Bupropion-induced inhibition of α7 nicotinic acetylcholine receptors expressed in heterologous cells and neurons from dorsal raphe nucleus and hippocampus

European Journal of Pharmacology 740 (2014) 103–111

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Molecular and cellular pharmacology

Bupropion-induced inhibition of α7 nicotinic acetylcholine receptors expressed in heterologous cells and neurons from dorsal raphe nucleus and hippocampus Elizabeth Vázquez-Gómez a, Hugo R. Arias b,n, Dominik Feuerbach c, Marcela Miranda-Morales d, Stefan Mihailescu a, Katarzyna M. Targowska-Duda e, Krzysztof Jozwiak e, Jesús García-Colunga d,nn a

Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México, México Department of Medical Education, California Northstate University College of Medicine, 9700W. Taron Dr., Elk Grove, CA 95757, USA Neuroscience Research, Novartis Institutes for Biomedical Research, Basel, Switzerland d Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus Juriquilla 3001, Querétaro 76230, México e Department of Chemistry, Laboratory of Medicinal Chemistry and Neuroengineering, Medical University of Lublin, Lublin, Poland b c

art ic l e i nf o

a b s t r a c t

Article history: Received 27 January 2014 Received in revised form 2 June 2014 Accepted 20 June 2014 Available online 9 July 2014

The pharmacological activity of bupropion was compared between α7 nicotinic acetylcholine receptors expressed in heterologous cells and hippocampal and dorsal raphe nucleus neurons. The inhibitory activity of bupropion was studied on GH3-α7 cells by Ca2 þ influx, as well as on neurons from the dorsal raphe nucleus and interneurons from the stratum radiatum of the hippocampal CA1 region by using a whole-cell voltage-clamp technique. In addition, the interaction of bupropion with the α7 nicotinic acetylcholine receptor was determined by [3H]imipramine competition binding assays and molecular docking. The fast component of acetylcholine- and choline-induced currents from both brain regions was inhibited by methyllycaconitine, indicating the participation of α7-containing nicotinic acetylcholine receptors. Choline-induced currents in hippocampal interneurons were partially inhibited by 10 mM bupropion, a concentration that could be reached in the brain during clinical administration. Additionally, both agonist-induced currents were reversibly inhibited by bupropion at concentrations that coincide with its inhibitory potency (IC50 ¼54 mM) and binding affinity (Ki ¼ 63 mM) for α7 nicotinic acetylcholine receptors from heterologous cells. The [3H]imipramine competition binding and molecular docking results support a luminal location for the bupropion binding site(s). This study may help to understand the mechanisms of actions of bupropion at neuronal and molecular levels related with its therapeutic actions on depression and for smoking cessation. & 2014 Elsevier B.V. All rights reserved.

Keywords: α7 nicotinic acetylcholine receptor Antidepressants Bupropion Dorsal raphe nucleus Hippocampus Molecular docking Chemical compounds studied in this article: Bupropion (PubChem CID: 62884)

1. Introduction Major depression and nicotine addiction are social devastating health situations. There is a high degree of comorbidity between mood disorders and tobacco dependence (Killen et al., 2003; Mackowick et al., 2012; Tsuang et al., 2012). Furthermore, the prevalence of nicotine dependence in patients with major depression is greater than among individuals who had never experienced major depression or with no psychiatric diagnosis (Glassman et al., n

Corresponding author. Tel.: þ 916 686 7300; fax: þ 916 686 7310. Corresponding author. Tel./fax: þ52 442 238 1063. E-mail addresses: [email protected] (H.R. Arias), [email protected] (J. García-Colunga). nn

http://dx.doi.org/10.1016/j.ejphar.2014.06.059 0014-2999/& 2014 Elsevier B.V. All rights reserved.

1990). Persons that present an adverse impact to smoking cessation treatment have increased risk of experiencing mild to severe states of depression (Covey et al., 1998). Nicotinic acetylcholine receptors are widely distributed in the nervous system and are implicated in a variety of disorders, including depression and drug addiction (Arias, 2009; Changeux, 2010; Mineur and Picciotto, 2010; Shytle et al., 2002; Yakel, 2013). Neurons from the dorsal raphe nucleus and interneurons from the stratum radiatum of the hippocampal CA1 region express preand post-synaptic nicotinic acetylcholine receptors (Alkondon and Albuquerque, 1993; Garduño et al., 2012). Interestingly, serotonergic projections from the dorsal raphe nucleus to the hippocampus and nucleus accumbens (brain regions implicated in cognitive deficits, depression, and nicotine addiction) are modulated by

