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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 (●) or 10 μM (▲). 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 (●) or 100 μM (▲). 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. (A–D) 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. (A–C) 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 = 7–10 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 = 7–9/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.
Alqahtani,
The pentacyclic triterpenoids in herbal medicines and their pharmacological activities in diabetes and diabetic complications.
2013, Pubmed
Alqahtani,
The pentacyclic triterpenoids in herbal medicines and their pharmacological activities in diabetes and diabetic complications.
2013,
Pubmed
Ballatore,
Carboxylic acid (bio)isosteres in drug design.
2013,
Pubmed
Chen,
A chemically standardized extract of Ziziphus jujuba fruit (Jujube) stimulates expressions of neurotrophic factors and anti-oxidant enzymes in cultured astrocytes.
2014,
Pubmed
Crivori,
Predicting blood-brain barrier permeation from three-dimensional molecular structure.
2000,
Pubmed
Cuddapah,
Regulation of the Blood-Brain Barrier by Circadian Rhythms and Sleep.
2019,
Pubmed
Dani,
Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms of the central nervous system.
2007,
Pubmed
Domino,
Role of cholinergic mechanisms in states of wakefulness and sleep.
1968,
Pubmed
Duga,
Characterization of the genomic structure of the human neuronal nicotinic acetylcholine receptor CHRNA5/A3/B4 gene cluster and identification of novel intragenic polymorphisms.
2001,
Pubmed
Fulda,
Betulinic Acid for cancer treatment and prevention.
2008,
Pubmed
Karlin,
Emerging structure of the nicotinic acetylcholine receptors.
2002,
Pubmed
Kodama,
Acetylcholine and glutamate release during sleep-wakefulness in the pedunculopontine tegmental nucleus and norepinephrine changes regulated by nitric oxide.
1999,
Pubmed
Monti,
The role of dorsal raphe nucleus serotonergic and non-serotonergic neurons, and of their receptors, in regulating waking and rapid eye movement (REM) sleep.
2010,
Pubmed
Mowry,
Using the Xenopus Oocyte Toolbox.
2020,
Pubmed
,
Xenbase
Niwa,
Muscarinic Acetylcholine Receptors Chrm1 and Chrm3 Are Essential for REM Sleep.
2018,
Pubmed
Peng,
Anxiolytic effect of seed of Ziziphus jujuba in mouse models of anxiety.
2000,
Pubmed
Reitstetter,
Dependence of nicotinic acetylcholine receptor recovery from desensitization on the duration of agonist exposure.
1999,
Pubmed
Sahu,
Molecular interactions of the physiological anti-hypertensive peptide catestatin with the neuronal nicotinic acetylcholine receptor.
2012,
Pubmed
Scholey,
Exploring the Effect of Lactium™ and Zizyphus Complex on Sleep Quality: A Double-Blind, Randomized Placebo-Controlled Trial.
2017,
Pubmed
Shi,
Herbal Insomnia Medications that Target GABAergic Systems: A Review of the Psychopharmacological Evidence.
2014,
Pubmed
Stępnik,
In Silico Studies on Triterpenoid Saponins Permeation through the Blood-Brain Barrier Combined with Postmortem Research on the Brain Tissues of Mice Affected by Astragaloside IV Administration.
2020,
Pubmed
Teles-Grilo Ruivo,
Coordinated Acetylcholine Release in Prefrontal Cortex and Hippocampus Is Associated with Arousal and Reward on Distinct Timescales.
2017,
Pubmed
Vazquez,
Basal forebrain acetylcholine release during REM sleep is significantly greater than during waking.
2001,
Pubmed
Watson,
Neuropharmacology of Sleep and Wakefulness.
2010,
Pubmed
Yogeeswari,
Betulinic acid and its derivatives: a review on their biological properties.
2005,
Pubmed
Zant,
Cholinergic Neurons in the Basal Forebrain Promote Wakefulness by Actions on Neighboring Non-Cholinergic Neurons: An Opto-Dialysis Study.
2016,
Pubmed
Zhao,
Simultaneous determination of saponins and fatty acids in Ziziphus jujuba (Suanzaoren) by high performance liquid chromatography-evaporative light scattering detection and pressurized liquid extraction.
2006,
Pubmed
