Chung et al. 1
Inhibition of Na+/K+-ATPase by Antcins, the Unique Steroid-Like
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Compounds in Antrodia camphorata
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Tse-yu Chung,† Feng-Yin Li,‡ Chi-I Chang,§ Tzyy-Rong Jinn,#,* and Jason T. C. Tzen†,#,¶,*
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†
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University, Taichung, 40227, Taiwan
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§
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Technology, Pingtung 91201, Taiwan
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#
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Graduate Institute of Biotechnology and ‡Department of Chemistry, National Chung Hsing
Graduate Institute of Biotechnology, National Pingtung University of Science and
School of Chinese Medicine, China Medical University, 40402, Taichung, Taiwan
Agricultural Biotechnology Research Center, Academia Sinica, Taipei 11529, Taiwan
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Corresponding author: Dr. Jason T.C. Tzen, Graduate Institute of Biotechnology and
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Department of Chemistry, National Chung Hsing University, Taiwan. Tel: (+886)-4-2284
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-0328, Fax: (+886)-4-2285-3527, E-mail:tctzen@dragon.nchu.edu.tw or Dr. Tzyy-Rong Jinn,
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School of Chinese Medicine, China Medical University, Taiwan. Tel: (+886)-4-2205-3366,
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Fax: (+886)-4-2203-2295, E-mail:jinn@mail.cmu.edu.tw
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Chung et al. 2
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Abstract:
Inhibition of Na+/K+-ATPase by variable steroid-like compounds contributes to
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therapeutic effects of many cardiac Chinese medicinal products putatively via the same
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molecular mechanism triggered by cardiac glycosides.
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antcin A, B, C, H, and K isolated from Niuchangchih (Antrodia camphorata), a unique
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mushroom of Taiwan displayed inhibition on Na+/K+-ATPase.
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significantly higher inhibitory potency than the other four antcins though weaker than
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ginsenoside Rh2.
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stronger than antcin A) showed no detectable inhibitory potency.
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showed that antcins bound to Na+/K+-ATPase with the steroidal skeleton structurally
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upside-down in comparison with ginsenoside Rh2.
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attributed to steroidal hydrophobic interaction within the binding pocket and formation of
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three hydrogen bonds between its carboxyl group and two cationic residues around the cavity
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entrance of Na+/K+-ATPase.
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C-7 of the other four antcins leads to severe repulsion in the hydrophobic pocket, and thus
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significantly reduces inhibitory potency.
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compound exerting anti-inflammatory effect and enhancing blood circulation via two
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different molecular mechanisms.
Five major steroid-like compounds,
Antcin A exhibited
In contrast, cortisone (an analogous steroid with anti-inflammatory effect
Molecular modeling
Inhibitory potency of antcin A is
The presence of an additional carbonyl or hydroxyl group at
It is proposed that antcin A is a bi-functional
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Keywords: Antcin A; Antrodia camphorata; Cortisone; Ginsenoside; Na+/K+-ATPase
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Introduction
Niuchangchih (Antrodia camphorata), a unique mushroom of Taiwan, is restricted
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growing on the inner cavity of the endemic tree species, Cinnamomum kanehirai (Wu et al.,
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1997). Being a local species, Niuchangchih is used as a traditional medicine by the
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aborigines for the treatment of discomforts caused by alcohol drinking or exhausting (Su,
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2002). In the past few decades, fruiting bodies of Niuchangchih have been generally used as
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a folk medicine for the treatment of liver diseases, drug intoxication, diarrhea, abdominal
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pain, hypertension, and tumorigenic diseases (Chen et al., 2001; Geethangili and Tzeng,
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2001; Shen et al., 2004; Hseu et al., 2005; Liu et al., 2007; Ao et al., 2009; Huang et al.,
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2010). Among the therapeutic effects of Niuchangchih, anti-inflammatory effect has been
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well-recognized by the consumers, particularly those who suffer from liver dysfunction.
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Moreover, it has been recommended for the development of Niuchangchih as a healthy (or
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functional) food to regulate blood pressure on the basis of its therapeutic effects of
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anti-hypertension and vasorelaxation despite the fact that the exact molecular mechanisms
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responsible for these two effects are still unknown (Geethangili and Tzeng, 2001).