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nicotinic acetylcholine receptors (Aznar et al., 2005; Chang et al., 2011; Mineur and Picciotto, 2010). One common substance, bupropion, is widely used to clinically treat both depression and nicotine addiction (reviewed in Arias (2009), Dhillon et al. (2008), Dwoskin et al. (2006), and Paterson (2009)). Bupropion is a dual inhibitor of noradrenaline and dopamine transporters (Stahl et al., 2004), and also inhibits a variety of nicotinic acetylcholine receptor subtypes in a noncompetitive fashion (Arias et al., 2009, 2012; García-Colunga et al., 2011; Slemmer et al., 2000; reviewed in Arias (2009)). However, there are just few reports on the direct effect of bupropion on the electrical responses elicited by activation of nicotinic acetylcholine receptors in neurons (Alkondon and Albuquerque, 2005; Mansvelder et al., 2007). In this regard, the aim of this work was to study the inhibitory potency of bupropion for α7 nicotinic acetylcholine receptors expressed in heterologous cells and compare with the inhibition elicited on choline-induced currents (i.e., α7containing nicotinic acetylcholine receptors) in neurons from the dorsal raphe nucleus as well as in interneurons from the stratumradiatum of the hippocampal CA1 region. Finally, the structural interaction of bupropion with the α7 nicotinic acetylcholine receptor ion channel was determined by [3H]imipramine competition binding and molecular docking methods.

2. Materials and methods 2.1. Ca2 þ influx measurements in the GH3-α7 cell line The inhibitory potency of bupropion for α7 nicotinic acetylcholine receptors was determined by Ca2 þ influx experiments using the GH3-α7 cell line as previously described (Arias et al., 2010a). Briefly, 5  104 cells per well were seeded 48 h prior to the Ca2 þ influx experiment on black poly-L-lysine 96-well plates (Costar, Corning Inc., NY, USA) and incubated at 37 1C in a humidified atmosphere (5% CO2/95% air). The medium was changed 16 h before the experiment to 1% fetal bovine serum in HEPESbuffered salt solution (in mM): 130 NaCl, 5.4 KCl, 2 CaCl2, 0.8 MgSO4, 0.9 NaH2PO4, 25 glucose, 20 HEPES, and pH 7.4. On the day of the experiment, the medium was removed by flicking the plates and replaced with 100 ml HEPES-buffered salt solution/ 1% fetal bovine serum containing 2 μM fluo-4 (Molecular Probes, Eugene, OR, USA) in the presence of 2.5 mM probenecid (Sigma, Buchs, Switzerland). The cells were then incubated at 37 1C in a humidified atmosphere (5% CO2/95% air) for 1 h. Plates were flicked to remove excess of fluo-4, washed twice with HEPESbuffered salt solution/1% fetal bovine serum, and finally refilled with 100 ml of HEPES-buffered salt solution containing different concentrations of bupropion and pre-incubated for 5 min. Plates were then placed in the cell plate stage of the fluorimetric imaging plate reader (Molecular Devices, Sunnyvale, CA, USA). A baseline consisted of five measurements of 0.4 s for each record. (7 )‐ Epibatidine (0.1 mM) was then added from the agonist plate to the cell plate using the 96-tip pipettor simultaneously to fluorescence recordings for a total of 3 min. In parallel experiments, 0.1 mM (7)-epibatidine was co-injected with different concentrations of bupropion. The laser excitation and emission wavelengths were 488 and 510 nm, at 1 W, with a CCD camera opening of 0.4 s. The concentration–response data were curve-fitted by nonlinear least squares analysis using the Prism software (GraphPad Software, San Diego, CA). 2.2. Preparation of brain slices All procedures were carried out in accordance with the National Institute of Health Guide for Care and Use of Laboratory

