Quercetin Inhibits α3β4 Nicotinic Acetylcholine Receptor-Mediated Ion Currents Expressed in Xenopus Oocytes

Abstract

Quercetin mainly exists in the skin of colored fruits and vegetables as one of flavonoids. Recent studies show that quercetin, like other flavonoids, has diverse pharmacological actions. However, relatively little is known about quercetin effects in the regulations of ligand-gated ion channels. In the previous reports, we have shown that quercetin regulates subsets of homomeric ligand-gated ion channels such as glycine, 5-HT_3A and α7 nicotinic acetylcholine receptors. In the present study, we examined quercetin effects on heteromeric neuronal α3β4 nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding bovine neuronal α3 and β4 subunits. Treatment with acetylcholine elicited an inward peak current (I_ACh) in oocytes expressing α3β4 nicotinic acetylcholine receptor. Co-treatment with quercetin and acetylcholine inhibited I_ACh in oocytes expressing α3β4 nicotinic acetylcholine receptors. The inhibition of I_ACh by quercetin was reversible and concentration-dependent. The half-inhibitory concentration (IC₅₀) of quercetin was 14.9±0.8 µM in oocytes expressing α3β4 nicotinic acetylcholine receptor. The inhibition of I_ACh by quercetin was voltage-independent and non-competitive. These results indicate that quercetin might regulate α3β4 nicotinic acetylcholine receptor and this regulation might be one of the pharmacological actions of quercetin in nervous systems.

INTRODUCTION

Nicotinic acetylcholine receptors are members of the Cys-loop family of ligand-gated ion channels [1]. Currently, sixteen different nicotinic acetylcholine receptor subunits are known [2]. Muscle form of nicotinic acetylcholine receptor consists of α1β1γε. Activation of muscle form of nicotinic acetylcholine receptor initiates muscle contraction by inducing depolarization of neuromuscular junctions. Neuronal forms of nicotinic acetylcholine receptor consist of α (α2-7, α9, α10) and β (β2-4) and their activations are mainly involved in rapid synaptic transmissions in central and peripheral nervous systems [2]. Neuronal nicotinic acetylcholine receptor contain α2-6 subunits that are usually expressed as heteropentamers in combination with β2-4 subunits [3-6]. For example, the α3 and β4 subunits can form heteromeric receptors [7]. Although many nicotinic acetylcholine receptor subunits are expressed in the central and peripheral nervous systems, the distributions of α3β4 nicotinic acetylcholine receptor are mainly restricted to several tissues such as adrenal chromaffin cells [8,9]. The α3β4 nicotinic acetylcholine receptors play an important role for release of catecholamine releases [10].

Skin of colored fruits and vegetables contains a variety of flavonoids and quercetin is one of representative flavonoids (Fig. 1A) [11]. Quercetin exhibits multiple pharmacological activities in nervous and non-nervous systems [12-16]. However, the underlying cellular mechanisms of quercetin actions are relatively unknown, especially with regards to possible regulation of receptors or ionic channels involved in synaptic transmissions. Recently, we demonstrated that quercetin differentially regulates subsets of nicotinic acetylcholine receptor such as homomeric glycine, 5-HT_3A, α7 and α9α10 nicotinic acetylcholine receptors [17-20]. In the present study, we examined the effects of quercetin on heteromeric α3β4 nicotine acetylcholine receptor channel activity and report here that quercetin inhibits heteromeric α3β4 nicotine acetylcholine receptor channel activity with voltage-independent and non-competitive manner. These results indicate that quercetin might play a role for the regulation of nicotinic acetylcholine receptor channel activities.

Fig. 1.

Chemical structure of quercetin (A) and effect of quercetin (Que) in oocytes expressing α3β4 nicotinic acetylcholine receptors. Quercetin itself had no effect on I_ACh in oocytes expressing α3β4 nicotinic acetylcholine receptors (B).

METHODS

Materials

Bovine α3 and β4 nicotine acetylcholine receptor subunit cDNAs were kindly provided by Dr. S. Sala (Universidad Miguel Hernández-Consejo Superior de Investigaciones Cientificas, Spain). Quercetin (Fig. 1A) and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Preparation of Xenopus laevis oocytes and microinjection

X. laevis frogs were purchased from Xenopus I (Ann Arbour, MI, USA). Animal care and handling were in accordance with the highest standards of Konkuk university guidelines. To isolate oocytes, frogs were anesthetised with an aerated solution of 3-amino benzoic acid ethyl ester, and the ovarian follicles were removed. The oocytes were separated with collagenase followed by agitation for 2 h in a Ca²⁺-free medium containing 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 5 mM HEPES, 2.5 mM sodium pyruvate, 100 units/ml penicillin, and 100 µg/ml streptomycin. Stage V-VI oocytes were collected and stored in a ND96 medium (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, and 5 mM HEPES, pH 7.5) supplemented with 50 µg/ml gentamicin. The solution containing the oocytes was maintained at 18℃ with continuous gentle shaking and was replaced daily. Electrophysiological experiments were performed three to five days after oocyte isolation. For α3β4 nicotine acetylcholine receptor experiments, α3 and β4 nicotine acetylcholine receptor subunit-encoding cRNAs (40 nl) were co-injected into the animal or vegetal pole of each oocyte 1 day after isolation using a 10-µl microdispenser (VWR Scientific, West Chester, PA, USA) fitted with a tapered glass pipette tip (15~20 µm in diameter) [19].