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More than 30 steroid-like and triterpene compounds have been identified in fruiting
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bodies of Niuchangchih and represent the major constituents accounting for approximately
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60% of dry weight in this edible mushroom (Geethangili and Tzeng, 2001 and Ao et al.,
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2009). Among these steroid-like compounds, antcins, a unique group having ergostane
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skeletons, are only found in Niuchangchih so far. Anti-inflammatory, anti-insecticidal and
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cytotoxic activities have been observed in various antcins and their derivatives (Chen et al.,
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1995; Cherng et al., 1996; Shen et al., 2003; Chen et al., 2007;Shen et al., 2007; Male et al.,
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2008; Yeh et al., 2009). Recently, antcin A, a relatively abundant constituent in fruiting
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bodies of Niuchangchih, was demonstrated to be an active ingredient responsible for the
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anti-inflammatory effect by mimicking glucocorticoids and thus triggering the translocation
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of the glucocorticoid receptor into the nucleus to initiate the suppression of inflammation via
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gene regulation (Chen et al., 2011).
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Ginsenosides are steroid-like compounds with sugar moieties attached mostly at the
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C-3, C-6 or C-20 position (Li-Saw-Hee et al., 1998). To date, more than 80 ginsenosides
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have been identified and assumed to be the active ingredients responsible for many
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therapeutic effects of ginseng and sanqi (the roots of Panax ginseng and Panax notoginseng),
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two well-known traditional Chinese medicinal herbs (Zhu et al., 2004). It has been shown
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that the cardiac therapeutic effects of ginseng and sanqi are attributed to the effective
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inhibition of Na+/K+-ATPase by ginsenosides with sugar moieties attached only to C-3 of the
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steroidal skeleton (Chen et al., 2009). Comparably, inhibition of Na+/K+-ATPase by variable
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steroid-like compounds contributes to therapeutic effects of many cardiac Chinese medicinal
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products putatively via the same molecular mechanism triggered by cardiac glycosides, such
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as ouabain (Chen et al., 2010 and Chen et al., 2011). The inhibition of Na+/K+-ATPase leads
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to the accumulation of intracellular Na+, which in turn activates a Na+ /Ca2+ exchanger
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resulting in an increase of intracellular Ca2+ concentration (Chen et al., 2010). The elevated
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intracellular Ca2+ concentration causes an increased inotropism, accentuating the force of
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myocardial contraction by increasing the velocity and extent of sarcomere shortening, thus
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translating into increased stroke work for a given filling volume of pressure.
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In view of the highly structural similarity between antcins and ginsenosides (in both the
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steroidal skeleton and the head portion attached to C-17), we wondered if antcins may act as
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active ingredients responsible for the anti-hypertension and vasorelaxation effects of
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Niuchangchih by promoting blood circulation via the same molecular mechanism triggered
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by ginsenosides. In this study, Na+/K+-ATPase inhibitory potency of five major antcins
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isolated from fruiting bodies of Niuchangchih as well as two analogous steroids, cortisone
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and ganoderic acid A, was examined and compared to that of ginsenoside Rh2 and ouabain.
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Molecular modeling of the steroid-like compounds docking to Na+/K+-ATPase was applied
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to reveal the detailed interaction responsible for the observed difference in their
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Na+/K+-ATPase inhibition at molecular level.
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Materials and Methods
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Chemicals and Reagents
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Cortisone and ouabain were purchased from Sigma-Aldrich (St. Louis, MO, USA).
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Ganoderic acid A and ginsenoside Rh2 were obtained from Hyclone (Logan, UT, USA) and
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Scientific Pharmaceutical Elite Company (Taiwan), respectively. Phosphate assay kit was
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purchased from Amresco (USA).