Animals and were approved by the Institutional Animal Care Committee of the Universidad Nacional Autónoma de México, with an effort to minimize the number of animals used and their suffering. The experiments were performed on brain slices obtained from Wistar rats, 15–20 postnatal days. Animals were deeply anesthetized with isoflurane and decapitated. Their brains were quickly removed and placed into ice cold (4 1C) solution containing (in mM): 250 sucrose, 2.5 KCl, 1.2 NaH2PO4, 5 MgCl2, 0.5 CaCl2, 26 NaHCO3, 10 glucose, and pH 7.4. Coronal midbrain slices of 350 mm thick containing the dorsal raphe nucleus or hippocampal area were cut with a Vibratome Leica VT 1000 and submerged in artificial cerebrospinal fluid containing (in mM): 125 NaCl, 2.5 KCl, 1.23 NaH2PO4, 1 MgCl2, 2 CaCl2, 26 NaHCO3, 10 glucose, and pH 7.4. The slices were stabilized in this solution for at least 1 h before transferring a single slice to the recording chamber. All solutions were continuously bubbled with 95% O2 and 5% CO2 at room temperature. 2.3. Whole-cell patch-clamp recordings Individual slices were transferred into a tissue chamber and superfused throughout the experiment with artificial cerebrospinal fluid at a rate of 2–3 ml/min. Whole-cell voltage-clamp recordings (Hamill et al., 1981) were performed with a PC-ONE Patch/Whole Cell Clamp (Dagan Corporation, MN, USA), using an acquisition system Digidata 1440A driven with pClamp 10. Patch-clamp electrodes were made with borosilicate glass (Sutter Instrument, CA, USA) having a resistance of 3–7 MΩ when filled with either of two internal solutions: (in mM): 140 KCl, 5 NaCl, 1 MgCl2, 10 HEPES, 10 EGTA (adjusted pH 7.4 with KOH); or 140 Csgluconate, 2 MgCl2, 0.5 CaCl2, 10 HEPES, 5 EGTA, 2 MgATP (adjusted pH 7.4 with CsOH). Similar results were obtained by using indistinctly each internal solution. Experimental data were stored in a PC using a Digidata 1440A AD converter (Axon Instruments, Union City, CA, USA), at a sampling rate of 10 kHz. Individual neurons were visualized using an infrared video-microscopy system (BX51WI, Olympus Instruments, Japan) endowed with an 80  water immersion objective. Neurons selected for recording were located close the midline area of the dorsal raphe nucleus identified as the translucent area between the medial longitudinal fasciculus and the aqueduct; or in the stratum radiatum hippocampal CA1 area. All recorded cells were maintained at a potential of  70 mV. Brief puffs (2–5 psi, 500 ms) of 1 mM acetylcholine or 10 mM choline on dorsal raphe nucleus or hippocampal neurons were applied through a fine tip glass micropipette placed about 10 mm far from the recorded neuron, by using a microinjector (IM 300, Narisihige Comp., Japan). Different substances were added to the external solution (artificial cerebrospinal fluid) during all the experiments: 500 nM atropine to eliminate the muscarinic component, the glutamate receptor antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione (10 mM) and DL-2-amino-5-phosphonopentanoic acid (50 mM), the γ-aminobutyric acid (GABA) A receptor antagonist bicuculline (10 mM), and 200 nM tetrodotoxin to avoid arrival of action potentials. Repeated administrations of acetylcholine or choline were applied at 5-min intervals to allow recovery of nicotinic acetylcholine receptors from desensitization. The effects of bupropion were assessed by microinjections of acetylcholine or choline on the recording neurons before and after bupropion bath application. We used the pClamp 10 software (Molecular Devices, CA, USA) to measure the acetylcholine- and choline-induced currents in the absence or presence of burpropion, and Origin 9 (Microcal Software Inc., MA, USA) to analyze and graph the results. Data are presented as mean 7S.E.M. Comparison of the mean values was performed by the Student's t-test, with p o0.05 considered statistically significant.

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2.4. Bupropion-induced inhibition of [3H]imipramine binding to nicotinic acetylcholine receptors

α7

To localize the bupropion binding site in the α7 nicotinic acetylcholine receptor, [3H]imipramine was used as a probe for the antidepressant site as it was determined previously in different nicotinic acetylcholine receptors (Arias et al., 2010a, 2010b). In this regard, the effect of bupropion on [3H]imipramine binding to α7 nicotinic acetylcholine receptors was studied using SHSY5Yα7 cell membranes as described previously (Arias et al., 2010a). More specifically, nicotinic acetylcholine receptor membranes (1.5 mg/ml) were suspended in binding saline buffer (in mM: 50 Tris–HCl, 120 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, and pH 7.4) with 14 nM [3H]imipramine, and preincubated for 30 min at room temperature. Nonspecific binding was determined in the presence of 100 mM imipramine. The total volume was divided into aliquots, and increasing concentrations of bupropion were added to each tube and incubated for 2 h at room temperature. The radioligand bound to nicotinic acetylcholine receptors was then separated from free ligand by a filtration assay using a 48-sample harvester system with GF/B Whatman filters (Brandel Inc., Gaithersburg, MD, USA), previously soaked with 0.5% polyethylenimine for 30 min. The membrane-containing filters were transferred to scintillation vials with 3 ml of Bio-Safe II (Research Product International Corp, Mount Prospect, IL, USA), and the radioactivity was determined using a Beckman LS6500 scintillation counter (Beckman Coulter, Inc., Fullerton, CA, USA). The concentration–response data were fitted by nonlinear least squares analysis using the Prism software, and the corresponding ligand concentration that inhibits 50% binding (IC50) and the Hill coefficient (nH) was calculated. The observed IC50 values were subsequently transformed into inhibition constant (Ki) values using the Cheng–Prusoff relationship (Cheng and Prusoff, 1973) K i ¼ IC50 =f1 þ ð½½3 Himipramine=K imipramine Þg; d 3

