Voltage-clamp studies of sodium pump current and flux

描述(申请人提供)：本项目的目标是全面了解电生Na+/K+泵的离子转移机制。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(Toyota oshima等人，2000年)。人类α亚基(Ogawa and Toyota oshima，2002)和非洲爪哇卵母细胞Na+/K+泵(Rakowski and Sagar，2003)中可能存在的Na+和K+离子结合位点已经被提出，这是基于一种预测位点价位的经验方法。由于结果可以基于简单的静电考虑来解释，因此选择仅在靶向结合位点的结构上产生微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该会降低位置的结合能(亲和力)，并增加离子解结率。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+泵限制在整个泵周期的特定部分。由于泵工作在无K+的条件下，在ATP和ADP存在的情况下，泵被限制在其Na+/Na+交换模式。如果细胞内[Na]保持较高水平，内部Na+结合部位饱和，则可以研究细胞外Na+释放和重新结合的动力学(Holmgren等人，2000)。采用双微电极和切开卵母细胞电压钳技术测量稳态泵电流和稳态前暂态电流的动力学。然后将这些参数与在对照卵母细胞中获得的参数(内源性以及表达哇巴因抗性突变体RK2和DR的参数)进行比较。在与内源性β亚基cRNA共同注射的卵母细胞中，研究了所选突变的影响，这两个抗哇巴因的阿尔法亚基中的任何一个在感兴趣的位置进一步突变。以适合实验条件的哇巴因剂量(2-100mU/M)工作时，内源泵活性被阻断。通过共注射编码非洲爪哇卵母细胞泵的内源性β亚基的c-RNA，避免了α和β泵亚单位的混合异二聚体的形成。联合注射突变的α亚基和内源性的β亚基cRNA通常会产生比未注射或注水的对照卵母细胞高6到10倍的功能泵表达水平。不能泵出的突变将被测试，以确定它们是否对孢子毒素敏感，从而存在于表膜上。

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.