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Purification of Antcins
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Five major antcins (A, B, C, H, and K) were isolated from fruiting bodies of Niuchangchih as
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described previously (Chen et al., 2011). Fruiting bodies of Niuchangchih (Antrodia
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camphorata) (50 g) were extracted with methanol (3 × 2000 ml) at room temperature. The
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combined extract was evaporated under reduced pressure, suspended in H2O (1000 ml), and
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then extracted with EtOAc and n-BuOH (3 × 1000 ml) sequentially. The EtOAc fraction was
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subjected to silica gel chromatography (60 × 3.5 cm) using a stepwise gradient mixture of
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n-hexane and EtOAc as eluent. Ten fractions were collected as follows: 1 [3000 ml,
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n-hexane], 2 [2000 ml, n-hexane－EtOAc (19:1)], 3 [2000 ml, n-hexane－EtOAc (9:1)], 4
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[2000 ml, n-hexane－EtOAc (17:3)], 5 [2000 ml, n-hexane－EtOAc (8:2)], 6 [3000 ml,
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n-hexane－EtOAc (7:3)], 7 [3000 ml, n-hexane－EtOAc (5:5)], 8 [3000 ml, n-hexane－
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EtOAc (4:6)], 9 [3000 ml, n-hexane－EtOAc (2:8)], and 10 (4000 ml, EtOAc). Fraction 5
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further purified through a silica gel column (2 × 45 cm), eluted with CH2Cl2－EtOAc (30:1
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to 0:1) to obtain six fractions, 5A－5F. Fraction 5C was applied to semipreparative HPLC
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eluted with CH2Cl2－acetone (40:1) to yield antcin A. Fraction 6 was further
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chromatographed on a silica gel column (2 × 45 cm), eluted with CH2Cl2－EtOAc (15:1 to
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0:1) to resolve into five fractions, 6A－6E. Fraction 6B was subjected to semipreparative
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HPLC eluted with CH2Cl2－acetone (20:1) to yield antcin B. Fraction 6C was also subjected
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to semipreparative HPLC eluted with CH2Cl2－acetone (15:1) to yield antcin C. Fraction 7
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was further chromatographed on a silica gel column (2×45 cm), eluted with CH2Cl2－
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methanol (50:1) to afford seven fractions, 7A－7G. Fraction 7C was subjected to
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semipreparative HPLC eluted with CH2Cl2－acetone (9:1) to yield antcin H. Fraction 9 was
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further purified through a silica gel column (2×45 cm), eluted with CH2Cl2－MeOH (15:1) to
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obtain six fractions, 9A－9F. Fraction 9E was subjected to semipreparative HPLC eluted
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with CH2Cl2－i-propanol (9:1) to yield antcin K.
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Measurement of Na+/K+-ATPase Activity
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The activity of Na+/K+-ATPase was determined by measuring the amount of inorganic
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phosphate (Pi) liberated from ATP (Tzen et al., 2007). A commercial Na+/K+-ATPase from
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porcine cerebral cortex (Sigma, 0.3 units/mg) was incorporated into a reaction mixture of 500
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μl containing 1 μmol/ml ATP, 3μmol/ml MgCl2, 48 μmol/ml NaCl, 12 μmol/ml KCl, and 24
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μmol/ml Tris-HCl (pH 7.8); the enzymatic reaction was terminated by adding 250 l of 30%
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(w/v) trichloroacetic acid after the incubation period. After centrifugation at 10000  g for 10
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min at 4C, the supernatant was diluted 25-fold with deionized water and then added with 50
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μl color development reagent provided in the phosphate assay kit. After 30 min of incubation
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at room temperature, the color intensity was measured at 620 nm on SpectraMax M2 reader
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(Molecular Devices, USA). Sodium pump activity was expressed as μmol Pi liberated from
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ATP by 1 mg of Na+/K+-ATPase in 1 h.
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Data Analysis
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Data were expressed as mean ± SEM of 5 replicates and the analysis of variance (One-way
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ANOVA) was performed on SPSS 12.0 for Windows. Differences were considered
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statistically significant at P<0.05.
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Molecular Modeling
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The complex crystal structure of shark rectal gland Na+/K+-ATPase with bound ouabain
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(PDB code 3A3Y) was downloaded from Protein Data Bank and used as the template for
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molecular docking (Ogawa et al., 2009). In order to facilitate docking process, the  and 
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subunits of the Na+/K+-ATPase, as well as the water molecules and counter-ions surrounding
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the remaining  subunit were removed. The modified Na+/K+-ATPase after hydrogen
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saturation was minimized with CHARMm force field (Miller et al., 2008) using the Discover
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Studio 2.1 package (http://accelrys.com/products/discovery-studio/). The 2D structures of
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steroid-like compounds used in this study were constructed by using the ChemDraw program,
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and their corresponding 3D structures were converted by the Chem3D program
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(http://www.cambridgesoft.com/). The binding site for the steroid-like compounds in the
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Na+/K+-ATPase  subunit was defined as ouabain binding site among the extracellular loops
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linking transmembrane segments in the ouabain-Na+/K+-ATPase complex structure. Docking
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of the steroid-like compounds was performed in silico by employing the LigandFit
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(Venkatachalam et al., 2003) in the Discover Studio 2.1 package. Input ligands’
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conformations were generated by variable number of Monte Carlo Steps. These docking
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poses were scored by Ligscore1. In this study, hydrogen bonds and hydrophobic-hydrophilic
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repulsion between ligands and Na+/K+-ATPase are defined for the distance shorter than 2.5
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and 4 Å, respectively.
