Lamotrigine (LTG), an anticonvulsive drug, is often used for the treatment of a variety of epilepsies. In addition to block of sodium channels, LTG may act on other targets to exert its antiepileptic effect. In the present study, we evaluated the effects of LTG on neuronal nicotinic acetylcholine receptors (nAChRs) using the patch-clamp technique on human α4β2-nAChRs heterologously expressed in the SH-EP1 cell line and on native α4β2-nAChRs in dopaminergic (DA) neurons in rat ventral tegmental area (VTA). In SH-EP1 cells, LTG diminished the peak and steady-state components of the inward α4β2-nAChR-mediated currents. This effect exhibited concentration-, voltage- and use-dependent behavior. Nicotine dose-response curves showed that in the presence of LTG, the nicotine-induced maximal current was reduced, suggesting a noncompetitive inhibition. These findings suggest that LTG inhibits human neuronal α4β2-nAChR function through an open-channel blocking mechanism. LTG-induced inhibition in α4β2-nAChRs was more profound when preceded by a 2-min pretreatment, after which the nicotine-induced current was reduced even without coapplication of LTG, suggesting that LTG is also able to inhibit α4β2-nAChRs without channel activation. In freshly dissociated VTA DA neurons, LTG inhibited α4β2-nAChR-mediated currents but did not affect glutamate- or GABA-induced currents, indicating that LTG selectively inhibits nAChR function. Collectively, our data suggest that the neuronal α4β2-nAChR is likely an important target for mediating the anticonvulsive effect of LTG and the blockade of α4β2-nAChR possibly underlying the mechanism through which LTG effectively controls some types of epilepsy, such as autosomal dominant nocturnal frontal lobe epilepsy or juvenile myoclonic epilepsy.
Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. Mammalian neuronal nAChRs, which exist as a diverse family of proteins composed of various combinations of α (α2–α7, α9, α10) and β (β2–β4) subunits, are widely expressed throughout the central nervous system (Hogg et al., 2003). The heteromeric α4β2- and homomeric α7-nAChRs are two major functional subtypes of neuronal nAChRs in the brain (Hogg et al., 2003; Gotti and Clementi, 2004). Activation of neuronal nAChRs plays an important role in mediating not only normal cholinergic signaling but also pathogenic processes in diseases, such as Parkinson's disease, Alzheimer's disease, schizophrenia, and epilepsy (Hogg et al., 2003; Gotti and Clementi, 2004; Yang et al., 2009b).
The identification of specific mutations in the neuronal α4β2-nAChRs was a key finding for the understanding of the pathogenesis of the genetic form of epilepsy, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Genetic studies demonstrated that two kinds of gain of function of missense mutations in α4β2-nAChRs are involved in ADNFLE. The first site-specific mutation exists within the gene of the nAChR α4 subunit, where phenylalanine 248 is replaced by serine (Ser248Phe) (Steinlein et al., 1997). The second site-specific mutation, a Val287Met substitution, occurs within the gene of the nAChR β2 subunit (Phillips et al., 2001). Electrophysiological experiments have demonstrated that the above mutations in α4β2-nAChRs result in approximately 10-fold increases in affinity for ACh (Steinlein et al., 1997; Phillips et al., 2001). Furthermore, the Val287Leu missense mutant in the nAChR β2 subunit gene prolongs opening duration of α4β2-nAChR channels; this mutant is also found in patients with ADNFLE (De Fusco et al., 2000). It has been reported that tobacco habits modulate ADNFLE (Brodtkorb and Picard, 2006). A recent report has indicated that the 1674 +11 C>T polymorphism of the nAChR α4 subunit gene may play a critical role in the pathogenesis of juvenile myoclonic epilepsy (Rozycka et al., 2009). Thus, the mutations of neuronal nAChR α4 and/or β2 subunits may cause neuronal hyperexcitation and initiate epileptic seizures (De Fusco et al., 2000; Phillips et al., 2001; Bertrand et al., 2002).
