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.
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