Molecular Determinants of α3β4 Nicotinic Acetylcholine Receptors Inhibition by Triterpenoids

Abstract

In a previous work, we reported the regulatory role of the triterpenoids on 5-hydroxytryptamine (5-HT) 3 A receptors activity in Xenopus laevis oocytes (Eur. J. Pharmacol., 615, 2009, Lee et al.). In the present report, we studied the modulation of triterpenoids on the activity of the human nicotinic acetylcholine receptor type α3β4. Two-electrode voltage clamp experiments were used to test acetylcholine mediated inward current (I ACh ). Treatment with triterpenoids (dehydroeburicoic acid, 6α-hydroxypolyporenic acid C and pachymic acid) inhibited I ACh in a concentration dependent and reversible manner. The IC 50 values for pachymic acid, dehydroeburicoic acid, and 6α-hydroxypolyporenic acid C were 14.9, 37.7, and 20.9 µM, respectively. The inhibitory regulation of I ACh by each triterpenoid showed in a non-competitive manner on the activity of α3β4 nicotinic acetylcholine receptors. These results show that triterpenoids (pachymic acid, dehydroeburicoic acid, 6α-hydroxypolyporenic acid C) can be used as agents to modulate the activity of nicotinic acetylcholine receptor type α3β4. Furthermore, molecular docking studies of 6α-hydroxypolyporenic acid C on α3β4 nicotinic acetylcholine receptors in silico showed that this molecule interacted predominantly with residues at cavities in the α3 subunit and β4 subunit. This docking assays indicated four potential binding sites for this ligand in the extracellular region at sensor domain of α3β4 nicotinic acetylcholine receptors. In point mutagenesis of those whose alanine substitution, 6α-hydroxypolyporenic acid C potency decreased on W25A of α3 subunit or N109A of β4 subunit in both mutants. The double mutation of W25A of α3 subunit and N109A of β4 subunit was significantly attenuated inhibitory effects by 6α-hydroxypolyporenic acid C. All taken together, this study revealed that molecular basis of α3β4 nicotinic acetylcholine receptors by triterpenoids and provides a novel potent interaction ligand Key words triterpenoid; docking assay; ligand-gated ion channel; α3β4 nicotinic acetylcholine receptor Triterpenoids are classified as nature compounds and synthesized materials from triterpenes modified by squalene cyclization or acyclic carbon substitution in Fig. 1. Triterpenoids isolated from various plants are generally used for clinical purposes in Far East Asia. 1) In particular, triterpenoids showed inhibitory effects on tumor growth in the dermal tissue of mice with second step tumoral calcinosis and 12 tetradecanoyl-phobol acetate derived infection.2) Furthermore, triterpenoids like as pachymic acid and dehydrotumulosic acid potently modulated PLA2 from snake toxin.3) Pachymic acid with a methyl-group at the 24th carbon also showed antiemetic effects in amphibians and was purified from the fungus Fomitopsis. 4,5)The acetylcholine receptor widely distributed throughout the human body and it has been studied in neuronal and muscular systems. In particular, nicotinic acetylcholine receptors are activated by the agonist acetylcholine, allowing cation movement into cyto...

Targeted Screening Approach to Systematically Identify the Absorbed Effect Substances of Poria cocos in Vivo Using Ultrahigh Performance Liquid Chromatography Tandem Mass Spectrometry

“…All herb medicines were identified by Prof. Zhiqiang Liu (Changchun Institute of Applied Chemistry, Chinese Academy of Sciences). Eight reference standards of dehydrotrametenolic acid (1), 3α,16β-dihydroxylanosta-7,9 (11),24-trien-21-oic acid (2), polyporenic acid C (3), poricoic B (4), dehydrotumulosic acid (5), tumulosic acid (6), 3-O-acetyl-16αhydroxydehydrotrametenolic acid (7), and pachymic acid (8) were obtained from Purification Engineering Technology Research Center of Sichuan Province Natural Medicine (Sichuan China). NADPH (Nicotinamide Adenine Dinucleotide Phosphate) was purchased from R&D. The analytical-grade reagents, ethyl acetate, n-butanol, and absolute ethanol were provided by Beijing Chemical Works (Beijing China), and deionized water was purified using a Milli-Q water purification system (Milford, MA, USA).…”

“…It also was used for making biscuits, cakes and bread owing to its potential promotion benefits for health. These potential application of Poria cocos attribute to the continued considerable levels of attention. − It is worthwhile to analyze the absorbed substances of Poria cocos in vivo systematically.…”

Pharmacological profiles and therapeutic applications of pachymic acid (Review)

“…Specifically, it suppresses 5-hydroxytryptamine (5-HT)-stimulated inward currents, and inhibits 5-HT-stimulated inward current in Xenopus oocytes expressing the human 5-HT3A receptor. It also inhibits the acetylcholine-induced inward current in oocytes expressing nicotinic type α3β4 acetylcholine channel receptors ( 75 , 76 ). PA decreases allograft rejection in rats, protects peripheral blood lymphocytes from apoptosis by stabilizing the mitochondrial transmembrane potential and reduces the percentage of CD8 + lymphocytes ( 77 ).…”

Non-enzymatic formation of isoprene and 2-methyl-3-buten-2-ol (2-MBO) by manganese

“…Furthermore, volatile organic compounds in breath can be a biochemical probe providing both non-invasive and continuous information on the metabolic and physiological state of an individuals. In terms of human exhalation, isoprene accounts for up to 70% of total hydrocarbon removal via exhalation and may act as a noninvasive indicator of several metabolic effects in the human body 9 .…”

“…In plants and bacteria, isoprene is biosynthesized by isoprene synthase 10 – 12 whilst its formation mechanism in the human body remains unclear 9 . IspS and many other terpene synthases require Mg 2+ or Mn 2+ metal ions as a cofactor 13 – 17 .…”