Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop family of ligand-gated ion channels. The Cys-loop family also includes serotonin (5-HT₃), gamma-amino-butyric acid (GABA_A), and glycine receptors (Jensen et al., 2005). nAChRs have been divided into two types: a muscle type and a neuronal type (Dani and Bertrand, 2007). Neuronal nAChRs are widely expressed in the human central and peripheral nervous systems. Eleven different nAChR subunits are currently known, and subunits of nAChR α (α_2–10) and β (β_2–4) have been identified (Nashmi and Lester, 2006). Neuronal nAChRs containing α_2–6 subunits are usually expressed as heteromers in combination with β_2–4 subunits (Boulter et al., 1987; Karlin, 2002) and are found throughout the whole nervous system (Gotti and Clementi, 2004). In contrast, the α7 and α9 subunits can form homomeric receptors (Couturier et al., 1990; Elgoyhen et al., 1994; Karlin, 2002). Homomeric α7 nAChRs are the major binding site for α-bungarotoxin in the central nervous system of mammals and are predominantly expressed in the cortical and the limbic areas including the hippocampus. Homomeric α7 nAChRs are known to play an important role in normal brain function and development (Gotti et al., 2000).

In previous reports, we have shown that the application of the flavonoid quercetin inhibits 5-HT- and glycine-induced peak inward currents (I_5-HT and I_Gly) of mouse 5-HT_3A and human glycine α receptor channels expressed in Xenopus laevis oocytes, respectively. The observed inhibition of I_5-HT by quercetin was competitive and voltage-independent, whereas inhibition of I_Gly by quercetin appeared non-competitive and voltage-dependent (Lee et al., 2005; Lee et al., 2007). In addition, we have found that co- or pre-application of quercetin with acetylcholine (ACh) enhanced I_ACh in oocytes expressing human α7 nAChRs. This enhancement appeared independent of ACh concentration and voltage. Furthermore, quercetin enhanced Ca²⁺-mediated potentiation of I_ACh, which was observed to be dependent on extracellular Ca²⁺ concentration (Lee et al., 2010).

On the other hand, in addition to quercetin, quercetin glycosides are also compounds of low molecular weight and are mainly found in apples, tomatoes, gingko, other red fruits, and vegetables (Havsteen, 2002). In fruits and vegetables, quercetin naturally exists in glycosylated forms such as Rham 1, Rutin, Rham2, or other glycosidic forms (Azevedo et al., 2013). In previous studies using glycine and 5HT₃ receptors, we have shown that quercetin glycosides can regulate ligand-gated ion channel activity in a differential manner with respect to quercetin. The inhibition of the glycine receptor channel activity by quercetin glycosides was noncompetitive and voltage-sensitive, whereas the inhibition of 5-HT₃ receptor channel activity by quercetin glycosides was competitive and voltage-insensitive. Recently, we have also shown that quercetin glycosides inhibit GABA_C receptor channel activity in a non-competitive and membrane voltage-insensitive manner. However, relatively little is known about the effects of quercetin glycosides on α7 nAChR channel activity.

In this study, we investigated the regulation of α7 nAChR channel activity expressed in Xenopus oocytes by quercetin glycosides. We first expressed neuronal human α7 nAChR copy RNAs (cRNAs) in Xenopus oocytes and examined the effect of quercetin and quercetin glycosides on I_ACh. This system was employed because (1) Xenopus laevis oocytes have been used widely as a tool to express the membrane proteins encoded by exogenously administered cDNAs or cRNAs including receptors, ion channels, and transporters (Dascal, 1987); and (2) nAChR channels expressed in Xenopus oocytes by the injection of nAChR subunit cRNAs have been well studied and characterized (Chavez-Noriega et al., 1997).

We found that co- or pre-application of quercetin with ACh enhanced I_ACh, whereas co- or pre-application of quercetin glycosides inhibited I_ACh. The observed inhibition of I_ACh by quercetin glycosides was ACh concentration- and voltage-independent. Interestingly, quercetin-induced enhancement of I_ACh was attenuated by co-treatment of quercetin glycosides. Here, we demonstrate that the quercetin glycosides-induced regulation of α7 nAChR channel activity is different from that of quercetin. We further discuss the role of the carbohydrate portion of quercetin glycosides in the differential regulation of α7 nAChR channel activity.

