Eom S et al. (2020), Molecular Regulation of α3β4 Nicotinic Acetylch...

XB-ART-57106

Int J Mol Sci 2020 Jun 18;2112:. doi: 10.3390/ijms21124329.

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Molecular Regulation of α3β4 Nicotinic Acetylcholine Receptors by Lupeol in Cardiovascular System.

Eom S , Kim C , Yeom HD , Lee J , Lee S , Baek YB , Na J , Park SI , Kim GY , Lee CM , Lee JH .

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Cardiovascular disease (CVD) occurs globally and has a high mortality rate. The highest risk factor for developing CVD is high blood pressure. Currently, natural products are emerging for the treatment of hypertension to avoid the side effects of drugs. Among existing natural products, lupeol is known to be effective against hypertension in animal experiments. However, there exists no study regarding the molecular physiological evidence against the effects of lupeol. Consequently, we investigated the interaction of lupeol with α3β4 nicotinic acetylcholine receptors (nAChRs). In this study, we performed a two-electrode voltage-clamp technique to investigate the effect of lupeol on the α3β4 nicotine acetylcholine receptor using the oocytes of Xenopus laevis. Coapplication of acetylcholine and lupeol inhibited the activity of α3β4 nAChRs in a concentration-dependent, voltage-independent, and reversible manner. We also conducted a mutational experiment to investigate the influence of residues of the α3 and β4 subunits on lupeol binding with nAChRs. Double mutants of α3β4 (I37A/N132A), nAChRs significantly attenuated the inhibitory effects of lupeol compared to wild-type α3β4 nAChRs. A characteristic of α3β4 nAChRs is their effect on transmission in the cardiac sympathetic ganglion. Overall, it is hypothesized that lupeol lowers hypertension by mediating its effects on α3β4 nAChRs. The interaction between lupeol and α3β4 nAChRs provides evidence against its effect on hypertension at the molecular-cell level. In conclusion, the inhibitory effect of lupeol is proposed as a novel therapeutic approach involving the antihypertensive targeting of α3β4 nAChRs. Furthermore, it is proposed that the molecular basis of the interaction between lupeol and α3β4 nAChRs would be helpful in cardiac-pharmacology research and therapeutics.

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Species referenced: Xenopus laevis
Genes referenced: chrna3 chrnb4
GO keywords: acetylcholine receptor activity