Lamotrigine (LTG), a triazine compound, was approved by the United States Food and Drug Administration in 1994 for use as an anticonvulsive drug for treating epileptic patients (Matsuo, 1999) (Supplemental Fig. 1). There is an increasing body of evidence to suggest that LTG is an effective anticonvulsive drug for the treatment of different types of patients with epilepsy (Clemens et al., 2008). For instance, LTG effectively controls Lennox-Gastaut syndrome (Motte et al., 1997; Matsuo, 1999), juvenile myoclonic epilepsy (Buchanan, 1996), Angelman syndrome (Dion et al., 2007), primary generalized tonic-clonic seizures (Trevathan et al., 2006), and patients with partial seizures (Labiner et al., 2009). The evidence indicates that LTG may exert its antiepileptic effect by inhibiting sodium channels (Cheung et al., 1992; Zona and Avoli, 1997). However, thus far, the precise mechanisms underlying the LTG-induced anticonvulsive effect remain elusive, and it is possible that other targets also mediate such effect.
In the present study, we evaluate a novel target, the neuronal α4β2-nAChR, which may mediate LTG anticonvulsive effects. Our results indicate that LTG blocks both heterologously expressed and native α4β2-nAChRs. The pharmacological mechanisms of LTG-induced antagonism of α4β2-nAChR involve both open- and closed-channel block.
Materials and Methods
LTG (Supplemental Fig. 1), (−)nicotine, glutamate, GABA, and dopamine (DA) were purchased from Sigma-Aldrich (St. Louis, MO). RJR-2403 [(i)-N-methyl-4-(3-pyridinyl)-3-buten-1-amine oxalate] was purchased from Tocris Bioscience (Ellisville, MO).
Expression of Human Neuronal α4β2-nAChRs in SH-EP1 Human Epithelial Cells.
Human neuronal α4β2-nAChRs were heterologously expressed in native nAChR-null SH-EP1 cells, as described previously in detail (Eaton et al., 2003; Wu et al., 2006; Zheng et al., 2009). In brief, α4 and β2 subunits of human nAChRs were subcloned into pcDNA3.1-Zeocin or pcDNA3.1-hygromycin vectors to generate the pcDNA3.1/zeo-hα4 or pcDNA3.1/hygro-hβ2 construct, respectively. Then, α4 and β2 constructs were transfected into native nAChR-null SH-EP1 cells to create the SH-EP1-hα4β2 cell line by using electroporation (Gene Pulser; 960 μF, 0.20 kV/cm, t = 28–36 ms; Bio-Rad Laboratories, Hercules, CA). Cells were maintained as low passage number cultures (1–26 from our frozen stocks to ensure stable expression of phenotype) in Dulbecco's modified Eagle's medium (detailed in Eaton et al., 2003) and augmented with 0.25 mg/ml Zeocin (Invitrogen, Carlsbad, CA) and 0.4 mg/ml hygromycin B to maintain positive selection of transfectants. All cultures were passaged once weekly by splitting just confluent cultures 1/20–1/40 to ensure cells continued their proliferative growth.
Single DA Neuron Dissociation.
All animal procedures were carried out in agreement with the protocol approved by the Institutional Laboratory Animal Care and Use Committee of the Barrow Neurological Institute. Midbrain slices containing the ventral tegmental area (VTA) were prepared following the procedures described previously (Yang et al., 2009a). In brief, 2- to 3-week postnatal Wistar rats were anesthetized with isoflurane. After decapitation, the brain tissue was quickly removed from the skull and stored in cold artificial cerebrospinal fluid (ACSF) for 1 min. The ACSF contains 124 mM NaCl, 5 mM KCl, 24 mM NaHCO3, 1.3 mM MgSO4, 1.2 mM KH2 PO4, 2.4 mM CaCl2, and 10 mM glucose, which was continuously bubbled with 95% O2 and 5% CO2. Several coronal slices (400 μM thickness) were subsequently cut using a Vibratome 1000 Plus (The Vibratome Company, St. Louis, MO) and incubated in ACSF at room temperature (22 ± 1°C) for at least 90 min. Thereafter, the slices were treated with pronase (1 mg/6 ml; Calbiochem, San Diego, CA) at 31°C for 25 to 45 min. After enzyme treatment, the region of VTA was identified according to the rat brain atlas (Paxinos and Watson, 1998) and micropunched from the slice using a well-polished needle with an inverted microscope (Meiji Techno Co., Ltd., Tokyo, Japan). Each punched tissue fragment was transferred to a 35-mm culture dish filled with well oxygenated standard external solution containing 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM glucose, and 10 mM HEPES, saturated with 100% O2. The pH was adjusted to 7.4 with Tris-base (Yang et al., 2009b). The punched fragments were then mechanically dissociated using fire-polished micro-Pasteur pipettes under an inverted microscope (Olympus IX-70, Tokyo, Japan). The single, isolated neurons usually adhered to the bottom of the culture dish within 30 min, maintaining good morphology and function for at least 2 to 4 h.
