Cholesterol depletion inhibits Na+,K+-ATPase activity in a near-native membrane environment

Abstract

Cholesterol's effects on Na⁺,K⁺-ATPase reconstituted in phospholipid vesicles have been extensively studied. However, previous studies have reported both cholesterol-mediated stimulation and inhibition of Na⁺,K⁺-ATPase activity. Here, using partial reaction kinetics determined via stopped-flow experiments, we studied cholesterol's effect on Na⁺,K⁺-ATPase in a near-native environment in which purified membrane fragments were depleted of cholesterol with methyl-β-cyclodextrin (mβCD). The mβCD-treated Na⁺,K⁺-ATPase had significantly reduced overall activity and exhibited decreased observed rate constants for ATP phosphorylation (ENa₃⁺ → E2P, i.e. phosphorylation by ATP and Na⁺ occlusion from the cytoplasm) and K⁺ deocclusion with subsequent intracellular Na⁺ binding (E2K₂⁺ → E1Na₃⁺). However, cholesterol depletion did not affect the observed rate constant for K⁺ occlusion by phosphorylated Na⁺,K⁺-ATPase on the extracellular face and subsequent dephosphorylation (E2P → E2K₂⁺). Thus, partial reactions involving cation binding and release at the protein's intracellular side were most dependent on cholesterol. Fluorescence measurements with the probe eosin indicated that cholesterol depletion stabilizes the unphosphorylated E2 state relative to E1, and the cholesterol depletion-induced slowing of ATP phosphorylation kinetics was consistent with partial conversion of Na⁺,K⁺-ATPase into the E2 state, requiring a slow E2 → E1 transition before the phosphorylation. Molecular dynamics simulations of Na⁺,K⁺-ATPase in membranes with 40 mol % cholesterol revealed cholesterol interaction sites that differ markedly among protein conformations. They further indicated state-dependent effects on membrane shape, with the E2 state being likely disfavored in cholesterol-rich bilayers relative to the E1P state because of a greater hydrophobic mismatch. In summary, cholesterol extraction from membranes significantly decreases Na⁺,K⁺-ATPase steady-state activity.

Keywords: Na+/K+-ATPase; cholesterol; lipid-protein interaction; lipid-protein interactions; methyl-β-cyclodextrin; molecular dynamics; partial reaction kinetics; pre-steady-state kinetics; steady-state activity.

Figure 1.

Dependence of relative Na⁺,K⁺-ATPase (NKA) activity on normalized relative membrane cholesterol content. Cholesterol extraction was performed using sequentially higher concentrations of mβCD, and ATPase activity was measured after mβCD removal. Solid line, nonlinear least-squares fit of an exponential decay curve to the data, i.e. the fit function was y = y₀ + (y_max − y₀)· (1 − e^−kx). k is the reciprocal of the relative membrane cholesterol content at which the difference between the normalized Na⁺,K⁺-ATPase activity and its value at zero cholesterol content has dropped to 1/e of its initial value at zero cholesterol content. The half-saturating relative membrane cholesterol content was determined to be 0.15 (±0.10). The Na⁺,K⁺-ATPase activity of untreated membranes was 1671 (± 71) μmol of ATP hydrolyzed (mg of protein)⁻¹ h⁻¹ (n = 7).

Figure 2.

Effect of reintroduction of cholesterol through a cholesterol:mβCD complex to membrane fragments treated with different concentrations of mβCD (30, 45, and 60 mm). Measurements are given as relative activity to untreated controls and are an average of 3 measurements with the errors presented as standard deviations.

Figure 3.

Stopped-flow fluorescence transients of Na⁺,K⁺-ATPase from pig kidney noncovalently labeled with RH421. A and B show the kinetics of the E1Na₃⁺ → E2P transition of an untreated control (A) and membrane fragments treated with 60 mm mβCD (B). C and D show the kinetics of the E2K₂⁺ → E1Na₃⁺ transition of an untreated control (C) and membrane fragments treated with 60 mm mβCD (D). All experimental conditions are described under “Experimental procedures.”

Figure 4.

Fluorescence excitation spectra of eosin. The fluorescence intensity is given in arbitrary units. The upper panel represents eosin in the presence of Na⁺,K⁺-ATPase membrane fragments from which cholesterol was either extracted (solid line) or an untreated control (dotted line) in E2K₂⁺ buffer (30 mm imidazole, 10 mm KCl, 1 mm EDTA, pH 7.2). The lower panel represents eosin in the presence of Na⁺,K⁺-ATPase membrane fragments from which cholesterol was either extracted with mβCD (solid line) or an untreated control (dotted line) in E1Na₃⁺ buffer (30 mm imidazole, 130 mm NaCl, 5 mm MgCl₂, 1 mm EDTA, pH 7.2). The emission wavelength was 550 nm (+OG530 cutoff filter). The bandwidths for both excitation and emission were 5 nm. The eosin and Na⁺,K⁺-ATPase concentrations were 29 nm and 230 μg ml⁻¹ in every case. Cholesterol extraction was achieved by equilibrating the Na⁺,K⁺-ATPase membrane fragments with 60 mm mβCD for 2 h at 37 °C. The spectra were recorded at a temperature of 24 °C.

Figure 5.

a, Na⁺,K⁺-ATPase simulation systems: E1P·ADP·3Na⁺ (left, “E1,” PDB 3WGU) and E2·P_i·2K⁺ (right, “E2,” PDB 3B8E). b, bilayer thickness deviation is based on glycerol C to glycerol C distance between lipids in each leaflet, viewed from the cytoplasm. Deviation is relative to thickness far from the protein, with an average of 34.8 ± 0.2 Å for both states. c, axially-averaged bilayer thickness, defined as the glycerol-carbon-to-glycerol-carbon distance between lipids in each membrane leaflet. The error bars represent mean ± 1 S.E.

Figure 6.

Cholesterol density for each membrane leaflet relative to the mean far from the protein. The left column shows the distribution for E1P·ADP·3Na⁺ (PDB 3WGU, E1) and the right column E2·P_i·2K⁺ (PDB 3B8E, E2). Density is viewed from the cytoplasm. Reproducibility of the 4 simulations for each state is shown in Fig. S1. Sites with a cholesterol packing score of ≥0.25 for at least one state are indicated with letters A–M, with green letters for sites unique to one state (packing scores in Table S1). a, shows results for the extracellular side, whereas b shows results for the cytoplasmic site. c, radial free energy profile for cholesterol binding, shown in blue for extracellular leaflet and red for cytoplasmic leaflet. Darker colors represent the E1 state, whereas lighter colors represent the E2 state, as labeled.