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	Graphical Abstract
	Figure 1. Structure and regulatory effects of lupeol on the α3β4 nicotinic acetylcholine receptors. (A) Chemical structure of lupeol (LP). (B) Summary of inhibitory effects of cotreatment of lupeol with acetylcholine. (C) Typically, 100 μM acetylcholine was applied with or without 30 μM lupeol. Mecamylamine (MEC) is an antagonist of nicotinic acetylcholine receptors (nAChRs). MEC shown at concentration of 10 μM. Arrow, point were LP was not treated onto oocyte surface but injected into cells. Traces representative of 5–9 separate oocytes from 3–5 different frogs. IAch recorded at holding potential of −80 mV before lupeol treatment. Small blue circle, cation; red one, lupeol.
	Figure 2. Mechanism by which lupeol interacts with α3β4 nicotinic acetylcholine receptors. (A) Inward currents of trace for cotreatment of acetylcholine and lupeol in α3β4 nACh receptor. (B) Concentration–response relationship induced by cotreatment of lupeol in α3β4 nACh receptors. Each point represents mean ± SEM (n = 10–13/group). (C) Representative current–voltage relationship obtained by using voltage ramps from −100 to +60 mV at holding potential of −80 mV. Voltage steps treated with 100 μM acetylcholine alone, and cotreated with 30 μM lupeol with ACh (n = 7–9 from four different frogs). (D) IAch induced by various concentrations of acetylcholine (■) and cotreatment with 30 μM lupeol (●). Oocytes voltage-clamped at holding potential of −80 mV. Each point represents mean ± SEM (n = 10–13 from five different frogs).
	Figure 3. Computational molecular modeling of lupeol docked to α3β4 nicotinic acetylcholine receptor. (A,C) Side views of docked lupeol in complex with nACh α3β4 receptor. (B,D) Top view of docking model.
	Figure 4. Binding pocket view and docking results comparing wild type and mutant in lupeol docked to α3β4 nAChRs. (A) Lupeol located in binding pocket in extracellular area between Segments 1 and 2 of α3β4 nicotinic acetylcholine receptors. (B) Two-dimensional schematic presentation of predicted binding mode of lupeol in ligand-binding pocket. Ligands and important residues shown. (C,D) Binding interface and lupeol of wild type. (C) Four mutant channels in which mutations disturbed interaction of lupeol to varying degrees.
	Figure 5. Effect of lupeol on double-mutant α3β4 nicotinic acetylcholine receptors. (A–C) Inward currents of concentration–response relationship to coapplication of acetylcholine and lupeol with 10, 30, and 100 μM concentrations of oocytes expressing α3 (I37A) + Wild β4, Wild α3 + β4 (N132A), and α3β4 (I37A/N132A) nAChRs. (D) Concentration–response graphs showing effect of different concentrations of lupeol on mutant α3β4 nACh receptors in presence of 100 μM acetylcholine. Each point is mean ± SEM (n = 6–7 from three different frogs). Additional half-inhibitory concentration, Hill coefficient, and Imax values described in Table 1.
	Figure 1. Structure and regulatory effects of lupeol on the α3β4 nicotinic acetylcholine receptors. (A) Chemical structure of lupeol (LP). (B) Summary of inhibitory effects of cotreatment of lupeol with acetylcholine. (C) Typically, 100 μM acetylcholine was applied with or without 30 μM lupeol. Mecamylamine (MEC) is an antagonist of nicotinic acetylcholine receptors (nAChRs). MEC shown at concentration of 10 μM. Arrow, point were LP was not treated onto oocyte surface but injected into cells. Traces representative of 5–9 separate oocytes from 3–5 different frogs. IAch recorded at holding potential of −80 mV before lupeol treatment. Small blue circle, cation; red one, lupeol.
	Figure 2. Mechanism by which lupeol interacts with α3β4 nicotinic acetylcholine receptors. (A) Inward currents of trace for cotreatment of acetylcholine and lupeol in α3β4 nACh receptor. (B) Concentration–response relationship induced by cotreatment of lupeol in α3β4 nACh receptors. Each point represents mean ± SEM (n = 10–13/group). (C) Representative current–voltage relationship obtained by using voltage ramps from −100 to +60 mV at holding potential of −80 mV. Voltage steps treated with 100 μM acetylcholine alone, and cotreated with 30 μM lupeol with ACh (n = 7–9 from four different frogs). (D) IAch induced by various concentrations of acetylcholine (■) and cotreatment with 30 μM lupeol (●). Oocytes voltage-clamped at holding potential of −80 mV. Each point represents mean ± SEM (n = 10–13 from five different frogs).
	Figure 3. Computational molecular modeling of lupeol docked to α3β4 nicotinic acetylcholine receptor. (A,C) Side views of docked lupeol in complex with nACh α3β4 receptor. (B,D) Top view of docking model.
	Figure 4. Binding pocket view and docking results comparing wild type and mutant in lupeol docked to α3β4 nAChRs. (A) Lupeol located in binding pocket in extracellular area between Segments 1 and 2 of α3β4 nicotinic acetylcholine receptors. (B) Two-dimensional schematic presentation of predicted binding mode of lupeol in ligand-binding pocket. Ligands and important residues shown. (C,D) Binding interface and lupeol of wild type. (C) Four mutant channels in which mutations disturbed interaction of lupeol to varying degrees.
	Figure 5. Effect of lupeol on double-mutant α3β4 nicotinic acetylcholine receptors. (A–C) Inward currents of concentration–response relationship to coapplication of acetylcholine and lupeol with 10, 30, and 100 μM concentrations of oocytes expressing α3 (I37A) + Wild β4, Wild α3 + β4 (N132A), and α3β4 (I37A/N132A) nAChRs. (D) Concentration–response graphs showing effect of different concentrations of lupeol on mutant α3β4 nACh receptors in presence of 100 μM acetylcholine. Each point is mean ± SEM (n = 6–7 from three different frogs). Additional half-inhibitory concentration, Hill coefficient, and Imax values described in Table 1.

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