The SH-EP1 cells transfected with human α4β2-nAChRs were seeded 2 to 4 days before patch-clamp recording. During recording, a 35-mm culture dish was placed on the stage of an inverted microscope (Axiovert 200; Carl Zeiss Inc., Thornwood, NY). SH-EP1 cells were continuously perfused with standard external solution containing 120 mM NaCl, 3 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 25 mM glucose. The pH was adjusted to 7.4 with Tris-base (Wu et al., 2006; Zheng et al., 2009). Patch pipettes (1.5 × 100 mm; Narishige, Greenvale, NY), fashioned on a two-stage pipette puller (P-830; Narishige) with resistances of 3 to 5 MΩ, were filled with an internal solution containing 110 mM Tris-phosphate dibasic, 28 mM Tris-base, 11 mM EGTA, 2 mM MgCl2, 0.5 mM CaCl2, and 4 mM Na-ATP, pH 7.3. A lifted whole-cell current-recording technique was applied as previously (Yu et al., 2009; Zheng et al., 2009), in which the recorded cell was lifted from the bottom of the recording dish after formation of the conventional whole-cell recording mode. This procedure accelerated the solution exchange rate when tested drugs were applied through a computer-controlled drug perfusion system (SF-77B Perfusion Fast Step; Warner Instruments, Hamden, CT). The interval between drug applications (2 min) was optimized specifically to ensure stability of nAChR responsiveness (i.e., no functional rundown). Whole-cell currents were recorded using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA) at a holding potential of −60 mV. An access resistance <20 MΩ was accepted to start recording, and the series resistance was routinely compensated to 80%.
The perforated patch-clamp recordings were performed in dissociated VTA neurons (Wu et al., 2004; Yang et al., 2009a). In brief, freshly dissociated neurons were continuously superfused with standard external solution (detailed in neuron dissociation section). For perforated patch-clamp recording, the amphotericin B (Sigma-Aldrich) was diluted from a stock solution (40 mg/ml in dimethyl sulfoxide) to a final concentration of 200 μg/ml in the pipette solution containing 140 mM potassium gluconate, 10 mM KCl, 5 mM MgCl2, and 10 mM HEPES, pH adjusted to 7.2 with Tris-OH. Neurons were clamped at a holding potential of −60 mV by using the same amplifier and software as for SH-EP1 cells. Access resistance was usually reduced to lower than 60 MΩ within 10 min after gigohm-seal formation. Only an access resistance of <60 MΩ was accepted to start experiments. No series resistance compensation was applied for dissociated neurons. All patch-clamp recordings were performed at room temperature.
Data acquisition and analyses were performed using pClampfit 9.2 (Molecular Devices, Sunnyvale, CA), and results were plotted using Prism 3.0 (GraphPad Software Inc., San Diego, CA) or Origin 5.0 (OriginLab Corp., Northampton, MA).
Data Analysis and Statistics.
All statistical results are presented as mean ± S.E.M. Whole-cell currents induced by ligands were normalized to the corresponding controls, which were set as 100%, and the normalized values were used to carry out the statistical analysis and plot the data. Concentration–response curves were fitted to the Hill equation. Student's t test was used to assess whether the means of two groups were statistically different from one another. In the case of multiple group comparisons, we used analysis of variance (ANOVA). If significance was obtained, further analysis was carried out by Tukey's post hoc test. Statistical significance was attributed to values below 0.05 (p < 0.05).
LTG Suppresses Whole-Cell Currents Mediated by Human Neuronal α4β2-nAChRs Heterologously Expressed in SH-EP1 Cells.