Materials

Human wild-type α7 nAChR cDNA was kindly provided by Dr. S. Heinemann (Salk Institute, California, USA). Quercetin (Fig. 1) 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 Arbor, MI, USA). Animal care and handling were in accordance with the highest standards of institutional guidelines. To isolate oocytes, frogs were anesthetized 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°C with continuous gentle shaking and media was replaced daily. Electrophysiological experiments were performed five to six days after oocyte isolation, during which time the relevant chemicals were added to the media. α7 nAChR-encoding cRNAs (40 nL) were injected into the animal or vegetal pole of appropriate oocytes 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 diameter) (Lee et al., 2005).

Data recording

A custom-made Plexiglas net chamber was used for two-electrode voltage-clamp recordings, as previously reported (Lee et al., 2005). A single oocyte was constantly superfused with ND96 media in the absence or presence of acetylcholine or quercetin during recording. The microelectrodes filled with 3 M KCl giving 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 digitized using Digidata 1200A (Molecular Devices, Sunnyvale, CA, USA). Both stimulation and data acquisition were controlled using pClamp 8 software (Molecular Devices). For most electrophysiological experiments, the oocytes were clamped at a holding potential of −80 mV, and 300 ms voltage steps were applied from −100 to +50 mV to assess the relationship between current and voltage. Linear leak and capacitance currents were corrected by means of the leak subtraction procedure. Because α7 nAChRs have a high relative permeability to Ca²⁺ (Séguéla et al., 1993; Castro and Albuquerque, 1995), oocytes were incubated in 100 μM 1,2-Bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM) for 4 h before recording to avoid α7 nAChR-mediated endogenous Ca²⁺-activated Cl⁻ currents.

Data analysis

To obtain the concentration-response curves for quercetin and quercetin glycosides on the inward peak I_ACh mediated by α7 AChR, the I_ACh peak was plotted at different concentrations of quercetin and its glycosides. Origin software (OriginLab Corp., Northampton, MA, USA) was used to fit the plot to the Hill equation: I/I_max=1/[1+(ED₅₀/[A])^nH], where I_max is maximal current obtained from each ED₅₀ value of acetylcholine in wild-type receptors, ED₅₀ is the concentration of quercetin or quercetin glycoside required to increase/decrease the response by 50%, [A] is the concentration of quercetin or quercetin glycoside, and nH is the Hill coefficient. All values are presented as means ± S.E.M. The differences between the means of control and treatment data were determined using the paired t-test or a one-way ANOVA followed by Tukey test. A value of p<0.05 was considered to be statistically significant.

Effects of quercetin or quercetin glycosides on I_ACh in oocytes expressing α7 nAChRs

Treatment of ACh (200 μM) to oocytes injected with human α7 nAChR cRNA induced a large inward current (I_ACh) (Fig. 2A) but the application of ACh did not induce any inward current in H₂O-injected control oocytes (data not shown) (Lee et al., 2010). Although quercetin (100 μM) itself had no effect on oocytes expressing α7 nAChRs at a holding potential of −80 mV (data not shown), the co-application of quercetin with ACh enhanced I_ACh in oocytes expressing α7 AChR (Fig. 2A, n=9 from three different frogs). The co-application of quercetin with ACh induced an enhancement of I_ACh by 46.5 ± 6.5% (Fig. 2B, **p<0.005 versus unexposed controls). In addition, pre-application of quercetin (100 μM) alone for 30 s before co-application with ACh (200 μM) induced a much larger enhancement of I_ACh in oocytes expressing α7 nAChRs than the enhancement observed after co-application as we previously demonstrated (Fig. 2A, ^#p<0.005, compared to co-treatment) (Lee et al., 2010). Next, we examined the effects of quercetin glycosides on I_ACh. Quercetin glycosides (100 μM each) themselves showed no effect on oocytes expressing the α7 nAChRs at a holding potential of −80 mV. Co-application of quercetin glycosides with ACh decreased the amplitude of I_ACh reversibly (13.2 ± 2.7%, 15.0 ± 2.9%, and 4.7 ± 1.2% inhibition by Rham1, Rutin, and Rham2, respectively) (Fig. 2C). Pre-application of quercetin glycosides alone for 30 s before co-application with ACh induced a much larger inhibitory effect on I_ACh (39.4 ± 3.5%, 42.1 ± 4.7%, and 13.1 ± 4.5% inhibition by Rham1, Rutin, and Rham2, respectively) (Fig. 2B, 2C, n=8–11 from three different frogs). Thus, the I_ACh inhibitory potency order appeared where Rutin≈Rham1>Rham2, also indicating that the regulatory pattern of quercetin glycosides on α7 nAChR channel activity is different from that of quercetin (Fig. 2).

