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

Abstract

The vital gradients of Na(+) and K(+) across the plasma membrane of animal cells are maintained by the Na,K-ATPase, an αβ enzyme complex, whose α subunit carries out the ion transport and ATP hydrolysis. The specific roles of the β subunit isoforms are less clear, though β2 is essential for motor physiology in mammals. Here, we show that compared to β1 and β3, β2 stabilizes the Na(+)-occluded E1P state relative to the outward-open E2P state, and that the effect is mediated by its transmembrane domain. Molecular dynamics simulations further demonstrate that the tilt angle of the β transmembrane helix correlates with its functional effect, suggesting that the relative orientation of β modulates ion binding at the α subunit. β2 is primarily expressed in granule neurons and glomeruli in the cerebellum, and we propose that its unique functional characteristics are important to respond appropriately to the cerebellar Na(+) and K(+) gradients.

Figure 1. Electrophysiological properties of α1β1 and α1β2.

A simplified Post-Albers scheme with Na⁺ in purple and K⁺ in green is shown in (a). Difference curves in K⁺-free buffer with and without 10 mM ouabain for (b) α1β1,(c) α1β2 are shown. The curves were fitted with single exponentials, giving the voltage dependent (d) charge translocation from the off currents and (e) rate constants from the on currents. N = 3–10 with oocytes from at least two Xenopus laevis females. Data are represented as mean ± SD.

Figure 2. Expression and localization of the β1 and β2 isoforms in mouse brain.

(a) Western blot analysis of the indicated isolated mouse brain regions using antibodies against β2 and GAPDH. (b) Fluorescence immunohistochemistry of β1 and β2 in cerebellum co-stained with the neuronal marker NeuN. GCL: granule cell layer. P: Purkinje cell layer. ML: molecular layer. Scale bars represent 20 μm.

Figure 3. Charge translocation curves of chimeras and pocket mutants.

Charge translocation was determined for α1 coexpressed with (a) β chimeras, where the N-terminal, the C-terminal or the transmembrane region of β1 was replaced with that of β2, with (b) β mutants where smaller stretches in the transmembrane region of β1 were replaced with the corresponding β2 sequences, FK with AF N-terminally, AGI with TAM in the middle or the C-terminal 16 residues, or (c) a combination of the FK toAF , AGI to TAM and VSD to ISE (at the C-terminus, cf. Fig. S6) in β1 giving β1/3mut. N ≥ 5 with oocytes from at least two Xenopus laevis females. Data are represented as mean ± SD.

Figure 4. Molecular dynamics simulations of α1β1 and α1β2.

(a) Atomistic models of the α (white-grey) and β1 (blue) subunits, embedded in the POPC lipid membrane (contour shown in magenta). Several important residues of the β subunit are shown in spacefill. Note that 33–34 FK and 61–63 ISE residues are located at or near the membrane interface. Important residues forming ion binding sites I, II and III are shown as sticks, and bound sodium ions are shown as pink spheres. Water and the γ subunit are omitted for clarity, but included in the model. (b) Comparison of the helix tilt between the transmembrane helix of β1 (blue) and β2 (light green). The tilt is defined as the angle between the helix axis and the z-axis, which is perpendicular to the membrane surface. Presented values are the averages from the last 40 ns, with error estimations obtained with block averaging. (c,d) Interaction patterns between the β helix (blue β1 C) and light green β2 D)) and the M7 helix (yellow) and the C-terminus (orange) of the α subunit in α1β1 C) and α1β2 D). Interactions between selected residues (shown in licorice) are shown as purple springs, with minimum distances recorded in simulations (last 40 ns) indicated in italics. Hydrophobic carbon atoms are shown in cyan, oxygen atoms bearing partial negative charge are shown in red and nitrogen atoms bearing partial positive charge are shown in deep blue.

Figure 5. Flexibility of the α C-terminus with β1 or β2.

(a) Root mean square deviation (RMSD) of the C-terminus of α1 with β1 or β2 showing displacement in α1β2. (b) Root mean square fluctuations (RMSF) of heavy atom positions of the protein residues that form the C-terminal tail of α1, average of the last 60 ns of the MD trajectory.