Our key question was whether LTG produces any of its modulatory effects by acting on neuronal α4β2-nAChRs. To address this possibility, we initially screened and profiled the effects of different concentrations (0.001–1000 μM) of LTG on α4β2-nAChR-mediated currents. Under voltage-clamp recording mode at a holding potential of −60 mV, bath application of nicotine for 4 s induced an inward current consisting of peak and steady-state components (Fig. 1, Aa and Ba). For these experiments, we used 3 μM nicotine to activate α4β2-nAChRs, because this concentration is close to the EC50 concentration for human α4β2-nAChRs (Wu et al., 2006). Exposure of the recorded cell to 100 μM LTG for 2 min did not induce any detectable current (data not shown). However, either coapplication of 100 μM LTG with 3 μM nicotine (Fig. 1Bb) or pretreatment with LTG for 2 min, followed by coapplication of LTG with nicotine (Fig. 1Bc), significantly reduced nicotine-induced currents (Fig. 1C), and the inhibitory effect was reversible after washout for 4 min (data not shown). Compared with the control (3 μM nicotine-induced current as 100%), the peak and steady-state components of whole-cell current were 80.9 ± 7.2% (p < 0.05, n = 7) and 74.7 ± 5.5% (p < 0.05, n = 7) after coapplication of 100 μM LTG with 3 μM nicotine (Fig. 1C) and 38.9 ± 2.2% (p < 0.01, n = 7) and 16.7 ± 1.5% (p < 0.001, n = 7) after 2-min LTG pretreatment (100 μM) followed by coapplication of LTG with 3 μM nicotine (Fig. 1D). The concentration-response relationship curves showed that with a 2-min LTG pretreatment LTG inhibited nicotinic responses in a concentration-dependent manner (Fig. 1D). The values of IC50 and the Hill coefficient were 183 μM and 0.7 for the peak component and 72 μM and 0.8 for steady-state component, respectively. These results suggest that LTG inhibits human α4β2-nAChR-mediated currents, and the inhibitory effect is profound when preceded by a 2-min pretreatment. Additional experiments also showed that with longer (10-min) pretreatment, LTG exhibit more profound inhibition in α4β2-nAChR-mediated currents (Supplemental Fig. 2). In addition, the steady-state component of the nicotinic current is more sensitive to LTG inhibition than the peak current (steady state, 72 ± 21 μM versus peak, 183 ± 39 μM, p < 0.05).
LTG Inhibits α4β2-nAChR-Mediated Currents Using Different Protocols.
To further characterize the observed inhibitory effect exerted by LTG on α4β2 nAChR-mediated currents, three LTG application protocols were compared. In the first protocol, 100 μM LTG was coapplied with 3 μM nicotine (without pretreatment; Fig. 2A Right), resulting in the reduction of both the peak and steady-state components of the nicotine-induced current (peak, 84.0 ± 4.5%, p < 0.05; steady state, 67.8 ± 10.5%, n = 7, p < 0.05). In the second protocol, 100 μM LTG was applied for 2 min, followed by LTG and nicotine coapplication (with pretreatment; Fig. 2B, right). In this case, the measured peak and steady-state components of nicotine-induced currents were markedly reduced (peak, 60.6 ± 7.6%, p < 0.01; steady state, 32.8 ± 12.1%, n = 6, p < 0.001). Finally, the cells were exposed to 100 μM LTG for 2 min followed by application of nicotine alone (Fig. 2C, right). It is noteworthy that in this protocol the peak current was selectively reduced (peak, 77.6 ± 7.9%, p < 0.05; steady state, 87.8 ± 8.0%, n = 6, p > 0.05). Figure 2 summarizes the effects of LTG with different exposure protocols on nicotine-induced peak (Fig. 2D) and steady-state (Fig. 2E) currents. These results suggest that LTG could exert its most potent inhibitory effect on human α4β2-nAChR function with 2-min pretreatment followed by coapplication with nicotine.
LTG Inhibits α4β2-nAChR Function in a Noncompetitive Manner.
To elucidate whether the LTG-induced inhibition in α4β2-nAChR-mediated currents is mediated through a competitive or noncompetitive mechanism, we examined the nicotine concentration-response relationship with and without 100 μM LTG. Figure 3A shows the typical traces of nicotinic responses with different concentrations of nicotine, and Fig. 3B shows the typical traces of nicotinic responses with different concentrations of nicotine plus 100 μM LTG (with 2-min pretreatment). Nicotine potency was indistinguishable in the absence or presence of LTG (Log EC50 = −5.5 ± 0.16 M, n = 6 versus Log EC50 of −5.4 ± 0.14 M, n = 6, respectively; EC50 values obtained by assessing whole-cell peak current amplitudes; p > 0.05; Fig. 3C). However, the inhibitory effect of 100 μM LTG increased as the nicotine concentration increased (Fig. 3D). Therefore, LTG reduced current responses induced by high concentrations of nicotine without altering EC50 values, suggesting a noncompetitive mechanism of antagonism.