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Results
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Structural Comparison of Antcins and Some Structurally Analogous Compounds
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The structures of five major antcins (A, B, C, H, and K), ouabain (a cardiac glycoside),
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ginsenoside Rh2 (an active ingredient in ginseng and sanqi), cortisone (an analogous steroid
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with anti-inflammatory effect stronger than antcin A), and ganoderic acid A (an analogous
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steroid from a different medicinal fungus, Lingtzhi, Ganoderma lucidum) were shown in Fig.
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1. All the five antcins possess a carbonyl or hydroxyl group at C-3 position, a carbonyl group
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at C-11 position, and an identical head portion (an aliphatic chain with one carboxyl acid
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group at the end) attached to C-17 position. Among the five antcins, antcin A is distinct from
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the other four antcins which have an additional carbonyl or hydroxyl group at C-7 position.
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Comparable to antcins, the head portion of ginsenoside Rh2 possesses an aliphatic chain but
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with one hydroxyl group at C-20 instead of carboxyl acid group at the end. Distinctly, a sugar
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moiety is attached to C-3 of ginsenoside Rh2 in a manner similar to ouabain. Cortisone,
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having a steroidal skeleton nearly identical to antcin A, possesses a short hydrophilic head
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portion containing one carbonyl and one hydroxyl groups. Ganoderic acid A is very similar
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to antcins including both the steroidal skeleton and the aliphatic head portion but with an
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extra hydroxyl group at C-15 and an extra carbonyl group at C-22.
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Inhibition of Na+/K+-ATPase by Antcins
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To examine whether antcins as well as two structurally analogous compounds, cortisone and
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ganoderic acid A, may promote blood circulation via the same mechanism triggered by
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ouabain or ginsenosides, a commercial Na+/K+-ATPase from porcine cerebral cortex was
Chung et al. 9
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used to evaluate the inhibitory potency of these compounds. All the five antcins displayed
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more or less inhibition on Na+/K+-ATPase while cortisone and ganoderic acid A showed no
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detectable inhibitory potency in our assay condition (Fig. 2). Antcin A exhibited significantly
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higher inhibitory potency than the other four antcins. However, the inhibitory potency of
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antcin A was apparently weaker than that of ginsenoside Rh2 or ouabain. The IC50 value of
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antcin A (501.2 M) was approximately 9 or 800 times higher than that of ginsenoside Rh2
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(55.6 M) or ouabain (0.6 M) (Fig. 3).
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Molecular Modeling of Antcins Docking to Na+/K+-ATPase
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To reveal the observed difference in Na+/K+-ATPase inhibition at molecular level, all the
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compounds examined were subjected to molecular modeling and docking, as exemplified by
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antcin A, to the extracellular domain of Na+/K+-ATPase  subunit (Fig. 4). Inhibitory
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potency of antcin A is attributed to steroidal hydrophobic interaction within the binding
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pocket and formation of three hydrogen bonds between its carboxyl group and two cationic
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residues, R893 (forming one H-bond) and K912 (forming two H-bonds), around the cavity
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entrance of Na+/K+-ATPase (Fig. 5). In contrast, two hydrogen bonds are formed between
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two hydroxyl groups (attached to C-12 and C-20) of ginsenoside Rh2 and two residues
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(N129 and T804) of Na+/K+-ATPase, respectively. Detailed inspection showed that antcin A
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binds to the pocket of Na+/K+-ATPase with the steroidal skeleton structurally upside-down in
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comparison with the binding of ginsenoside Rh2. While the attached glycoside at C-3 of
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ginsenoside Rh2 occupies the entrance of binding pocket, the hydrophilic carbonyl group at
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C-3 of antcin A penetrates deeply in the hydrophobic cavity and repelled by hydrophobic
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residues, F323 and F790 of Na+/K+-ATPase. This repulsion presumably explains why antcin
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A possesses lower inhibitory potency on Na+/K+-ATPase than ginsenoside Rh2.
