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Molecules
2021 May 01;269:. doi: 10.3390/molecules26092659.
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Molecular Regulation of Betulinic Acid on α3β4 Nicotinic Acetylcholine Receptors.
Lee S
,
Jung W
,
Eom S
,
Yeom HD
,
Park HD
,
Lee JH
.
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Betulinic acid (BA) is a major constituent of Zizyphus seeds that have been long used as therapeutic agents for sleep-related issues in Asia. BA is a pentacyclic triterpenoid. It also possesses various anti-cancer and anti-inflammatory effects. Current commercially available sleep aids typically use GABAergic regulation, for which many studies are being actively conducted. However, few studies have focused on acetylcholine receptors that regulate wakefulness. In this study, we utilized BA as an antagonist of α3β4 nicotinic acetylcholine receptors (α3β4 nAChRs) known to regulate rapid-eye-movement (REM) sleep and wakefulness. Effects of BA on α3β4 nAChRs were concentration-dependent, reversible, voltage-independent, and non-competitive. Site-directed mutagenesis and molecular-docking studies confirmed the binding of BA at the molecular level and showed that the α3 subunit L257 and the β4 subunit I263 residues affected BA binding. These data demonstrate that BA can bind to a binding site different from the site for the receptor's ligand, acetylcholine (ACh). This suggests that BA may be an effective antagonist that is unaffected by large amounts of ACh released during wakefulness and REM sleep. Based on the above experimental results, BA is likely to be a therapeutically useful sleep aid and sedative.
Figure 2
Confirmation of α3β4 nAChRs current-voltage relationship and non-competitive action of BA. (A) Confirmation of the interaction between the response of α3β4 nAChRs and different concentrations of BA using a voltage ramp protocol from −100 to +60 mV. The membrane holding potential was −80 mV. The applied ACh concentration was fixed at 100 μM (n = 8–10 from four different frogs). (B) After applying various concentrations of ACh, induced internal current and non-competitive action of BA were observed. The concentration of BA applied was 3 μM (â—) or 10 μM (â–²). The holding potential of the oocyte voltage-clamp was −80 mV. Each point represents mean ± SEM (n = 7–9/group). (C) Double-mutant α3β4 nAChRs (α3: L257A, β4: I263A) and the application of different concentrations of BA using a voltage ramp protocol from −100 to +60 mV. The membrane holding potential was −80 mV. The applied ACh concentration was fixed at 100 μM (n = 7–9 from five different frogs). (D) Applying various concentrations of ACh and BA to double-mutant α3β4 nAChRs (α3: L257A, β4: I263A). The concentration of BA applied was 10 μM (â—) or 100 μM (â–²). The holding potential of the oocyte voltage-clamp was −80 mV. Each point represents mean ± SEM (n = 7–9/group).
Figure 3
Molecular interaction of BA based on the docking model of α3β4 nAChRs in 3D structure. (AâD) A view of the BA interaction site on α3β4 nAChRs from the side and top. This structure was constructed based on the structure shown in the protein data bank (PDB) (ID 5T90).
Figure 4
Molecular docking model of BA with wild-type and double-mutant α3β4 nAChRs. (A) Binding pocket site in the transmembrane domain of α3β4 nAChRs. BA docked to the transmembrane domain close to the ion-permeable pore. (B) The predicted binding site of BA in a 2D schematic. (C) The binding interaction energy of BA and residues in wild-type α3β4 nAChRs. (D) The binding interaction energy of BA and residues in five mutant channels of α3β4 nAChRs.
Figure 5
Inhibition of BA by α3β4 nAChRs mutant types. (AâC) Concentration-response of inward current depending on mutant type. ACh and BA were co-applied. The applied ACh concentration was fixed at 100 μM while BA concentrations included 10, 30, and 100 μM (n = 7â10 from four different frogs). (D) Graph represents the inhibition of BA activity by mutant type at various concentrations. Each point is presented as mean ± SEM (n = 7â9/group). Detailed values are shown in Table 1.
Figure 1. Chemical structure of betulinic acid (BA) and activation manner on bovine α3β4 nicotinic acetylcholine receptors expressed in Xenopus oocytes. (A) Structure of BA. (B) Inhibition of IACh on bovine α3β4 nAChRs by mecamylamine (MEC), a nicotinic acetylcholine receptor antagonist, and BA. ACh, MEC, and BA were administered at 2 mL per min. The applied concentration of ACh was 100 μM. MEC and BA were administered at 10 μM. The trace indicated inward current in the result of a co-applied and induced reversible inward current (n = 6–8 from four different frogs). (C) Concentration-relationship results induced by co-treatment with ACh and BA for α3β4 nAChRs. The applied ACh concentration was fixed at 100 μM (n = 8–10 from five different frogs). (D) The inward current induced by the concentration-relationship of BA and MEC. The inhibition percentage curve of BA and MEC in α3β4 nAChRs was fitted with the Hill equation. Each point represents the mean ± SEM (n = 7–9/group). The oocyte voltage-clamp holding potential was −80 mV.
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