LTG-Induced Inhibition in α4β2-nAChRs Is Voltage-Dependent.
To further elucidate the mechanism of LTG inhibition of α4β2 nAChR, we examined the effect of LTG on α4β2-nAChR-mediated currents at different holding potentials (VHs). As shown in Fig. 4A, 100 μM LTG (2-min pretreatment) reduced α4β2-nAChR-mediated peak currents at different VHs. The results showed that within this range of holding potentials LTG reduced the peak currents to 51.4 ± 6.8, 44.5 ± 9.9, and 41.6 ± 9.8%, respectively, at the VH of −60, −100, and 0 mV, respectively (p < 0.01 compared with nicotinic responses without LTG). Statistical analysis showed no significant differences in LTG inhibition when the VH was held at 0, −60, and −100 mV (p > 0.05, ANOVA; Fig. 4Ba), suggesting that LTG-induced inhibition in the peak component of α4β2-nAChR-mediated currents is independent of transmembrane voltage. Conversely, significant differences in the steady-state component were observed at different VHs. After exposure to LTG (2-min pretreatment), the amplitude of steady-state currents was 6.3 ± 6.1, 24.8 ± 9.9, and 40.1 ± 9.1% at the VH of 0, −100, and −60 mV, respectively. The difference in steady-state currents was statistically significant between 0 and −60 mV (p < 0.05, n = 4) and between 0 and −100 mV (p < 0.05, n = 4, Fig. 4Bb). Furthermore, Tukey post hoc comparisons of the three groups revealed that inhibition in steady-state currents occurred in a voltage-dependent manner (p < 0.05, ANOVA, n = 4, Fig. 4Bb). Taken together, these results suggest that LTG is likely to affect the peak and steady-state components of α4β2-nAChR-mediated currents through different mechanisms: LTG inhibits peak current independently of the VH but inhibits steady-state currents in a VH-dependent manner.
LTG-Induced Inhibition of α4β2-nAChRs Is Use-Dependent.
The next series of experiments addressed the question of whether LTG inhibits α4β2-nAChR function in a use-dependent manner. First, we examined nicotine-induced currents by repetitively exposing them to 3 μM nicotine for 4 s at 2-min intervals (Fig. 5A, inset), and the results demonstrated that the α4β2-nAChR-mediated currents did not show any functional rundown (Fig. 5A). Then, we used the same protocol to test whether LTG inhibition exhibited use dependence. SH-EP1 cells were repetitively exposed to 3 μM nicotine (4-s exposure at an interval of 2 min) in the presence of 100 μM LTG for 10 min. Under this experimental condition, nicotine-induced currents were gradually reduced by LTG in a time-dependent manner (Fig. 5B). The amplitudes of the peak currents at the initial and final exposure to LTG were 77.9 ± 9.8 and 10.9 ± 3.8% of control, respectively (Fig. 5D, black columns). After full recovery of nicotine-induced currents with a ∼10-min washout, the same recorded SH-EP1 cell was continually exposed to 100 μM LTG for 10 min, but nicotine was only applied at 0 and 10 min (without repetitive application of nicotine) (Fig. 5C). The results (n = 4) show that LTG reduced the amplitude of peak currents to 86.7 ± 4.7 and 30.6 ± 7.1% of control at the beginning (0 min) and end (10 min) of LTG exposure, respectively (Fig. 5D, open columns). Statistical analysis demonstrated that LTG produced a more profound inhibition in the peak currents induced by repetitive challenges of nicotine (10.9 ± 3.8% of control) than that induced by nonrepetitive challenges of nicotine (30.6 ± 7.1% of control) in the same recorded SH-EP1 cell (p < 0.05, comparison between the two groups, Fig. 5C, n = 4). These results suggest that LTG inhibits α4β2-nAChR-mediated whole-cell currents in a use-dependent manner.
LTG Inhibits Native α4β2-nAChRs in DAergic Neurons Freshly Dissociated from the Rat VTA.
The results reported above demonstrate that LTG works as an antagonist at human neuronal α4β2-nAChRs in SH-EP1 cells. However, the recordings were made on heterologously transfected α4β2-nAChRs rather than on native α4β2-nAChRs. Thus, it was interesting to test whether LTG could also exert its inhibitory effects on native α4β2-nAChRs. Given that functional α4β2-nAChRs are well identified in DA neurons of rat VTA (Yang et al., 2009a), we tested the effects of LTG on native α4β2-nAChRs in DA neurons acutely dissociated from rat VTA.