Quercetin enhances I_ACh, while quercetin glycosides inhibit I_ACh in a concentration-dependent manner

In concentration-dependent experiments with quercetin, pre-application with quercetin for 30 s enhanced I_ACh in a con centration-dependent manner in oocytes expressing α7 nA ChRs (Fig. 3C). Pre-application of quercetin at 3, 10, 30, 100, and 300 μM increased I_ACh by 5.9 ± 0.9, 23.1 ± 3.0, 52.9 ± 4.2, 98.7 ± 7.3, and 113.4 ± 11.9% in oocytes expressing α7 AChRs, respectively. Thus, the apparent EC₅₀ of I_ACh for quercetin pre-application was 35.1 ± 3.8 μM (n=10–11, with samples taken from three different frogs for each point; Fig. 3C). In concentration-dependent experiments with quercetin glycosides, pre-application with quercetin glycosides for 30 s inhibited I_ACh in a concentration-dependent manner in oocytes expressing α7 nAChRs (Fig. 3D). For instance, pre-application of Rham1 inhibited I_ACh by 1.2 ± 0.3, 6.8 ± 0.8, 16.2 ± 1.1, 41.9 ± 3.5, and 53.9 ± 4.1% at 3, 10, 30, 100, and 300 μM in oocytes expressing α7 AChRs, respectively. Pre-application of Rutin inhibited I_ACh by 2.9 ± 0.9, 8.9 ± 0.8, 21.8 ± 1.8, 45.7 ± 3.7 and 63.1 ± 4.6% at 3, 10, 30, 100, and 300 μM in oocytes expressing α7 AChRs, respectively. Pre-application of Rham2 inhibited I_ACh by 0.7 ± 0.3, 1.3 ± 0.4, 4.1 ± 1.1, 16.3 ± 1.9 and 23.2 ± 2.6% at 3, 10, 30, 100, and 300 μM in oocytes expressing α7 AChRs, respectively. The apparent IC_50s of I_ACh were 56.4 ± 8.4, 70.4 ± 3.6, 71.3 ± 6.7 μM for Rham1, Rutin, and Rham2 pre-application in oocytes expressing the α7 AChR receptor, respectively (n=10–11, with samples taken from three different frogs for each point; Fig. 3D). These results indicate that quercetin and quercetin glycosides regulate α7 nAChR channel activity in a differential manner.

Concentration-dependent effect of ACh and the relationship between current and voltage in the quercetin- or quercetin glycoside-mediated effects on I_ACh

Similar to our previous report (Lee et al., 2010), when we analyzed the effect of quercetin on I_ACh evoked by different ACh concentrations, we found that pre-application of quercetin with different concentrations of ACh did not significantly shift the concentration-response curve of ACh to the left (EC₅₀ values were changed from 70.8 ± 9.6 to 63.5 ± 7.5 μM, *p<0.08, while the Hill coefficient changed from 1.1 to 1.2) in oocytes expressing α7 nAChRs (Fig. 4), suggesting that quercetin did not affect the ACh binding.

Next, to further study the mechanism by which quercetin glycosides inhibit I_ACh, we first analyzed the effects of quercetin glycosides on I_ACh evoked by different ACh concentrations in oocytes expressing the α7 nAChR (Fig. 4). Pre-application of quercetin glycosides for 30 s with different concentrations of ACh did not significantly shift the dose-response curve of ACh. The apparent EC₅₀ values were 70.8 ± 9.6, 78.6 ± 7.1, 76.1 ± 6.5, and 80.6 ± 6.5 μM for ACh alone, ACh + Rham1, ACh + Rutin, and ACh + Rham2, respectively, and the Hill coefficients were 1.1 ± 0.1, 1.0 ± 0.2, 1.0 ± 0.1, and 1.1 ± 0.1, respectively. Thus, Rham1, Rutin, and Rham2 significantly inhibited the I_ACh elicited, independent of ACh concentration (n=9–12 from three different frogs) (Fig. 4). These results show that quercetin increases I_ACh, whereas quercetin glycosides inhibit I_ACh and that these effects probably occur in a non-competitive manner.

