K-ATP 酶释放 Na 的时间过程， 以及Na吸留状态的形成和破坏。 Na 易位事件将在时间上与 磷酸化中间体的形成和分解， 蛋白质构象状态的变化，以及 跨膜电荷运动。 2）我们还将尝试发现 鉴定参与阳离子运输的氨基酸，通过 用光敏有机阳离子标记 Na, K-ATP 酶 在 Na 或 K 位点处被遮挡。 3) 之前的调查 已经表明，除了 Na 的 α 和 β 亚基之外， K-ATP酶，一种低分子量膜蛋白，假定的γ 亚基，由哇巴因衍生物光标记， 与酶特异性结合。 我们建议进一步 通过免疫学和生化来表征该蛋白质 技术以及通过 cDNA 克隆的分离 编码其肽序列。 这些分子探针将 用于检测不同组织中的表达水平， 推导肽的氨基酸序列并确定其 通过体外表达发挥功能作用。 4）最后，我们计划 利用分子生物学技术研究生物合成 Na、K-ATP酶的组装、加工和分选。 我们将 从编码 Na、K- 亚基的 cDNA 克隆转录 mRNA ATPase，并将该 mRNA 注射到非洲爪蟾卵母细胞中。 免疫荧光和免疫沉淀将用于 确定单个或组合运输的要求 亚基到细胞表面，以及确定 内源性非洲爪蟾成分的参与。