Voltage-clamp studies of sodium pump current and flux

描述（由申请人提供）：本项目的目的是全面了解生电Na+/K+泵的离子移位机制。Na+/K+泵的结构可以通过基于SERCA-1A的原子分辨率结构的同源建模来预测（Toyoshima等人，2000年）。基于预测位点价的经验方法，提出了人（Ogawa和Toyoshima，2002）和非洲爪蟾卵母细胞Na+/K+泵（Rakowski和萨加尔，2003）α亚基中的推定Na+和K+离子结合位点。由于可以基于简单的静电考虑来解释结果，因此选择仅在靶向结合位点的结构中产生小变化的候选突变用于进一步研究。例如，中心到中心距离的增加应降低位点的结合能（亲和力）并增加离子解结合速率。为了测试这些预测，可以通过控制离子和核苷酸浓度将Na+/K+泵限于整个泵循环的特定部分。通过在无K+条件下和在ATP和ADP两者存在下操作，泵被限制于其Na+/Na+交换模式。如果细胞内[Na]保持高，则内部Na+结合位点饱和，并且可以研究细胞外Na+释放和再结合的动力学（Holmgren等人，2000年）。稳态泵电流和前稳态瞬态电流的动力学使用双微电极或切开的卵母细胞电压钳技术测量。然后将这些参数与对照卵母细胞（内源的，以及表达哇巴因抗性突变体RK 2和DR的那些）中获得的参数进行比较。在共注射内源性β亚基cRNA的卵母细胞中研究所选突变的作用，并且这两种哇巴因抗性α亚基中的任一种在感兴趣的位点进一步突变。通过在适合于实验条件的剂量（2- 100 μ/M）下工作来阻断内源性泵活性。通过共注射编码非洲爪蟾卵母细胞泵的内源性β亚基的c-RNA，避免了α和β泵亚基的混合异源二聚体的形成。共注射突变的α和内源性β亚基cRNA通常产生比未注射或注水的对照卵母细胞中发现的高6至10倍的功能性泵表达水平。将测试不能泵送的突变，以确定它们是否对海葵毒素敏感并因此存在于表面膜中。

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.