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

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 α3β4, α7 and α9α10. 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 (I(ACh)) in oocytes expressing both muscle-type of nicotinic acetylcholine receptors and co-treatment of quercetin with acetylcholine inhibited I(ACh). Pre-treatment of quercetin further inhibited I(ACh) in oocytes expressing adult and fetal muscle-type nicotinic acetylcholine receptors. The inhibition of I(ACh) by quercetin was reversible and concentration-dependent. The IC(50) of quercetin was 18.9±1.2 µM in oocytes expressing adult muscle-type nicotinic acetylcholine receptor. The inhibition of I(ACh) 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.

Keywords: Flavonoids; Muscle-type nicotinic acetylcholine receptors; Quercetin; Xenopus oocyte.

Fig. 1

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

Fig. 2

Effect of quercetin (Que) on I_ACh in oocytes expressing α1β1δγ 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 I_ACh 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 I_ACh 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 I_ACh 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 I_ACh 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 I_ACh. 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) I_ACh 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 I_ACh 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/y_max=[Quercetin]/[Quercetin]+K_1/2), where y_max, the maximum inhibition (89.1±2.2% and 19.3±1.3 from pre-treatment and co-treatment, respectively, mean±S.E.M.) and K_1/2 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 I_ACh 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 I_ACh 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 I_ACh. (A) The representative traces were obtained from α1β1δε nicotinic acetylcholine receptors expressed in oocytes. I_ACh 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 I_ACh 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).