A novel kinetic model based on the Albers-Post scheme that mimics the behaviour of the Na+, K+-ATPase in situ

Glutathionylation of the β1 subunit of the Na+, K+-ATPase is a relatively recently reported posttranslational modification that regulates activity via intracellular oxidant signalling pathways. The determination of which partial reactions of the overall ATPase reaction cycle are modified has important ramifications regarding structure, interactions between the ATPase and its surrounding lipid membrane environment and functional aspects, especially under pathophysiological conditions. In this study we incorporate glutathionylation into a current kinetic model of the ATPase determined from the study of partial reactions in the pig kidney enzyme. We use voltage clamped cardiomyocyte and protein immunoblotting data to determine the probable sites of glutathionylation within the enzyme cycle. Using the nomenclature of the Albers-Post scheme results from K+ activated transients indicate that both E2P and the E1K states of the ATPase cycle are highly susceptible to glutathionylation. Results are corroborated by protein immunoblotting experiments on purified pig kidney enzyme. The kinetic model with glutathionylation incorporated faithfully replicates a series of K+ activated transient experiments in cardiomyocytes and experiments altering intracellular oxidant status by including superoxide dismutase in the patch pipette. The incorporation of glutathionylation into the ATPase cycle is the first ever kinetic model that has the ability to reproduce voltage clamped cardiomyocyte experimental data. This novel model offers the possibility of redefining the origin of several experimental observations such as K+ activated transients, apparent changes to intracellular Na+ affinities and the ATPase response to changes in intracellular redox balance.