Data recording

A custom-made Plexiglas net chamber was used for two-electrode voltage-clamp recordings, as previously reported [19]. A single oocyte was constantly superfused with a recording solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, and 10 mM HEPES, pH 7.5) in the absence or presence of glutamate or quercetin during recording. The microelectrodes were filled with 3 M KCl and had a resistance of 0.2~0.7 MΩ. Two-electrode voltage-clamp recordings were obtained at room temperature using an Oocyte Clamp (OC-725C, Warner Instrument) and were digitised using Digidata 1,200 A (Molecular Devices, Sunnyvale, CA, USA). Stimulation and data acquisition were controlled using pClamp 8 software (Molecular Devices). For most electrophysiological data, the oocytes were clamped at a holding potential of -80 mV. For current and voltage (I-V) relationship, voltage ramps were applied from -100 to +60 mV for 300-ms. In the different membrane-holding potential experiments, the oocytes were clamped at the indicated holding potentials. Linear leak and capacitance currents were corrected by means of the leak subtraction procedure.

Data analysis

To obtain the concentration-response curve for the effect of quercetin on the inward peak I_ACh mediated by the α3β4 nicotinic acetylcholine receptor, the I_ACh peak was plotted at different concentrations of quercetin. Origin software (OriginLab Corp., Northampton, MA, USA) was used to fit the plot to the Hill equation: I/I_max=1/[1+(IC₅₀/[A])^nH], where I_max was maximal current obtained from each ED₅₀ value of acetylcholine in wild-type receptors, IC₅₀ was the concentration of quercetin required to decrease the response by 50%, [A] was the concentration of quercetin, and nH was the Hill coefficient. All values were presented as mean±S.E.M. The differences between the means of control and treatment data were determined using the unpaired Student's t-test. A value of p<0.05 was considered to be statistically significant.

RESULTS

Effect of quercetin on I_ACh in oocytes expressing heteromeric α3β4 nicotinic acetylcholine receptors

The addition of acetylcholine to the bathing solution induced a large inward current in oocytes injected with α3β4 nicotinic acetylcholine receptor, indicating that this nicotinic acetylcholine receptor was functionally expressed in this system (Fig. 1B). Quercetin itself had no effect in oocytes expressing α3β4 nicotinic acetylcholine receptors at a holding potential of -80 mV (Fig. 1B). But co- and pre-treatment with quercetin and acetylcholine inhibited I_ACh in oocytes expressing α3β4 nicotinic acetylcholine receptors (Fig. 2A and B, n=9 from three different frogs). The inhibition of I_ACh by quercetin in oocytes expressing α3β4 nicotinic acetylcholine receptors was reversible with a negligible desensitization (Fig. 2A). Thus, these results suggest the possibility that quercetin regulates α3β4 nicotinic acetylcholine receptor channel activity, although quercetin itself had no effect on α3β4 nicotinic acetylcholine receptor channel activity.

Fig. 2.

Effect of quercetin (Que) on I_ACh in oocytes expressing α3β4 nicotinic acetylcholine receptors. (A) Acetylcholine (ACh, 100 µM) was first applied and then acetylcholine was co- or pre-applied with quercetin (Que, 30 µM). Thus, co- and pre-application of quercetin with acetylcholine 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. (B) Co- or pre-application of quercetin did not affect differently on I_ACh. (C) I_ACh in oocytes expressing α3β4 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 co-applied with acetylcholine. (D) % 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-application of quercetin with acetylcholine. 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 (97.8±1.7%, mean±S.E.M.) and K_1/2 is the concentration for half-maximum inhibition (14.9±0.8 µM, 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).

Concentration-dependent effect of quercetin on I_ACh in oocytes expressing α3β4 nicotinic acetylcholine receptor

Since pre-treatment of quercetin did not induce further inhibition on I_ACh in oocytes expressing α3β4 nicotinic acetylcholine receptor compared to co-treatment, in next experiments we examined quercetin effects on I_ACh after co-treatment of quercetin with acetylcholine. In concentration-response experiments, co-treatment of quercetin with acetylcholine inhibited I_ACh in a concentration-dependent manner in oocytes expressing α3β4 nicotinic acetylcholine receptor (Fig. 2C). The IC₅₀ of I_ACh was 14.9±0.8 µM in oocytes expressing α3β4 nicotinic acetylcholine receptors (n=9~12 from three different frogs) (Fig. 2D).

