Curcumin modulation of Na,K-ATPase: phosphoenzyme accumulation, decreased K+ occlusion, and inhibition of hydrolytic activity

Abstract

1 Curcumin, the major constitute of tumeric, is an important nutraceutical that has been shown to be useful in the treatment of many diseases. As an inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase, curcumin was shown to correct cystic fibrosis (CF) defects in some model systems, whereas others have reported no or little effects on CF after curcumin treatment, suggesting that curcumin effect is not due to simple inhibition of the Ca(2+)-ATPase. 2 We tested the hypothesis that curcumin may modulate other members of the P(2)-type ATPase superfamily by studying the effects of curcumin on the activity and kinetic properties of the Na,K-ATPase. 3 Curcumin treatment inhibited Na,K-ATPase activity in a dose-dependent manner (K(0.5) approximately 14.6 microM). Curcumin decreased the apparent affinity of Na,K-ATPase for K(+) and increased it for Na(+) and ATP. Kinetic analyses indicated that curcumin induces a three-fold reduction in the rate of E1P --> E2P transition, thereby increasing the steady-state phosphoenzyme level. Curcumin treatment significantly abrogated K(+) occlusion to the enzyme as evidenced from kinetic and proteolytic cleavage experiments. Curcumin also significantly decreased the vanadate sensitivity of the enzyme. 4 Thus, curcumin partially blocks the K(+) occlusion site, and induces a constitutive shift in the conformational equilibrium of the enzyme, towards the E1 conformation. 5 The physiological consequences of curcumin treatment previously reported in different epithelial model systems may, at least in part, be related to the direct effects of curcumin on Na,K-ATPase activity.

Scheme 1

Minimum reaction cycle of Na,K-ATPase.

Figure 1

Curcumin inhibition of ATPase activity. The ouabain-dependent Na,K-ATPase activity was measured in the presence of 20 mM histidine buffer, pH 7.0, 3 mM MgCl₂, 100 mM NaCl, 20 mM KCl, 3 mM ATP, and the indicated concentrations of curcumin. Data are expressed as percentage of control, measured in the absence of curcumin. A sigmoid dose–response equation was fitted to the data, giving a K_0.5 for curcumin inhibition of 14.64±1.12 μM. Data are mean±s.e.m. of duplicate measurements. Representative of three independent experiments is shown, and each experiment gave similar result.

Figure 2

Substrate-dependent ATPase activity of control- and curcumin-treated enzymes. Ouabain-dependent ATPase activity was measured in the presence of 20 mM histidine buffer, pH 7.0, 3 mM MgCl₂ without or with 25 μM curcumin. Data are mean±s.e.m. of duplicate measurements and expressed as percentage of 100% control. Representative of three independent experiments is shown, and each experiment gave similar result. (a) Na⁺-stimulated ATP hydrolysis in the presence of 10 μM ATP, in the absence of K⁺, and in the presence of the indicated Na⁺ concentrations is shown. Analysis of the data using a sigmoid dose–response curve gave a K_0.5(Na⁺)=4.52±1.10 mM for control- and 4.08±1.06 mM for curcumin-treated enzyme, P-value <0.0001. Curcumin treatment resulted in ∼60% inhibition of hydrolytic activity. (b) K⁺-stimulated ATP hydrolysis measured in the presence of 130 mM Na⁺, 10 μM ATP, and the indicated K⁺ concentrations. Analysis of the data using a hyperbolic function gave a K_0.5(K⁺)=51.8±1.0 μM for control- and 177±2.0 μM for curcumin-treated enzyme, P-value <0.0001. Curcumin treatment resulted in ∼21% inhibition of hydrolytic activity. (c) The effect of curcumin on the K_0.5(K⁺)/V_max is shown. The K_0.5(K⁺)/V_max was estimated from K⁺-activation curves measured at the indicated curcumin concentrations. (d) ATP-stimulated ATP hydrolysis measured in the presence of 130 mM Na⁺, 20 mM K⁺, and the indicated ATP concentrations. Analysis of the data using a Michaelis–Menten equation gave a K_0.5(ATP)=130.0±0.006 μM for control- and 90±0.009 μM for curcumin-treated enzyme, P-value <0.05. Curcumin treatment resulted in ∼40% inhibition of hydrolytic activity.