ð1Þ 3

where [[ H]imipramine] is the initial concentration of [ H]imipramine, and Kimipramine corresponds to the dissociation constant of d [3H]imipramine for the α7 nicotinic acetylcholine receptor (1 μM; Arias et al., 2010a). The calculated Ki and nH values are summarized in Table 1. 2.5. Molecular docking of bupropion enantiomers to the α7 nicotinic acetylcholine receptor ion channel The model of the α7 nicotinic acetylcholine receptor was built applying homology modeling methods using the cryo-electron microscopy structure of the Torpedo nicotinic acetylcholine receptor determined at  4 Å resolution (PDB ID 2BG9) (Unwin, 2005) as a template, as described previously (Arias et al., 2009, 2010a, 2010b, 2012). Modeller 9.9 was used to obtain 100 homology

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models, and subsequently, their Discrete Optimized Protein Energy (DOPE) profiles (Eswar et al., 2006) were assessed. The best model was subjected to model quality assessment, using the web-based tools of Verify3D (Eisenberg et al., 1997) and ProCheck (Laskowski et al., 1993). Bupropion enantiomers, in the protonated and neutral states, were prepared using Spartan 10V.1.1.0 (Wavefunction, Inc., Irvine, CA, USA), and the molecular docking simulations were performed using the same protocol as reported previously (Arias et al., 2012). 2.6. Materials [3H]Imipramine (47.5 Ci/mmol) was obtained from PerkinElmer Life Sciences Products, Inc. (Boston, MA, USA) and stored in ethanol at 20 1C. (7 )-epibatidine hydrochloride and tetrodotoxin citrate were obtained from Tocris Bioscience (Ellisville, MO, USA). Fetal bovine serum was purchased from Gibco BRL (Paisley, UK). (7)-bupropion hydrochloride [( 7)-2-(tert-butylamino)-1(3-chlorophenyl)propan-1-one], imipramine hydrochloride, acetylcholine chloride, atropine, DL-2-amino-5-phosphonopentanoic acid, bicuculline methobromide, choline chloride, 6-cyano-7nitroquinoxaline-2,3-dione disodium salt hydrate, and methyllycaconitine citrate hydrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Salts were of analytical grade.

3. Results 3.1. Inhibitory potency of bupropion at receptors

α7 nicotinic acetylcholine

The potency of ( 7)-epibatidine to activate α7 nicotinic acetylcholine receptors was first determined by assessing the fluorescence change in GH3-α7 cells after ( 7)-epibatidine stimulation (Fig. 1). The observed EC50 value (ligand concentration producing 50% Ca2 þ influx) for ( 7)-epibatidine (52 74 nM; Table 1) is in the same concentration range as those previously determined for α7 nicotinic acetylcholine receptors (Arias et al., 2010a). Pre-incubation with bupropion subsequently inhibited the (7)epibatidine-induced nicotinic acetylcholine receptor activation (Fig. 1). Comparing the calculated IC50 with values from previous studies (Arias et al., 2009, 2012; reviewed in Arias (2009)), it is apparent that bupropion has low selectivity and low potency for the α7 nicotinic acetylcholine receptor subtype (Table 1). However, the inhibitory potency increased 3.3-fold when the α7 nicotinic acetylcholine receptors were inhibited by bupropion in the presence of the agonist (co-injection protocol). The observed nH value is close to unity using the pre-incubation protocol, whereas it is higher than unity using the co-injection protocol (Table 1). This indicates that bupropion pretreatment inhibits α7 nicotinic

Table 1 Activation potency of ( 7 )-epibatidine as well as inhibitory potency and binding affinity of ( 7 )-bupropion at α7 nicotinic acetylcholine receptors. Protocol

Conformational state

Ca2 þ influx: ( 7 )-epibatidine only Activated Ca2 þ influx: 5 min preincubation with bupropion followed by activation with ( 7 )- Mix of different states epibatidine for several seconds Mix of activated and Ca2 þ influx: Co-injection of (7 )-epibatidine and bupropion for several seconds desensitized states Mainly resting state [3H]Imipramine competition binding a b c

These values were obtained from Fig. 1. The Hill coefficient. These values were obtained from Fig. 6.

