Tuning of the Na,K-ATPase by the beta subunit.

The Na,K-ATPase (NaKA) transports three Na+ from inside the cell to the outside coupled to auto-phosphorylation from ATP and two K+ from outside to inside coupled to auto-dephosphorylation in a reaction cycle where the conformations with high Na+ affinity are termed E1 and those with high K+ affinity are termed E2 (Fig. 1a)1,2,3,4,5,6. The ATP-driven reaction proceeds against the concentration gradients of both ions and generates steep electrochemical gradients across the plasma membrane that are used for a variety of cellular processes including neuronal signalling and secondary active transport7,8. The 3:2 stoichiometry of ion transport means that the activity of the pump can be determined from the steady-state current it generates, and under restricting conditions, individual voltage-sensitive steps in the catalytic cycle can be monitored. The extracellular translocation of each of the three Na+ is voltage-dependent, and the relatively slow pre-steady-state charge movement associated with the third Na+ can readily be recorded if the NaKA is restricted to binding and releasing Na+ by omission of extracellelular K+,9,10. The pre-steady-state currents reflect the voltage-dependent E1P-E2P transition (Fig. 1a)9, where the probability of the NaKA being in the Na+-occluded E1P state is highest at negative membrane potentials and in the outward-open E2P state highest at positive membrane potentials. Figure 1 Electrophysiological properties of α1β1 and α1β2. The minimal pump has two subunits, α and β, and can further interact with a γ (FXYD) subunit. Humans express four isoforms of α, three of β, and seven of FXYD11, while insects have a single functional α subunit and several β subunits. Deletion or mutation of the β subunit can have severe consequences. In Drosophila, the β subunits regulate sight and hearing12, and in mice, deletion of the gene encoding β2 gene causes motor disabilities, and the animals die a few weeks after birth13. In humans, changes in the expression pattern of β2 have been linked to glioma14. Different α/β combinations were previously shown to have different apparent K+ affinities15, especially α2β2 has very low apparent K+ affinity, but a high turn-over-rate, and is suggested to be specifically geared for K+-clearance in hippocampal glia cells16 and in fast-twitch glycotic muscle fibers17,18. In brain, the expression profile for β2-encoding mRNA indicates high expression in cerebellum19, and protein stainings show β2 in Purkinje cells with α2, α3 and β1, and in granule cells and glomeruli with α1, α3 and β120. In cerebellum, about 60% of the ATP consumption is estimated to be used to maintain the ionic gradients required for signalling, and almost 70% of that ATP is used by the granule cells21, so the NaKA activity in granule cells is clearly a dominant factor in the overall energy consumption in the cerebellar cortex. We have investigated the molecular and functional role of β2 and find that it significantly influences the E1P-E2P equilibrium with any of the α subunits studied. To determine the molecular mechanism of β’s functional effect, we constructed chimeras of β1 and β2, which pinpointed the transmembrane domain as the main determinant for the observed electrophysiological characteristics. Molecular dynamics (MD) simulations suggest that the transmembrane helices of β1 and β2 have different tilt angles, and we propose that the tilt angle of β can influence the relative stability of the Na+ occluded E1P state.