In the current-voltage relationship, the membrane potential, which was held at −80 mV, and a voltage ramp was applied from −100 to +50 mV for 300 ms. In the absence of ACh, the inward current (at −100 mV) and the outward current (at +50 mV) were negligible (data not shown). Treatment of ACh to in oocytes expressing the α7 nAChR induced a mainly inward current and outward current at negative- and positive voltages, respectively. Pre-application of quercetin with ACh enhanced both inward and outward currents. The reversal potential was near 0 mV for both ACh alone and for ACh with quercetin. This indicates that Na⁺ and Ca²⁺ are main charge carriers (Revah et al., 1991; Galzi et al., 1992). In addition, the pre-application of quercetin with ACh further increased currents but did not appear to affect α7 nAChR channel properties as quercetin addition did not change the reversal potential of the α7 nAChR (Fig. 5A). Pre-application of quercetin glycosides combined with ACh treatment gave greater inhibition of both inward and outward currents than those achieved when ACh and quercetin glycosides were applied together. The reversal potential was also near −0 mV when ACh was used alone, as well as in ACh+quercetin glycoside treatments (Fig. 5A). In addition, the enhancement of quercetin or the inhibitory effects of quercetin glycosides on I_ACh in oocytes expressing α7 nAChRs did not appear to be membrane voltage-sensitive. Quercetin increased I_ACh by 98.5 ± 5.4%, and 95.6 ± 6.3%, at −120 and −30 mV, respectively. Rham1, Rutin and Rham2 inhibited I_ACh by 41.2 ± 3.8%, 44.9 ± 2.2%, and 16.7 ± 3.7%, respectively, at −120 mV, and by 38.9 ± 4.9%, 48.1 ± 2.1%, and 18.7 ± 4.6%, respectively, at −30 mV (n=10–12, from three different frogs). These results indicate that quercetin enhances I_ACh, while quercetin glycosides inhibit I_ACh in a voltage-insensitive manner (n=10–12, from three different frogs; Fig. 5B).

Effects of quercetin glycosides on quercetin-induced I_ACh enhancement

The above results indicate that quercetin glycosides may be novel regulators of α7 nAChR channel activity and that their actions could be different from those of quercetin. Therefore, we investigated the effect of quercetin on I_ACh after co-treatment of quercetin glycosides. As shown in Fig. 6, the enhancing effect of quercetin on I_ACh was significantly attenuated in the presence of Rutin, Rham1 and Rham2. The order of potency for the quercetin attenuating effects was Rutin> Rham1>Rham2. The above results show that quercetin-mediated enhancement of I_ACh could be affected by the presence of quercetin glycosides.

Quercetin is a flavonoid that shows diverse effects in nervous and non-nervous systems (Kandaswami and Middleton, 1994; Harborne and Williams, 2000). For example, quercetin protects the central nervous system against oxidative effects and exerts effects on analgesia, locomotor activity and sleep (Speroni and Minghetti, 1988; Picq et al., 1991; Oyama et al., 1994), as well as having anticonvulsant, sedative, and anxiolytic effects (Marder et al., 1996; Medina et al., 1997; Griebel et al., 1999; Yao et al., 2010). Quercetin glycosides are natural forms of quercetin that are found in colored fruit and vegetables (Fig. 1) (Murota and Terao, 2003; Nemeth and Piskula, 2007). In previous reports, we have shown that quercetin and quercetin glycosides can regulate the activity of several types of ligand-gated ion channels, but the relationship between quercetin and quercetin glycosides and α7 nAChR regulation has not been well characterized.

In the present study, we have investigated the effects of quercetin glycosides on human α7 nAChRs heterologously expressed in Xenopus oocytes. We found that: (1) pre-application of quercetin with ACh induced a large enhancement of I_ACh in a reversible and concentration-dependent manner; (2) quercetin glycoside pre-application with ACh inhibited I_ACh; (3) quercetin-mediated enhancement of I_ACh was non-competitive and membrane potential independent, while quercetin glycosides inhibit I_ACh in a non-competitive manner and are also membrane potential independent; and (4) quercetin-induced enhancement of I_ACh was attenuated in the presence of quercetin glycosides. These results indicate that quercetin and quercetin glycosides show opposite effects on α7 nAChR channel activity and quercetin glycosides are different from quercetin in their regulation of α7 nAChRs. Structural differences between quercetin and quercetin glycosides suggest that the differential regulation of α7 nAChR channel activity might be due to the carbohydrate components.