The DA neuronal phenotype was identified using the following criteria at the beginning of our patch-clamp recordings, as used elsewhere (Xiao et al., 2009; Yang et al., 2009a): 1) slow and regular spontaneous action potential firing at a frequency of approximately 1 to 3 Hz (Supplemental Fig. 3Ca); 2) the depression of spontaneous firing by 10 μM DA (Supplemental Fig. 3Ca); 3) expressing a prominent inwardly rectified current (IH) induced by hyperpolarizing voltage steps (from −60 to −120 mV in steps of 10 mV, supplemental Fig. 3Da); and 4) tyrosine hydroxylase staining showed positive reaction (Supplemental Fig. 3A). Supplemental Fig. 3B, Cb and Db represents the features of non-DA neurons. Our previous observations have demonstrated that the above electrophysiological criteria are reliable markers for identifying VTA DA neurons (Yang et al., 2009a).
To activate native α4β2-nAChRs in the DA neurons, RJR-2403 (30 μM), a selective agonist of α4β2-nAChRs, was used (Yang et al., 2009a). In most neurons tested, RJR-2403 induced an inward current at a VH of −60 mV, and this current was sensitive to a selective α4β2-nAChR antagonist, dihydro-β-erythroidine (1 μM; data not shown), suggesting that the α4β2-nAChRs are mediated by RJR 2403-induced currents (Fig. 6A). The effect of LTG (100 μM) on native α4β2-nAChRs was examined next. Bath-applied LTG alone eliminated action potential firing, suggesting that LTG blocks Na+ channels (supplemental Fig. 4). It is noteworthy that a coapplication of LTG (100 μM) with RJR-2403 (30 μM) did not show significant inhibition in either peak or steady-state components of RJR-2403-induced currents (peak and steady-state were 97.1 ± 4.1 and 90.4 ± 9.1% of control, respectively; p > 0.05, n = 4; Fig. 6B, open columns). However, with 2-min pretreatment, LTG significantly reduced both peak and steady-state currents to 54.1 ± 5.9% and 22.0 ± 8.7% of control (p < 0.01, n = 4; Fig. 6B, filled columns). To compare the effects of LTG on native and transfected α4β2-nAChRs, we also examined the effects of LTG (100 μM) on 30 μM RJR-2403-induced currents in transfected SH-EP1 cell line (Supplemental Fig. 5), and the results showed a similar effect of LTG. These results suggest that with appropriate pretreatment LTG also significantly inhibits native α4β2-nAChRs in VTA DAergic neurons.
α4β2-nAChR Is a Specific Target for Mediating the Effect of LTG on VTA DAergic Neurons.
In a last series of experiments, we investigated whether LTG has any effect on non-nicotinic neurotransmitter receptors, mainly glutamatergic and GABAergic receptors. We examined the effects of LTG on 1 mM glutamate- or 0.1 mM GABA-induced current at the VH of −60 mV. With a 2-min pretreatment, 100 μM LTG had no detectable effect on either glutamate- or GABA-induced currents (Fig. 7, A and B). Compared with controls, the peak amplitudes were 101.2 ± 4.9% (p > 0.05, n = 4) and 96.1 ± 5.2% (p > 0.05, n = 4) for glutamate- and GABA-induced currents, respectively, and the steady-state amplitudes were 95.6 ± 5.6% (p > 0.05, n = 4) and 95.9 ± 7.6% of control (p > 0.05, n = 4) for glutamate- and GABA-induced currents, respectively (Fig. 7C). These results suggest that the neuronal nAChR, rather than glutamatergic or GABAergic receptors in the VTA DAergic neurons, is a main target mediating the effect of LTG.
In this study, we have demonstrated that LTG, an anticonvulsive drug, inhibits human neuronal α4β2-nAChR function in a concentration-, voltage-, and use-dependent manner. It is also shown that LTG selectively inhibits neuronal nAChRs but not other ligand-gated ion channels tested. These findings suggest that the LTG-induced inhibition in α4β2-nAChRs may be an important mechanism underlying its anticonvulsive action against the JME, thus shedding some light on new antiepileptic drug design and development. LTG also exhibits potential for the development of compounds having antagonist effects on α4β2-nAChRs, useful both for the study of nAChR pharmacology and anticonvulsive treatment in cases such as ADNFLE and JME.