Current-voltage relationship and voltage-independent inhibition in oocytes expressing α3β4 nicotinic acetylcholine receptors by quercetin

As shown in Fig. 3, the current-voltage relationship induced by acetylcholine with voltage steps from -100 to +60 mV showed a slight rectification at positive potentials in oocytes expressing α3β4 nicotinic acetylcholine receptor. The reversal potential of α3β4 nicotinic acetylcholine receptors was Vr=-11.2±2.4 mV (means±S.E.M., n=6 from three different frogs). Co-treatment with quercetin and acetylcholine did not modify the reversal potential of α3β4 nicotinic acetylcholine receptor with a reduction of I_ACh (n=6 from three different frogs). The inhibitory effect of quercetin on I_ACh in oocytes expressing α3β4 nicotinic acetylcholine receptors was independent of the membrane holding potential (Fig. 3B). Thus, quercetin inhibited I_ACh by 65.4±4.1, 65.7±1.7, 66.8±5.7, and 65.8±2.8% at -120, -90, -60, and -30 mV membrane holding potential in oocytes expressing α3β4 nicotinic acetylcholine receptor, respectively (Fig. 3B; n=9~12, from three different frogs).

Fig. 3.

Current-voltage relationship and voltage-independent inhibition by quercetin. (A) Current-voltage relationships of I_ACh inhibition by quercetin (Que) in α3β4 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 α3β4 nicotinic acetylcholine receptors by quercetin. Inset; the values were obtained from the receptors in the absence or presence of 20 µM quercetin at the indicated membrane holding potentials.

Noncompetitive inhibition of α3β4 nicotinic acetylcholine receptors by quercetin

To study further the mechanism by which quercetin inhibits I_ACh in oocytes expressing α3β4 nicotinic acetylcholine receptors, we analyzed the effect of 20 µM quercetin on I_ACh evoked by different acetylcholine concentrations in oocytes expressing α3β4 nicotinic acetylcholine receptors (Fig. 4). Co-application of quercetin with different concentrations of acetylcholine did not shift the dose-response curve of acetylcholine to the right (ED₅₀, from 81.5±2.3 to 91.2±6.5 µM and Hill coefficient, from 1.25 to 1.37) in oocytes expressing α3β4 nicotinic acetylcholine receptors, indicating that quercetin regulates α3β4 nicotinic acetylcholine receptor channel activity with non-competitive manner (n=9~12 from three different frogs) (Fig. 4).

Fig. 4.

Concentration-dependent effects of acetylcholine on quercetin-mediated inhibition of I_ACh. (A) The representative traces were obtained from α3β4 nicotinic acetylcholine receptors expressed in oocytes. I_ACh of the upper and lower panels were elicited from concentration of 30 µM ACh and 1 mM ACh at a holding potential of -80 mV, respectively. (B) Concentration-response relationships for ACh in the α3β4 nicotinic acetylcholine receptors treated with ACh (3~1,000 µM) alone or with ACh plus co-application of 20 µM quercetin. The I_ACh of oocytes expressing the α3β4 nicotinic acetylcholine receptors was measured using the indicated concentration of ACh in the absence (□) or presence (○) of 20 µM quercetin (Que). 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=9~12/group).

DISCUSSION

In the present study, we demonstrated that (1) co- or pre-treatment with quercetin and acetylcholine inhibited I_ACh in oocytes expressing bovine ±3β4 nicotine acetylcholine receptor in reversible and concentration-dependent manner, (2) the inhibition of I_ACh by quercetin occurred in a non-competitive and voltage-independent manner in oocytes expressing ±3β4 nicotinic acetylcholine receptors, indicating that quercetin could be associated with the inhibitory regulator on I_ACh in oocytes expressing ±3β4 nicotinic acetylcholine receptors (Fig. 3 and 4).

From the present results, however, it is unclear precisely how quercetin acts to inhibit I_ACh in oocytes expressing ±3β4 nicotinic acetylcholine receptor. One possible mechanism is that quercetin may act as open channel blocker of ±3β4 nicotinic acetylcholine receptors but this may be not the case because the inhibitory effect of quercetin on I_ACh in oocytes expressing ±3β4 nicotinic acetylcholine receptors was not voltage-dependent (Fig. 3). It is known that open channel blockers such as local anesthetics or hexamethonium are strongly voltage dependent, due to the charge that they carry in the transmembrane electrical field [21-23].