It will be questioned how structural differences between quercetin and quercetin glycosides induce differential regulations of α7 nAChR channel activity. One possibility is that the carbohydrates attached to quercetin might cause a different behavior in the regulations of α7 nAChRs. For example, quercetin enhances I_ACh, whereas quercetin glycosides inhibit I_ACh of α7 nAChR (Fig. 3). The other is that the number or different size of carbohydrate attached to quercetin might also induce differential effects on I_ACh of α7 nAChRs. Rham1 or Rutin with one or two carbohydrates more potently inhibited I_ACh than Rham2, which is tri-glycosylated forms of quercetin (Fig. 3). In addition, quercetin glycosides attenuated the quercetin-induced enhancement of I_ACh. Thus, although we found in the present study that carbohydrate component of quercetin could play important roles in the regulations of ligand-gated ion channel such as α7 nAChR, we do not know exactly how carbohydrate(s) attached to quercetin cause an opposite on I_ACh of α7 nAChRs or differential effects on I_ACh of α7 nAChRs and how quercetin glycosides decreases the quercetin-induced enhancement of I_ACh. Further study will be required to elucidate molecular mechanisms how carbohydrate(s) attached to quercetin contribute to quercetin-induced regulations of α7 nAChR.

The activation of the α7 nAChR is known to be linked to many physiological conditions (Khiroug et al., 2003; Gotti and Clementi, 2004; Gilbert et al., 2009). The α7 nAChR is widely expressed throughout the central nervous systems, including the cortical and limbic areas of the brain. Clearly, the α7 nAChR plays an important role in normal brain function because α7 nAChR dysfunction is associated with neurological disorders such as learning and memory loss, Alzheimer’s disease, schizophrenia, and epilepsy (Chini et al., 1994; Léna and Changeux, 1997; Weiland et al., 2000; Changeux and Edelstein, 2001). However, relatively little is known about the effects of quercetin glycosides on α7 nAChR function. In the present study, we found that quercetin glycosides inhibited I_ACh and furthermore, Rutin and Rham1 could attenuate the quercetin-induced enhancement of I_ACh. Interestingly, the inhibitory effects of quercetin glycosides on I_ACh as well as the attenuation of quercetin-induced enhancement of I_ACh, was observed to be more potent with pre-treatment before ACh addition. Currently we cannot explain the exact physiological or pharmacological role of quercetin glycosides or the reason for their behavior being different from that of quercetin in α7 nAChR regulation. It is known that dietary quercetin glycosides are usually metabolized in two ways. First, they are deglycosylated to the aglycone quercetin; and second, they remain as quercetin glycosides without further metabolism (Havsteen, 2002; Lee et al., 2010). Further studies will be required to elucidate the role of quercetin glycosides in the in vivo regulation of the α7 nAChR.

In previous studies, we have shown that quercetin inhibits 5-HT₃ receptor-gated ion currents through interactions within the pre-transmembrane domain I. Furthermore, quercetin can inhibit or potentiate glycine receptor-gated ion currents through interactions with the amino acid Ser256 residue in transmembrane domain II (Lee et al., 2005; Lee et al., 2007). Examination of the effects of quercetin glycosides shows an inhibition of GABA_C receptor channel activity in the order of Rutin>Rham1>Rham2 (Kim et al., 2015). In the present study, we found that quercetin glycosides also inhibited α7 nAChR-gated ion currents with the same order of efficacy. Thus, although the Cys-loop family of ligand-gated ion channels such as glycine, GABA_C, 5-HT₃, and α7 nAChR all form homomeric receptors, quercetin and quercetin glycosides regulate these homomeric receptors in a different manner.

In conclusion, we found that quercetin increased I_ACh, while quercetin glycosides inhibited I_ACh in Xenopus oocytes expressing α7 nAChRs. The present results indicate that quercetin and quercetin glycosides act differently in the regulation of α7 nAChRs. Finally, these results also suggest that quercetin and quercetin glycosides exhibit their differential regulation of α7 nAChR channel activity through their structural differences.

This research was supported by the Bio & Medical Technology Development Program of the NRF funded by the Korean government, MSIP(2015M3A9E3052336).

This paper was also written as part of Konkuk University’s research support program for its faculty on sabbatical leave in 2015 to S.M. Lee.