Korean J Physiol Pharmacol.  2011 Aug;15(4):195-201. 10.4196/kjpp.2011.15.4.195.

Inhibitory Effects of Quercetin on Muscle-type of Nicotinic Acetylcholine Receptor-Mediated Ion Currents Expressed in Xenopus Oocytes

Affiliations
  • 1Department of Physiology, College of Veterinary Medicine and Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Korea. synah@konkuk.ac.kr
  • 2Department of Clinical Pharmacology and Therapeutics, University of Ulsan College of Medicine, Seoul 138-736, Korea.

Abstract

The flavonoid quercetin is a low molecular weight compound generally found in apple, gingko, tomato, onion and other red-colored fruits and vegetables. Like other flavonoids, quercetin has diverse pharmacological actions. However, relatively little is known about the influence of quercetin effects in the regulation of ligand-gated ion channels. Previously, we reported that quercetin regulates subsets of nicotinic acetylcholine receptors such as alpha3beta4, alpha7 and alpha9alpha10. Presently, we investigated the effects of quercetin on muscle-type of nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding human fetal or adult muscle-type of nicotinic acetylcholine receptor subunits. Acetylcholine treatment elicited an inward peak current (IACh) in oocytes expressing both muscle-type of nicotinic acetylcholine receptors and co-treatment of quercetin with acetylcholine inhibited IACh. Pre-treatment of quercetin further inhibited IACh in oocytes expressing adult and fetal muscle-type nicotinic acetylcholine receptors. The inhibition of IACh by quercetin was reversible and concentration-dependent. The IC50 of quercetin was 18.9+/-1.2 microM in oocytes expressing adult muscle-type nicotinic acetylcholine receptor. The inhibition of IACh by quercetin was voltage-independent and non-competitive. These results indicate that quercetin might regulate human muscle-type nicotinic acetylcholine receptor channel activity and that quercetin-mediated regulation of muscle-type nicotinic acetylcholine receptor might be coupled to regulation of neuromuscular junction activity.

Keyword

Flavonoids; Quercetin; Muscle-type nicotinic acetylcholine receptors; Xenopus oocyte

MeSH Terms

Acetylcholine
Adult
Flavonoids
Fruit
Ginkgo biloba
Humans
Inhibitory Concentration 50
Ligand-Gated Ion Channels
Lycopersicon esculentum
Molecular Weight
Neuromuscular Junction
Onions
Oocytes
Quercetin
Receptors, Nicotinic
RNA, Complementary
Vegetables
Xenopus
Acetylcholine
Flavonoids
Ligand-Gated Ion Channels
Quercetin
RNA, Complementary
Receptors, Nicotinic

Figure

  • Fig. 1. Chemical structure of quercetin (A) and its effect in oocytes expressing α1β1δε nicotinic acetylcholine receptors (B). Quercetin had no effect on IACh in oocytes expressing α1β1δε nicotinic acetylcholine receptors.

  • Fig. 2. Effect of quercetin (Que) on IACh in oocytes expressing α1β1 δγ and α1β1δε muscle-type nicotinic acetylcholine receptors. (A) Acetylcholine (100 μM) was first applied and then acetylcholine was co- or pre-treated with quercetin (100 μM). Thus, co-treatment of quercetin with acetylcholine and pre-treatment of quercetin before acetylcholine application inhibited IACh in oocytes expressing α1β1δγ nicotinic acetylcholine receptors. (B) Acetylcholine (100 μM) was first applied and then acetylcholine was co- or pre-treated with quercetin (100 μM). Thus, co-treatment of quercetin with acetylcholine and pre-treatment of quercetin before acetylcholine application inhibited IACh in oocytes expressing α1β1δε nicotinic acetylcholine receptors. The resting membrane potential of oocytes was about –35 mV and oocytes were voltage-clamped at a holding potential of –80 mV prior to drug application. Traces are representative of six separate oocytes from three different frogs. (C) Summary of % inhibition by quercetin of IACh was calculated from the average of the peak inward current elicited by acetylcholine alone before quercetin and the peak inward current elicited by acetylcholine alone after co- and pre-treatment of quercetin with acetylcholine. Each point represents the mean±S.E.M. (n=9∼12 from three different frogs).

  • Fig. 3. Dose-dependent effect of quercetin on IACh in oocytes expressing α1β1δε nicotinic acetylcholine receptors. (A) Acetylcholine (100 μM) was first applied and then acetylcholine was pre-treated with different concentration of quercetin. Thus, co-treatment of quercetin with acetylcholine and pre-treatment of quercetin before acetylcholine application inhibited IACh. The resting membrane potential of oocytes was about –35 mV and oocytes were voltage-clamped at a holding potential of –80 mV prior to drug application. Traces are representative of six separate oocytes from three different frogs. (A) IACh in oocytes expressing α1β1δε nicotinic acetylcholine receptors was elicited at – 80 mV holding potential with indicated time in the presence of 100 μM acetylcholine and then the indicated concentration of quercetin was pre-treated for 30 s before acetylcholine. Traces are representative of 6∼9 separate oocytes from three different frogs. (B) Percent inhibition by quercetin of IACh was calculated from the average of the peak inward current elicited by acetylcholine alone before quercetin and the peak inward current elicited by acetylcholine alone after co-treatment of quercetin with acetylcholine and pre-treatment of quercetin before acetylcholine application. The continuous line shows the curve fitted according to the equation. y/ymax=[Quercetin]/[Quercetin] +K1/2), where ymax, the maximum inhibition (89.1±2.2% and 19.3±1.3 from pre-treatment and co-treatment, respectively, mean±S.E.M.) and K1/2 is the concentration for half-maximum inhibition (18.9±1.3 and 44.3±1.1 μM from pre-treatment and co-treatment, respectively, mean±S.E.M.), and [Quercetin] is the concentration of quercetin. Each point represents the mean±S.E.M. (n=9∼12 from three different frogs).

  • Fig. 4. Current-voltage relationship and voltage-independent inhibition by quercetin. (A) Current-voltage relationships of IACh inhibition by quercetin in α1β1δε nicotinic acetylcholine receptors. Representative current-voltage relationships were obtained using voltage ramps of –100 to +60 mV for 300 ms at a holding potential of –80 mV. Voltage steps were applied before and after application of 100 μM acetylcholine in the absence or presence of 20 μM quercetin. (B) Voltage-independent inhibition of IACh in the α1β1δε nicotinic acetylcholine receptors by quercetin. The values were obtained from the receptors in the absence or presence of 20 μM quercetin at the indicated membrane holding potentials.

  • Fig. 5. Concentration-dependent effects of acetylcholine on quercetin-mediated inhibition of IACh. (A) The representative traces were obtained from α1β1δε nicotinic acetylcholine receptors expressed in oocytes. IACh of the upper and lower panels were elicited from concentration of 10 μM and 300 μM acetylcholine at a holding potential of –80 mV, respectively. (B) Concentration-response relationships for acetylcholine in the α1β1δε nicotinic acetylcholine receptors applied with acetylcholine (1∼300 μM) alone or with acetylcholine plus pre-application of 20 μM quercetin. The IACh of oocytes expressing the α1β1δε nicotinic acetylcholine receptors was measured using the indicated concentration of acetylcholine in the absence (☐) or presence (❍) of 20 μM quercetin. Oocytes were exposed to ACh alone or to ACh with quercetin. Oocytes were voltage-clamped at a holding potential of –80 mV. Each point represents the mean±S.E.M. (n=8 ∼ 12/group).


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