Another possibility is that quercetin may work as a competitive inhibitor by inhibiting acetylcholine binding to its binding site(s) in ±3β4 nicotinic acetylcholine receptors. In competition experiments, we observed that the presence of quercetin did not shift the concentration of acetylcholine in oocytes expressing ±3β4 nicotinic acetylcholine receptors without changing the Hill coefficient (Fig. 4). Thus, the non-competitive modulation of ±3β4 nicotinic acetylcholine receptor channel activity by quercetin shows that quercetin might have different binding or interaction site(s) as a non-competitive inhibitor at ±3β4 nicotinic acetylcholine receptors.

The third and last possibility is that quercetin might have its binding sites for the regulation of ±3β4 nicotinic acetylcholine receptor. In previous reports, we have demonstrated that the regulatory effects of quercetin on homomeric glycine, 5-HT₃ and ±7 nicotinic acetylcholine receptor channel activities were attenuated or abolished after site-directed mutations of amino acid residues of pre-transmembrane domain of glycine and 5-HT₃ receptor or Ca²⁺-binding sites of ±7 nicotinic acetylcholine receptor [17-19]. Based on present results and previous reports, it is likely that quercetin achieves its effect through direct interactions with ±3β4 nicotinic acetylcholine receptors. However, there might be some difficulties to construct mutant ±3β4 nicotinic acetylcholine receptors to confirm quercetin binding sites compared to the previous homomeric ligand-gated ion channels, since ±3β4 nicotinic acetylcholine receptors consist of heteromeric ± and β subunits, In future, further investigations will be required to identify quercetin binding site(s) on ±3β4 nicotinic acetylcholine receptors.

Since subsets of nicotinic acetylcholine receptor channels play an important role for fast synaptic transmissions in postsynaptic sites, there are several reports on the regulations of nicotinic acetylcholine receptor channel activity using natural compounds such as flavonoids and polyphenol. Grønlien et al showed that genistein, one of flavonoids, inhibits ±7 nicotinic acetylcholine receptor channel activity in oocytes expressing ±7 nicotinic acetylcholine receptor [24]. Zhang et al showed that nobiletin, one of flavones inhibited catecholamine release by acetylcholine, whereas nobiletin stimulate catecholamine release via activation of Ca²⁺ channels or Na⁺/Ca²⁺ exchangers [25]. In addition, Shinohara et al demonstrated that resveratrol, one of grape polyphenol exhibited a profound effect by inhibiting on ±3β4 nicotinic acetylcholine receptor and polyphenols of Rubus coreanum also inhibited catecholamine release from adrenal medulla [26,27]. On the other hand, in the previous studies we found that quercetin enhanced ±7 nicotinic acetylcholine receptor channel activity with extracellular Ca²⁺-independent manner, whereas quercetin inhibited ±9±10 nicotinic acetylcholine receptor channel activity. In the present study, we found that quercetin inhibited I_ACh in oocytes expressing ±3β4 nicotinic acetylcholine receptors. Taking together of previous reports and present results, these results indicate that quercetin might be a differential regulator of nicotinic acetylcholine receptors.

On the other hand, the previous reports have shown that various agents such as serotonin, strychnine, Ca²⁺ channel blockers, polyamines, steroids such as progesterone and hydrocortisone, ethanol, and metal ion like Zn²⁺, regulate muscle or neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes [28-35]. Interestingly, the mechanism (i.e., voltage dependence or competition with acetylcholine for binding site) by which these substances regulate nicotinic acetylcholine receptors depend on the receptor subunit composition. The previous and present studies showed that quercetin inhibited heteromeric ±3β4 and ±9±10 nicotinic acetylcholine receptor channel activities, whereas quercetin enhanced homomeric ±7 nicotinic acetylcholine receptors. These results show the possibility that the differential effects of quercetin on subset of nicotinic acetylcholine receptor might be also due to different receptor subunit composition. In addition, since these nicotinic acetylcholine receptors may play an important role in modulating the neurotransmitter release or neuronal cell excitability induced by acetylcholine in pre-synaptic or post-synaptic site(s), quercetin-mediated differential regulations of subsets of nicotinic acetylcholine receptor channel activity might contribute to a diverse range of neuropharmacological effects of quercetin [12,13].

In summary, we found that quercetin inhibited I_ACh in oocytes expressing bovine neuronal ±3β4 nicotinic acetylcholine receptors. Since ±3β4 nicotinic acetylcholine receptors are closely involved in neurotransmitter release in adrenal chromaffin cells, these inhibitory effects of quercetin on I_ACh in oocytes expressing bovine neuronal ±3β4 nicotinic acetylcholine receptor might provide the single cellular basis for one of mechanisms for the pharmacological effects of quercetin.

ABBREVIATIONS

5-HT_3A

5-hydroxytryptamine 3A

ACh

acetylcholine

Que

quercetin