Voltage-clamp studies of sodium pump current and flux

钠泵电流和通量的电压钳研究

• 批准号: 6773516
• 负责人: ROBERT F RAKOWSKI
• 金额: $ 26.41万
• 依托单位: OHIO UNIVERSITY ATHENS
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-05-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6773516
• 关键词: Xenopus; Xenopus oocyte; acrylamides; adenosine diphosphate; adenosine triphosphate; enzyme mechanism; gene mutation; intracellular; ion transport; marine toxins; membrane potentials; microelectrodes; potassium ion; sodium ion; sodium potassium exchanging ATPase; voltage /patch clamp

DESCRIPTION (provided by applicant): The objective of this project is to obtain a comprehensive understanding of the mechanism of ion translocation by the electrogenic Na+/K+ pump. The structure of the Na+/K+ pump can be predicted by homology modeling based on the atomic resolution structure of SERCA-1A (Toyoshima et al., 2000). Putative Na+ and K+ ion binding sites in the alpha subunit of the human (Ogawa and Toyoshima, 2002) and Xenopus oocyte Na+/K+ pump (Rakowski and Sagar, 2003) have been proposed based on an empirical method to predict site valence. Since the results can be interpreted based on simple electrostatic considerations, candidate mutations that only produce small changes in the structure of the targeted binding site are selected for further study. For example, increases in center-to-center distance should decrease the binding energy (affinity) of the site and increase the ion unbinding rate. To test these predictions the Na+/K+ pump can be limited to specific parts of the overall pump cycle by controlling ion and nucleotide concentrations. By operating in K+-free conditions and in the presence of both ATP and ADP the pump is restricted to its Na+/Na+ exchange mode. If intracellular [Na] is kept high internal Na+ binding sites are saturated and the kinetics of extracellular Na+ release and rebinding can be studied (Holmgren, et al., 2000). The kinetics of steady state pump current and pre-steady state transient current are measured using either the two-microelectrode or cut open oocyte voltage clamp techniques. The parameters are then compared with those obtained in control oocytes (endogenous, and those expressing ouabain resistant mutants RK2 and DR). The effect of the selected mutations are studied in oocytes co-injected with endogenous beta subunit cRNA and either of these two ouabain resistant alpha subunits further mutated at the sites of interest. Endogenous pump activity is blocked by working at a dose of ouabain (2-100mu/M) appropriate for the experimental conditions. The formation of mixed heterodimers of alpha and beta pump subunits is avoided by co-injection of c-RNA coding for the endogenous beta subunit of the Xenopus oocyte pump. Co-injection of mutated alpha and endogenous beta subunit cRNA typically produces functional pump expression levels that are 6 to 10 times higher than those found in uninjected or water-injected control oocytes. Mutations that fail to pump will be tested to determine if they are sensitive to palytoxin and hence present in the surface membrane.

描述（由申请人提供）：该项目的目的是全面了解电致Na+/K+泵离子转运的机制。Na+/K+泵的结构可以通过基于SERCA-1A原子分辨率结构的同源建模来预测（Toyoshima et al., 2000）。人类（Ogawa and Toyoshima, 2002）和非洲爪鼠卵母细胞Na+/K+泵（Rakowski and Sagar, 2003）的α亚基中的Na+和K+离子结合位点已根据预测位点价的经验方法提出。由于结果可以基于简单的静电因素进行解释，因此选择仅对目标结合位点的结构产生微小变化的候选突变进行进一步研究。例如，中心到中心距离的增加会降低位点的结合能（亲合力），增加离子的解结合率。为了验证这些预测，可以通过控制离子和核苷酸浓度将Na+/K+泵限制在整个泵循环的特定部分。通过在无K+条件下运行，在ATP和ADP的存在下，泵被限制在Na+/Na+交换模式。如果细胞内[Na]保持高水平，则内部Na+结合位点饱和，可以研究细胞外Na+释放和再结合的动力学（Holmgren等，2000）。采用双微电极或切开卵母细胞电压钳技术测量稳态泵电流和预稳态瞬态电流的动力学。然后将这些参数与对照卵母细胞（内源性和表达乌巴因耐药突变体RK2和DR的卵母细胞）中获得的参数进行比较。在共注射内源性β亚基cRNA的卵母细胞中，研究了选定突变的影响，这两个抗瓦巴因α亚基中的任何一个在感兴趣的位点进一步突变。内源性泵活性通过在适合实验条件的剂量（2-100mu/M）下工作而被阻断。通过共同注射编码非洲爪蟾卵母细胞泵内源性β亚基的c-RNA，避免了α和β泵亚基混合异源二聚体的形成。共同注射突变的α和内源性β亚基cRNA通常会产生比未注射或注射水的对照卵母细胞高6至10倍的功能性泵表达水平。不能泵出的突变将被测试，以确定它们是否对孢毒素敏感，从而存在于表面膜中。