Figure 3

Vanadate sensitivity of control- and curcumin-treated enzymes. The hydrolytic activities of control- and curcumin-treated enzymes were measured in the presence of 130 mM NaCl, 10 mM KCl, 3 mM MgCl₂, 1 mM ATP, and the indicated vanadate concentrations. Data are normalized to 100% control (in the absence of curcumin), and expressed as percentage of the activity measured in the absence of vanadate. Data are mean±s.e.m. of duplicate measurements. Representative of two independent experiments is shown, and each experiment gave similar result. A sigmoid dose–response curve was fitted to the data points, which gave a K_0.5 for vanadate inhibition of 3.26±0.015 μM for control-, and 13.16±0.017 μM for curcumin-treated enzyme (P-value <0.001).

Figure 4

Curcumin- and Na⁺-dependent accumulation of EP. (a) Steady-state EP level was measured in the presence of 20 mM Na⁺ and the indicated concentrations of curcumin, as described in Methods. Data are expressed as percentage of control, measured in the absence of curcumin. (b) Phosphorylation was measured in the presence of the indicated concentrations of Na⁺ plus either DMSO (control), 25 μM curcumin, or 25 μM oligomycin, as indicated in the figure. Data are expressed as percentage of control, measured in the presence of 1 mM NaCl, and in the absence of curcumin and oligomycin. Fitting of the initial Na⁺-dependent stimulation of EP accumulation using a hyperbolic function gave maximum steady-state phosphorylations of 288.7±32.3, 664.3±52.37, and 798.0±21.9% for control-, curcumin-, and oligomycin-treated enzymes, respectively. The K_0.5's for the initial Na⁺ activation were 3.915±1.07, 2.805±0.627, and 0.742±0.11 mM for control-, curcumin-, and oligomycin-treated enzymes, respectively (P-value <0.0001). Data are mean±s.e.m. of triple determinations. Representative of three independent experiments is shown, and each experiment gave similar result.

Figure 5

The E1P → E2P reaction in control- and curcumin-treated enzymes. (a) Phosphorylated enzyme was chased at 0°C with 1 mM ATP and 1 mM ADP, followed by acid quenching at the indicated time intervals. A two-phase exponential decay function was fitted to the date points, giving the rate constants corresponding to the slow and fast components as follows: control, 0.04±0.01 and 0.23±0.10 s⁻¹; curcumin-treated, 0.01±0.003 and 0.83±0.22 s⁻¹. The slow-decaying component was analyzed using linear regression model to estimate the initial amount of E2P, giving intercept with the ordinate axis of 74.48±6.88 for control- and 55.43±5.94 for curcumin-treated enzyme, corresponding to the initial fractional amounts of E2P phosphoform as percentage of total. (b) Dephosphorylation of enzyme phosphorylated at 600 mM Na⁺ (see Methods) was initiated at 0°C by chasing with 2 mM ATP and 5 mM KCl, followed by acid quenching at the indicated time intervals. A monoexponential decay function was fitted to the data points, giving rate constants of 1.99±0.05 s⁻¹ for control-, and 0.68±0.001 s⁻¹ for curcumin-treated enzyme. Data are mean±s.e.m. of triple determinations. Representative of two independent experiments is shown, and both experiments gave similar results.

Figure 6

Spontaneous and K⁺-dependent dephosphorylation of control- and curcumin-treated enzymes. (a) Spontaneous dephosphorylation of E2P (E2P → E1) was performed by first phosphorylating the enzyme as described in Methods. Dephosphorylation was studied at 0°C by diluting the EP into 1.5 mM Tris–ATP and 3 mM MgCl₂, followed by acid quenching at the indicated time intervals. Analysis of the data using a monoexponential decay function gave rates of EP decay of 30.44±1.8 and 14.58±1.21 min⁻¹ for control- and curcumin-treated enzyme, respectively. (b) K⁺-induced dephosphorylation was studied at 0°C by diluting the EP into 1.5 mM Tris–ATP and 1 mM K⁺, followed by acid quenching at the indicated time intervals. Analysis of the date using a monoexponential decay function gave a rate of EP decay of 193.86±13.10 and 206.94±11.34 min⁻¹ for control- and curcumin-treated enzyme, respectively. Data are mean±s.e.m. of triple determinations. Representative of two independent experiments is shown, and each experiment gave similar result.

Figure 7

K⁺ occlusion in control- and curcumin-treated enzymes. (a) K⁺ occlusion was measured as described in Methods. Data are expressed as percentage of maximum EP level, as previously described (Daly et al., 1996). (b) Immunoblot showing time-dependent accumulation of the ‘19 kDa fragment' in control- and curcumin-treated membranes, as indicated. Control- and curcumin-treated membranes were incubated with K⁺, submitted to trypsin treatment for the indicated time intervals, loaded on SDS gels, and blotted using antibody specific for the C-terminus of the α-subunit, as described previously (Mahmmoud et al., 2000).