Our study shows that only high concentrations of LTG (above 100 μM, without pretreatment) caused a significant reduction in both the peak and steady-state currents of α4β2-nAChRs. After 2-min pretreatment, LTG inhibited the peak and steady-state currents in a concentration-dependent manner, with IC50 values of 183 and 72 μM for peak and steady-state components, respectively (Fig. 1). We recognize that the concentration of LTG (100 μM, close to its IC50 concentration) used in this study is approximately 2- to 4-fold higher than the plasma levels seen in clinical practice, which are approximately 24 to 41 μM (Doose et al., 2003; Beck et al., 2006). However, compared with the effects of LTG on its well known target Na+ channels, IC50 is 100 μM at a holding potential of −60 mV (Xie et al., 1995). Its affinity to block α4β2-nAChR function is within the range of LTG pharmacology (IC50 values of 183 and 72 μM for peak and steady state). These initial results suggest that LTG exerts an inhibitory mode of action on neuronal nAChRs.
Pharmacological experiments demonstrated that LTG-induced inhibition in human α4β2-nAChR function is voltage- and use-dependent. This in turn suggests that LTG suppression of nicotine-induced currents involves an agonist-induced transition of nAChRs to conformations having higher affinity for the antagonist and/or allowing free access to its binding site(s) (Zhao et al., 2004); it is reasonable to conclude that this transition is to an open-channel state. In addition, we found that when LTG was coapplied with nicotine the peak current of whole-cell current responses was modified to a much lesser extent than the steady-state component (Fig. 2A), again suggesting a possible open-channel block with relatively slow association kinetics. Pretreatment with LTG followed by coapplication of LTG plus nicotine (Fig. 2B) induced a more profound inhibition of nicotinic responses, suggesting that binding sites on the nAChRs are at least partially accessible in the closed state. Finally, LTG pretreatment only (i.e., without coapplication of nicotine) significantly inhibited the peak currents (Fig. 2C), suggesting that LTG association with its site of action on the nAChRs does not require channel activation. It is possible that more than one type of binding sites for LTG is available at nAChRs in the closed state, with each site having a different affinity for the antagonist. It is tempting to suggest that the inhibition exerted by the drug when acting as an open-channel blocker favors the transition to the closed state rather than to a desensitized state. On the other hand, when LTG acts as a closed-channel blocker it is possible that the binding site(s) of the drug is close enough or can modify the conformation of the agonist binding site, thus preventing free agonist from binding to the receptor and resulting in the observed diminution of peak currents while normal transitions to desensitized state(s) take place.
The fact that LTG association with its site(s) of action on the nAChR may occur without channel activation is not consistent with a use-dependent channel-block mechanism of inhibition, in that such agents can enter the ion channel only in the open agonist-bound state (MacDonald et al., 1987). It is possible, however, that LTG is able to bind to a site within the lumen of the ion channel but above the ion channel gate proper (i.e., in the vestibule). The N-methyl-d-aspartate receptor blocker memantine, referred to as a partially trapping channel blocker, has been proposed to act at two sites: one below the channel gate, and thus susceptible to trapping block, and one above the ion channel gate, and thus not subject to trapping block (Blanpied et al., 1997). The fact that pre-exposure of LTG plus coapplication of LTG with nicotine (Fig. 2B) diminishes the peak and steady-state currents to a greater extent than the “coapplication only” protocol (Fig. 2A) opens up the possibility that LTG produces a trapping channel block. The concentration-response relationship of nicotine-induced currents in the presence of LTG clearly shows a reduction of that peak of nicotinic response at high nicotinic concentration (Fig. 3, A and D), suggesting a noncompetitive mechanism of antagonism. Collectively, the findings of the present study suggest that LTG has access to the α4β2-nAChR in both the resting and activated states.
The possibility that LTG operates via an open-channel blocking mechanism was evaluated by testing whether the inhibition was voltage-dependent. It was found that LTG inhibition of nicotine-activated currents was independent of membrane voltage between −100 and 0 mV, with no statistically significant differences being observed between the amplitudes of the peak currents (Fig. 4B). Again, this does not rule out the possibility of an open-channel blocking mechanism; such a posit would entail binding of LTG to a site within the ion channel but beyond the influence of the membrane electric field (Marszalec and Narahashi, 1993). Actually, voltage did influence the steady state of nicotine-induced currents, which were significantly smaller between 0 mV and negative holding potentials. Previous experiments performed by one of our groups have demonstrated a clear interaction between LTG and adult muscle-type nAChR (Vallés et al., 2007, 2008). In Vallés et al. (2008), application of fluorescence spectroscopy techniques led for the first time to the demonstration that LTG is able to modify the equilibrium between the basic conformational states of the AChR (resting, open, close, and desensitized). In the present experiments, nAChRs are highly purified, and no Na+ channels are present in the samples; thus, voltage-dependent effects of Na+ can be safely discarded.