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Among the five antcins examined, antcin A showed the best affinity with the binding
pocket of Na+/K+-ATPase; similar binding interaction with Na+/K+-ATPase was observed for
Chung et al. 10
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the other four antcins, and thus exemplified by antcin B (Fig. 5). The presence of an
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additional carbonyl or hydroxyl group at C-7 of the other four antcins leads to severe
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repulsion in the hydrophobic pocket, and thus significantly reduces their inhibitory potency
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on Na+/K+-ATPase. To minimize this severe repulsion, only one hydrogen bond is formed
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between the carboxyl group of the other four antcins and K912 of Na+/K+-ATPase. In spite of
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the expense of reducing the number of hydrogen bond from three to one, the
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hydrophilic-hydrophobic repulsion of the other four antcins is still higher than that of antcin
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A in the binding pocket of Na+/K+-ATPase; the carbonyl groups at C-3, C-7 and C-11 of
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antcin B are repelled by L132, I323, F790, and L800 of Na+/K+-ATPase. Evidently, the
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repulsion caused by an additional hydrophilic group at C-7 of steroidal skeleton within the
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binding pocket of Na+/K+-ATPase is the main factor why the other four antcins possess lower
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inhibitory potency than antcin A (Fig. 2).
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Hydrophilic-hydrophobic repulsion is also observed between two carbonyl groups at
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C-3 and C-11 of cortisone and two hydrophobic residues F790 and L800 of Na+/K+-ATPase
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(Fig. 5). However, in contrast with the three hydrogen bonds formed between the head
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portion of antcin A and Na+/K+-ATPase, no hydrogen bond is formed between the head
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portion of cortisone and Na+/K+-ATPase. The lack of forming hydrogen bonds as well as
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repulsion in the binding pocket of Na+/K+-ATPase probably explains why no inhibitory
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potency was detected for cortisone. The docking result of ganderic acid A was similar to that
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of antcin B. One hydrogen bond is formed between the carboxyl group of ganoderic acid A
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and K912 of Na+/K+-ATPase. However, more severe repulsion in the binding pocket of
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Na+/K+-ATPase is observed for ganoderic acid A than antcin B; the carbonyl and hydroxyl
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groups at C-3, C-7, C-11, and C-15 of ganoderic acid A are repelled by V329, L132, F323,
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F790, and F793 of Na+/K+-ATPase.
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Discussion
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Being a local species exclusively grown in Taiwan, Niuchangchih has never been evaluated
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and documented in any of the traditional Chinese medicinal books. Indeed, Niuchangchih
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was regarded as a folk medicine and popularly used as a functional food only in the past few
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decades. In this study, five major antcins isolated from fruiting bodies of Niuchangchih were
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demonstrated to possess inhibitory potency on Na+/K+-ATPase. In light of this observation,
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we propose that the anti-hypertension and vasorelaxation effects of Niuchangchih may be
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partly, at least, attributed to its abundant antcins by promoting blood circulation via the same
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molecular mechanism triggered by cardiac glycosides and many analogous steroid-like
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compounds in Chinese herbs, that is, accentuating the force of myocardial contraction by
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elevating calcium concentration via the inhibition of Na+/K+-ATPase. Relatively, antcin A is
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a moderate Na+/K+-ATPase inhibitor weaker than ginsenoside Rh2, ursolic acid and
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oleanolic acid while the other four antcins are weak ones comparable to saikosaponin A,
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cholic acid, sarsasapogenin, polygalacic acid, jujuboside B, and glycyrrhizin (Chen et al.,
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2010).
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Originally seeing the highly structural similarity between antcins and ginsenosides, we
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intuitionally speculated that antcins and ginsenosides might bind to Na+/K+-ATPase in a
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similar manner and thus possess comparable inhibitory potency. However, the outcome was
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definitely surprising though all the five antcins examined in this study possessed inhibitory
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potency on Na+/K+-ATPase. In contrast with all the steroid-like compounds, e.g., ginsenoside
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Rh2, responsible for the therapeutic effects of many cardiac Chinese medicinal products
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examined so far (Chen et al., 2011), antcins bind to the hydrophobic pocket of
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Na+/K+-ATPase with the steroidal skeleton structurally upside-down. The reverse of steroidal
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skeleton is presumably due to the presence of an additional carboxyl acid group at the end of
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the aliphatic head portion attached to C-17 of antcins. Unlike the stable interaction between
Chung et al. 12
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the aliphatic head portion of ginsenoside Rh2 and the hydrophobic residues around the
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bottom of binding cavity in Na+/K+-ATPase, the additional carboxyl acid group in antcins
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seems to be severely repelled in the cavity leading to the steroidal skeleton turned reversely.