EC50a (nM)

IC50a (μM)

nHa,b

Ki c (μM)

nHb,c

52 74 –

– 3.69 7 0.18 – 1797 20 1.08 7 0.04 –

– –

–

547 7

1.48 70.07 –

–

–

–

–

637 10 0.767 0.09

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acetylcholine receptor activation by a non-cooperative mechanism, suggesting that bupropion stabilizes the closed state of the receptor, whereas bupropion inhibits the recently activated receptor by a cooperative mechanism. This latter mechanism suggests

the existence of more than one binding site for bupropion and/or the combination of several inhibitory processes (e.g., openchannel blockade and induced receptor desensitization), as it has been observed for bupropion and its derivatives in muscle nicotinic acetylcholine receptors (Arias et al., 2009, 2012; reviewed in Arias (2009)). 3.2. Dorsal raphe and hippocampal neurons express functional and non-α7 nicotinic acetylcholine receptors

Fig. 1. Effect of bupropion on (7 )-epibatidine-induced Ca2 þ influx in GH3-α7 cells. Increased concentrations of (7 )-epibatidine (■) activate α7 nicotinic acetylcholine receptors. Subsequently, cells were pre-treated (5 min) with several concentrations of bupropion followed by addition of 0.1 μM ( 7 )-epibatidine (○), or alternatively different concentrations of bupropion were co-injected with 0.1 μM ( 7 )-epibatidine (□). The response was normalized to the maximal ( 7 )-epibatidine response which was set as 100%. The plots show the results of 27 (■), 11 (○), and 8 (□) separate determinations. Error bars correspond to the S.D. The calculated EC50, IC50, and nH values are summarized in Table 1.

α7

To compare the inhibitory activity of bupropion on α7 nicotinic acetylcholine receptors expressed in heterologous cells with those endogenously expressed in neurons, we subsequently determined its pharmacological activity in neurons from the dorsal raphe nucleus and in interneurons from the stratum radiatum of the hippocampal CA1 region. The identity of endogenous nicotinic acetylcholine receptors was determined by comparing the electrical activity elicited by the unspecific agonist acetylcholine with that for the selective agonist choline, as well as by the inhibitory effect provoked by methyllycaconitine, a selective antagonist for α7 nicotinic acetylcholine receptors (Alkondon et al., 1992, 1997), on these agonist-induced electrical responses. The local application of 1 mM acetylcholine or 10 mM choline onto hippocampal and dorsal raphe nucleus neurons generated an inward current that decayed in the presence of the agonist, due to receptor desensitization (Fig. 2A). Of 15 recorded dorsal raphe nucleus neurons, 11 presented acetylcholine-induced currents,

Fig. 2. Functional activity of α7-containing nicotinic acetylcholine receptors in dorsal raphe nucleus and hippocampal neurons. (A) Upper traces correspond to acetylcholine (ACh) induced currents recorded in hippocampal (Hipp, left) and dorsal raphe nucleus (DRN, right) neurons. Lower traces correspond to choline (Ch) induced currents recorded in hippocampal (left) and DRN (right) neurons. (B) Inhibition of acetylcholine- and choline-induced currents by 5 nM methyllycaconitine (MLA). The upper acetylcholine-induced currents were recorded from a hippocampal CA1 interneuron (n¼ 5), whereas the lower choline-induced currents from a dorsal raphe nucleus neuron (n¼3). Ion currents on the left correspond to controls, middle currents were obtained in the presence of methyllycaconitine after 7 min of preincubation, and the records on the right illustrate the recovered responses after 30 min of wash out methyllycaconitine. The holding potential was  70 mV. During all the experiments, slices were continuously perfused with the external solution plus (in mM): 10 6-cyano-7-nitroquinoxaline-2,3-dione, 50 DL-2-amino-5-phosphonopentanoic acid, 0.2 tetrodotoxin, 0.5 atropine, and 10 bicuculline. The time of agonist application (500 ms, 2–5 psi) is illustrated by a line above the left upper record in (A). Calibration bars: 100 pA applies for the record having the scale; 50 pA for the rest of the records.

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ranging from 30 to 400 pA; whereas in 42 out of 50 hippocampal neurons tested the acetylcholine-induced currents ranged from 10 to 512 pA. On the other hand, choline-induced currents were present in 11 out of 26 dorsal raphe nucleus neurons, ranging from 20 to 400 pA; and in 31 out of 37 hippocampal interneurons recorded, ranging from 20 to 525 pA. The high variability in the amplitude of agonist-induced currents may indicate the presence of different nicotinic acetylcholine receptor subtypes with variable expression among neurons. A fast component in acetylcholineinduced currents (Fig. 2A, upper records) and a fast decaying inward current generated by choline (Fig. 2A, bottom records) were observed in dorsal raphe nucleus and hippocampal neurons. Afterward, methyllycaconitine was applied at concentrations between 5 and 20 nM. The choline-induced current was completely inhibited by 20 nM methyllycaconitine (n ¼4; data not shown). In addition, 5 nM methyllycaconitine completely inhibited the fast, but not the slow, inward component of the acetylcholineinduced currents (Fig. 2B, upper records), whereas the cholineinduced currents were reversibly inhibited (Fig. 2B, lower records). These results indicate that both the fast acetylcholine- and the total choline-induced currents were due to the activation of α7containing nicotinic acetylcholine receptors.

