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...

Transient receptor potential (TRP) channel has critical actions as conditional sensors in primary afferent neurons. We studied the regulatory action of gomisin A on TRPV1 channel current in this report. Schisandra chinensis contains bioactive compounds such as the gomisin derivatives and their related compounds. Coapplication with gomisin A inhibited the capsaicin-mediated inward peak current. This inhibitory effect of gomisin A on capsaicin-induced inward current showed concentration-dependence and was reversible. The half maximal inhibitory concentration of gomisin A was 62.7 ± 8.4 µM. In addition, this inhibition occurred in a noncompetition regulation mode and voltage insensitive manner. Furthermore, molecular docking studies of gomisin A on TRPV1 showed that it interacted predominantly with residues at cavities in the segments 1 and 2 of each subunit. Four potential binding sites for this ligand in the extracellular region at sensor domain of TRPV1 channel were identified. Point mutagenesis studies were undertaken, and gomisin A potency decreased for both the Y453A and N467A mutants. The double mutation of Y453 and N467 significantly attenuated inhibitory effects by gomisin A. In summary, this study revealed the molecular basis for the interaction between TRPV1 and gomisin A and provides a novel potent interaction ligand.

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