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With the reverse steroidal structure, the carboxyl acid group of antcins forms three hydrogen
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bonds with two cationic residues around the entrance of binding pocket while the carbonyl
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group at C-3 of antcins located deeply in the hydrophobic cavity suffers repulsion. In this
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point of view, sugar attachment at C-3 of steroidal skeleton that enhances Na+/K+-ATPase
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inhibitory potency of ginsenoside Rh2 and several other steroid-like compounds is likely to
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cause more severe hydrophilic-hydrophobic repulsion and thus not tolerable for antcins when
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binding to the hydrophobic pocket of Na+/K+-ATPase.
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In our recent study, antcin A was shown to be responsible for the anti-inflammatory
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effect of Niuchangchih via the same molecular mechanism triggered by glucocorticoids, i.e.,
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triggering the translocation of the glucocorticoid receptor into the nucleus to initiate the
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suppression of inflammation via gene regulation (Chen et al., 2011). In this study, we
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showed that antcin A should be competent to enhance blood circulation via effective
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inhibition of Na+/K+-ATPase. Regarding these two therapeutic effects, antcin A
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out-competes the other four antcins examined presumably due to the same feature, i.e., lack
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of hydrophilic groups attached to C-7, which is surrounded by hydrophobic residues in the
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binding pocket of glucocorticoid receptor or Na+/K+-ATPase. Indeed, the binding modes of
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antcin A to these two proteins are similar; the aliphatic head portion and the carbonyl group
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at C-3 are located in the entrance and the bottom of binding pocket, respectively.
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Coincidentally, the therapeutic capability of antcin A for anti-inflammatory effect is roughly
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10 times weaker than that of cortisone, and the Na+/K+-ATPase inhibitory potency of antcin
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A is roughly 10 times weaker than that of ginsenoside Rh2. Presumably, antcin A is a
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bi-functional compound with both therapeutic effects relatively mild. It will be interesting to
Chung et al. 13
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see if antcin A can be developed into a unique drug or functional food supplement on the
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basis of its dual biological functions.
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Acknowledgements
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The work was supported by a grant from the National Science Council, Taiwan, ROC (No
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98-2622-B-005-007-CC3 to J.T.C. Tzen).
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Legends
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Figure 1. Chemical structures of ouabain, ginsenoside Rh2, five major antcins (A, B, C, H,
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and K) of Niuchangchih, cortisone, and ganoderic acid A.
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Figure 2. Inhibition of porcine Na+/K+-ATPase by 0.2 mM of the 9 steroid-like compounds
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shown in Figure 1. Inhibitory potency of these compounds was observed as the reduction of
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Pi liberation released from ATP by a constant amount of commercial porcine
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Na+/K+-ATPase. Data represent mean±SEM of 5 replicates. bP<0.05, cP<0.01 vs control
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group (CON; deionized water only).
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Figure 3. Inhibitory potency of antcin A, ginsenoside Rh2 and ouabain on porcine
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Na+/K+-ATPase. Inhibitory potency of various concentrations of antcin A, ginsenoside Rh2
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and ouabain was observed as the reduction of Pi liberation released from ATP by a constant
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amount of commercial porcine Na+/K+-ATPase.
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Figure 4. Modeling of antcin A docking to the binding pocket of Na+/K+-ATPase  subunit.
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The amino acid residues around the binding pocket of Na+/K+-ATPase are shown in ribbon
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structure, and antcin A in ball-and-stick (A). Three K+ binding sites are shown in blue balls;
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two of them are located in the enlarged box (B).
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Figure 5. Detailed molecular interaction between the binding pocket of Na+/K+-ATPase and
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ginsenoside Rh2, antcin A, antcin B, cortisone, or ganderic acid A. (Top panels) Modeling is
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displayed for ligand compounds, ginsenoside Rh2, antcin A, antcin B, cortisone, and
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ganderic acid A binding to Na+/K+-ATPase  subunit. Amino acid residues around the
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binding pocket of Na+/K+-ATPase are shown in ribbon structure, and ligand compounds are
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illustrated in ball-and-stick structure with their carbon backbones colored in green. (Middle
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panels) Amino acid residues of Na+/K+-ATPase close to ligand compounds (ball-and-stick
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structure with green carbon backbones) are shown in line structure with hydrophobic residues
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colored in purple. (Bottom panels) Interactions between Na+/K+-ATPase and ligand
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compounds are depicted in simple drawings. Amino acid residues of Na+/K+-ATPase
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involved in formation of hydrogen bonds and hydrophobic-hydrophilic repulsion are shown
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as blue and pink squares, respectively. Distances of hydrogen bonds (green dash lines) and
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repulsion (red dash lines) between ligands and Na+/K+-ATPase are indicated.
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