3.3. Bupropion inhibits nicotinic acetylcholine receptors from dorsal raphe nucleus and hippocampal neurons To evaluate the direct interaction of bupropion with nicotinic acetylcholine receptors, the effects of bupropion were explored on both acetylcholine- and choline-induced currents in the dorsal raphe nucleus and CA1 hippocampal region from rat acute slices. Focal 1 mM acetylcholine applications (2–5 psi, 500 ms) were evaluated before, during, and after the acute bath application of 50 mM bupropion during 7–8 min. The acetylcholine-induced currents were inhibited during and even after bupropion exposure, and partially recovered after washing out bupropion (Fig. 3). Similar results were obtained on choline-induced currents in the dorsal raphe nucleus and CA1 hippocampal region (Fig. 4). In a set of experiments, the inhibitory effects of bupropion on choline-induced currents were compared to those mediated by

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methyllycaconitine and α-bungarotoxin, two selective α7 nicotinic acetylcholine receptor antagonists (Alkondon et al., 1992; Couturier et al., 1990). The choline-induced current in hippocampal interneurons was inhibited by 50 mM bupropion. The ratio between the inhibited choline-induced current by bupropion and the control current in hippocampal interneurons was 0.52 7 0.04 (n ¼6). Then, after recovery of bupropion-inhibited cholineinduced current, 5 nM methyllycaconitine (n ¼3) or 20 nM α-bungarotoxin (n¼ 3) for 15 min was added to the bath perfusion. The choline-induced current was completely inhibited by both antagonists (Fig. 4A and B), confirming the involvement of α7-containing nicotinic acetylcholine receptors. To determine whether the observed bupropion-induced inhibition is elicited at clinically relevant concentrations (i.e., 20 mM; reviewed in Arias (2009)), bupropion was tested at this concentration level (10–20 mM). The results show that bupropion effectively inhibits choline-induced currents at these concentrations (Fig. 4C and D). Accordingly, the ratio between the inhibited cholineinduced current by 20 mM bupropion and the control current in hippocampal interneurons was 0.66 70.06 (n ¼3), whereas the ratio for the acetylcholine-induced current inhibited by 10 mM bupropion in dorsal raphe nucleus neurons was 0.72 70.04 (n ¼ 3). A comparison of the results between acetylcholine- and choline-induced currents in the dorsal raphe nucleus and CA1 hippocampal region indicates a similar inhibitory activity exerted by 50 mM bupropion (Fig. 5). All data were calculated considering the maximal inhibitory effect of bupropion and the maximal recovery (see Figs. 3 and 4). The height of the columns represents the amplitude of the agonist-induced currents in the presence of bupropion normalized to control agonist-induced currents (considered as the unity). The obtained ratios were statistically different (po0.05): 0.4870.05 and 0.4770.04 for the acetylcholineinduced currents in dorsal raphe nucleus and hippocampal neurons, respectively; and 0.4870.04 for the choline-induced currents in hippocampal interneurons. Very few dorsal raphe nucleus neurons (two from 11) responded to choline with amplitudes high enough to evaluate confidently the inhibition elicited by bupropion (with a ratio of 0.46). 3.4. Binding affinity of bupropion for the receptor ion channel

α7 nicotinic acetylcholine

The binding affinity of bupropion for the α7 nicotinic acetylcholine receptor ion channel was determined by using [3H]imipramine as a probe for the ion channel as described previously (Arias et al., 2010a). Bupropion inhibits the specific binding of [3H]imipramine to the α7 nicotinic acetylcholine receptor in a concentration-dependent fashion (Fig. 6). The calculated binding affinity of bupropion for the [3H]imipramine binding site(s) at α7 nicotinic acetylcholine receptors in the resting state (Ki ¼66 μM; Table 1) is relatively lower than that determined at other nicotinic acetylcholine receptor subtypes (Arias et al., 2009, 2012; reviewed in Arias (2009)). The calculated nH value for bupropion is close to unity, indicating that this antidepressant inhibits [3H]imipramine binding to α7 nicotinic acetylcholine receptors in a non-cooperative manner, and suggesting that bupropion binds to the luminal [3H]imipramine site by a steric mechanism. In turn, this result supports the idea that bupropion interacts within the α7 nicotinic acetylcholine receptor ion channel, as it was previously showed for muscle nicotinic acetylcholine receptors (Arias et al., 2009, 2012; reviewed in Arias (2009)). Fig. 3. Bupropion decreases acetylcholine-induced currents in dorsal raphe nucleus neurons. (A) Representative acetylcholine (ACh) induced currents from eight separate experiments, recorded at 4-min intervals. (B) The amplitude of acetylcholine-induced currents as a function of time, before, during, and after the application of bupropion. Ion currents were elicited by a 5-psi, 500-ms puff of 1 mM acetylcholine. The timing of bupropion application is indicated by the thick black lines.

3.5. Molecular docking of bupropion enantiomers to the α7 nicotinic acetylcholine receptor Since bupropion is 95% protonated at physiological pH [pKa ¼ 8.7 70.4; calculated by the ACD/ADME Suite software (Advanced

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Fig. 4. Bupropion decreases choline-induced currents in hippocampal CA1 interneurons. (A, C) Representative choline (Ch) induced currents recorded at 5-min intervals from six and three separate experiments, respectively. (B, D) The amplitude of choline-induced currents is plotted as a function of time, before, during, and after the application of bupropion. (A, B) α-bungarotoxin was added to bath perfusion after recovery of bupropion-inhibited choline-induced currents (n¼ 3). Ion currents were elicited by a 5-psi, 500-ms puff of 10 mM choline. The timing of bupropion and α-bungarotoxin application is indicated by the thick black lines.

Chemistry Development, Inc., Toronto, Canada)], the molecular docking is shown for the protonated state. The docking results indicate that both bupropion enantiomers interact with two sites:

a site close to the extracellular mouth of the receptor, and another in the middle of the channel lumen (Fig. 7). In the former site, (S)(þ)-bupropion interacts predominantly by van der Waals contacts

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Fig. 5. Bupropion inhibits acetylcholine- and choline-induced currents in dorsal raphe nucleus (DRN) and hippocampal (Hipp) neurons. The height of the columns (IExp/ICtrl) represents the minimal amplitude of acetylcholine (ACh) or choline (Ch) induced currents by the action of bupropion normalized to those in the absence of bupropion considered as the unity (IBP/ICtrl, light gray columns); and the maximal recovery of the acetylcholine- or choline-induced currents after washing out bupropion compared to those in the absence of bupropion (IRec/ICtrl, dark gray columns). Data are the mean 7 S.E.M. The numeral in parenthesis indicates the number of experiments; the Student's t-test, np o 0.05.

Fig. 6. Bupropion-induced inhibition of [3H]imipramine binding to α7 nicotinic acetylcholine receptors. Nicotinic acetylcholine receptor membranes (1.5 mg/ml) were equilibrated (2 h) with 14 nM [3H]imipramine, and increasing concentrations of bupropion. Nonspecific binding was determined at 100 mM imipramine. The plot is the combination of two separate experiments, each one in triplicate, where the error bars correspond to the S.D. The IC50 and nH values were obtained by nonlinear least-squares fit and the Ki value was calculated using Eq. (1) and summarized in Table 1.

with residues at position 200 (i.e., Glu259) (outer ring), 170 (i.e., Leu256) (nonpolar ring), 140 (i.e., Phe253), and 130 (i.e., Val252) (valine ring). In the latter site, (S)-(þ )-bupropion interacts by van der Waals contacts with Phe253, Val252, and Leu248 at the leucine ring (position 90 ) (Fig. 7B). In addition, the ligand is stabilized by three hydrogen bonds, two formed between its amino moiety and the hydroxyl groups of two Ser249 (position 100 ) and a third one formed between its oxygen and the same hydroxyl groups. The (R)-(  )-enantiomer only interacts with the site located in the middle of the ion channel (data not shown).

4. Discussion Since we do not have a clear picture of how bupropion produces its pharmacological activity at neuronal level, the inhibitory activity of this antidepressant was compared between

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heterologous cells and hippocampal interneurons and dorsal raphe nucleus neurons. Additional radioligand binding and molecular docking experiments were performed to determine its binding site location. Our Ca2 þ influx results (Table 1), and the fact that the bupropion-inhibited choline-induced current in hippocampal interneurons and dorsal raphe nucleus neurons is sensitive to the selective antagonists methyllycaconitine and α-bungarotoxin indicate that bupropion inhibits, at least partially, α7 nicotinic acetylcholine receptors. These results are in agreement with previous studies (Slemmer et al., 2000; reviewed in Arias (2009)), indicating that, although with low potency, bupropion inhibits α7 nicotinic acetylcholine receptors at clinically relevant concentrations (10–20 μM) (Fig. 4C and D). This evidence coincides with the attained brain concentration of bupropion ( 20 mM; reviewed in Arias (2009)). Interestingly, the fact that ketamine produces rapid and sustained antidepressant effects in patients with severe and treatment-resistant depression (Cornwell et al., 2012), and that its metabolites strongly inhibit α7 nicotinic acetylcholine receptors (Moaddel et al., 2013), supports the possibility that α7 nicotinic acetylcholine receptors are target for structurally different antidepressants. The docking results suggest that both bupropion enantiomers bind to the α7 nicotinic acetylcholine receptor channel, more specifically between the valine (position 130 ) and the leucine (position 90 ) rings (Fig. 7), concurrent with that observed in other nicotinic acetylcholine receptor subtypes (Arias et al., 2009, 2012). Interestingly, the bupropion site overlaps that for imipramine (Arias et al., 2009, 2010a, 2010b, 2012), which is in agreement with the radioligand competition results, indicating that bupropion binds to the [3H]imipramine binding site at the α7 nicotinic acetylcholine receptor (Table 1). Some of the dorsal raphe nucleus neurons, containing pre- and post-synaptic functional α4β2 and α7 nicotinic acetylcholine receptors, project to the nucleus accumbens and hippocampus, impacting serotonin release (Aznar et al., 2005; Chang et al., 2011). Furthermore, during nicotine withdrawal, a persistent serotonergic tone is observed increasing anxiety (Cheeta et al., 2001). This serotonin increase, in turn, augments dopaminergic transmission (Benloucif et al., 1993). In this regard, bupropion may increase dopamine levels through inhibition of nicotinic acetylcholine receptors in dorsal raphe nucleus neurons by modulating serotonin release. All these neurochemical actions could explain, at least partially, the clinical use of bupropion for depression and smoking cessation. We also found that bupropion inhibits α7-containing nicotinic acetylcholine receptors expressed in GABAergic interneurons from the CA1 hippocampus area. The activation of α7 nicotinic acetylcholine receptors expressed in interneurons can produce inhibition or disinhibition of pyramidal GABAergic neurons (Ji and Dani, 2000; Banerjee et al., 2012). Consequently, the observed bupropion-induced inhibition might indirectly stimulate (i.e., disinhibit) pyramidal neurons. In this regard, we speculate that this mechanism can be part of the antidepressant activity elicited by bupropion. The results that reduced GABA levels in plasma and cerebrospinal fluid are pronounced in treatment-resistant depression patients (Luscher et al., 2011), and that reduced GABAergic activity can induce diverse mood disorders (Earnheart et al., 2007) may support our conjecture. We conclude that bupropion, at concentrations that could be reached in the brain during clinical administration, inhibits agonist-induced currents mediated by α7 nicotinic acetylcholine receptors in dorsal raphe nucleus neurons as well as in interneurons from the stratum radiatum of the hippocampal CA1 area, and may modulate serotonin signaling and GABA synaptic transmission, respectively. This study may help to understand the

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Fig. 7. Molecular docking of protonated (S)-(þ )-bupropion within the α7 nicotinic acetylcholine receptor ion channel. (A) Side view of the two binding modes of (S)( þ)-bupropion within the ion channel: one closer to the extracellular mouth (gray) and another located in the middle of the ion channel (black). Residues from each ring are shown explicitly in stick mode. (B) Detailed view of the two binding sites. (S)-( þ)-bupropion interacts with a site located closer to the extracellular mouth, predominantly by van der Waals contacts with residues at position 200 (i.e., Glu259) (outer ring), 170 (i.e., Leu256) (nonpolar ring), 140 (i.e., Phe253), and 130 (i.e., Val252) (valine ring). (S)( þ)-bupropion also interacts with a site located in the middle of the ion channel, by van der Waals contacts with Phe253, Val252, and Leu248 at the leucine (position 90 ) ring. In addition, the ligand is stabilized by three hydrogen bonds (see black arrows), two formed between its amino moiety and the hydroxyl groups of two Ser249 (position 100 ), and a third one formed between its oxygen and the same hydroxyl groups. All nonpolar hydrogens atoms are hidden. The ligands are rendered in ball (A) or stick (B) mode. Subunits are shown in secondary structure mode. One subunit was removed for clarity. All other segments (A) or the M2 transmembrane helices (B) are shown in light gray.

mechanisms of actions of bupropion at neuronal and molecular levels related with its therapeutic actions on depression and for smoking cessation.

from DGAPA, UNAM. We are grateful to Martín García Servín for his assistance in taking care of the rats.

References Acknowledgments This work was supported by a Grant from Dirección General de Asuntos del Personal Académico (DGAPA), UNAM, IN201313 (to J.G.C.), and by the TEAM research subsidy from the Foundation for Polish Science (TEAM 2009/4-5) (to K.J.). E.V.G. was a postdoctoral fellow

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