In conclusion, LTG may inhibit α4β2-nAChRs function via a dual open-channel and close-channel blocking mechanism. Furthermore, the inhibitory action of LTG is restricted to cholinergic receptors; our experiments discarded the possibility that GABAergic or glutamatergic receptors are involved.
Functional alterations in nAChRs, and in particular gain of function of α4β2-nAChRs, play an important role in the pathogenesis of some forms of epilepsy (e.g., ADNFLE and JME) (Steinlein et al., 1997; Phillips et al., 2001; Rozycka et al., 2009), although the exact nature of this role is largely unclear. It is possible that gain-of-function α4β2-nAChRs enhance neurotransmitter release resulting from an increase in the activation of presynaptic nAChRs or directly induce hyperexcitability of the postsynaptic neuron as a result of the activation of supersensitive postsynaptic nAChRs (De Fusco et al., 2000). Blocking the function of α4β2-nAChRs may thus constitute a new target for the treatment of some forms of epilepsy.
Our present findings show that LTG blocks the transfected human neuronal α4β2-nAChRs in SHEP-1 cells as well as the native rat neuronal α4β2-nAChRs in VTA DAergic neurons. On the basis of these results, we speculate that LTG may also inhibit those gain-of-function α4β2-nAChR mutants found in the above-mentioned forms of epilepsy, although further study of α4β2-nAChR mutants is called for. It is likely that clinical doses of LTG might effectively antagonize the function of gain-of-function nAChR mutants, whose sensitivity to nicotinic ligands is higher (Steinlein et al., 1997; Phillips et al., 2001). The blockade of these mutant receptors, or restoration of the nAChR function to normal levels, could prevent the neuronal hyperexcitability induced by mutant nAChRs and in turn help to control seizure activity, while (at this low concentration) producing limited inhibition of normal nAChRs. Thus, in addition to inhibition of sodium channels, inhibition of nAChRs may be an important mechanism underlying LTG's antiepileptic effect because 1) LTG blocks both sodium channels and nAChRs with similar affinity; 2) as mentioned before, the α4β2-nAChRs in ADNFLE and juvenile myoclonic epilepsy exhibit gain of function, which may increase sensitivity to LTG; and 3) LTG may, like lipophilic drugs (e.g., nicotine), accumulate and achieve levels 10-fold higher in the brain compared with serum levels (Ghosheh et al., 2001). This could be the case with LTG; therefore, serum levels of 5 to 20 μM could reach 10-fold higher levels in the brain, which are certainly enough to effectively block α4β2 AChRs. This double-target pharmacological property may make LTG different from other sodium channel-blocking anticonvulsants, enabling it to be effectively used to treat juvenile myoclonic epilepsy (Buchanan, 1996). As previously stated, a common characteristic of ADNFLE mutations is that nAChRs display increased ACh sensitivity, causing a gain of function (Moulard et al., 2001; Bertrand et al., 2002). In ADFLE, sensitized mutant nAChRs respond excessively to ACh, which is released during rapid-eye-movement sleep and at waking, thereby mediating seizures (Magnusson et al., 1996). Thus, one could hypothesize that a potential beneficial effect of LTG could be the blockage of an overactive mutant receptor.
We thank Dr. Paul A. St. John for help in performing some immunohistochemical experiments.
This work was supported in part by the Shantou University Internal Seed Fund. F.J.B. is the holder of the United Nations Educational, Scientific and Cultural Organization Chair of Biophysics and Molecular Neurobiology.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- nicotinic acetylcholine receptor
- ventral tegmental area
- autosomal dominant nocturnal frontal lobe epilepsy
- juvenile myoclonic epilepsy
- (E)-N-methyl-4-(3-pyridinyl)-3-buten-1-amine oxalate
- artificial cerebrospinal fluid
- analysis of variance.
- Received June 7, 2010.
- Accepted August 2, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics