Interaction of Bupropion with Muscle-Type Nicotinic Acetylcholine Receptors in Different Conformational States\xe2\x80\xa0
To characterize the binding sites and the mechanisms of inhibition of bupropion on muscle-type nicotinic acetylcholine receptors (AChRs), structural and functional approaches were used. The results established that bupropion: (a) inhibits epibatidine-induced Ca2+ influx in embryonic muscle AChRs, (b) inhibits adult muscle AChR macroscopic currents in the resting/activatable state with ~100-fold higher potency compared to that in the open state, (c) increases desensitization rate of adult muscle AChRs from the open state and impairs channel opening from the resting state, (d) inhibits [3H]TCP and [3H]imipramine binding to the desensitized/carbamylcholine-bound Torpedo AChR with higher affinity compared to the resting/\xce\xb1-bungarotoxin-bound AChR, (e) binds to the Torpedo AChR in either state mainly by an entropy\xe2\x80\x93driven process, and (f) interacts with a binding domain located between the serine (position 6\xe2\x80\x99) and valine (position 13\xe2\x80\x99) rings, by a network of van der Waals, hydrogen bond, and polar interactions. Collectively our data indicate that bupropion first binds to the resting AChR, decreasing the probability of ion channel opening. The remnant fraction of open ion channels is subsequently decreased by accelerating the desensitization process. Bupropion interacts with a luminal binding domain shared with PCP that is located between the serine and valine rings, and this interaction is mediated mainly by an entropy-driven process.
<!>Materials<!>Preparation of AChR native membranes<!>Preparation of the cellular membrane affinity chromatography (CMAC) column and chromatographic system<!>Cells expressing different muscle-type AChRs<!>Ca2+ influx measurements in TE671 cells<!>Patch-clamp recordings in HEK293 cells expressing mouse adult muscle AChRs<!>Bupropion-induced inhibition of [3H]TCP and [3H]imipramine binding to Torpedo AChRs in different conformational states<!>Determination of the binding kinetics for bupropion using the CMAC-Torpedo AChR column<!>Thermodynamic parameters of Torpedo AChR-bupropion interactions<!>Docking and molecular dynamics of R- and S-bupropion in muscle-type AChR ion channels<!>Bupropion inhibits epibatidine-mediated Ca2+ influx in TE671 cells<!>Bupropion inhibits macroscopic AChR currents from resting and open states by different mechanisms<!>Bupropion-induced inhibition of [3H]TCP or [3H]imipramine binding to AChRs in different conformational states<!>Binding kinetic parameters for bupropion determined by column-immobilized Torpedo AChRs<!>Thermodynamic parameters for bupropion interacting with Torpedo AChRs<!>Molecular dynamics of bupropion interacting with Torpedo and mouse muscle AChR ion channels<!>DISCUSSION<!>Molecular mechanisms of inhibition mediated by bupropion<!>Characterization of the bupropion binding site<!>
<p>Bupropion is an antidepressant that is also being marketed as an aid to smoking cessation (1,2). The proposed mechanism of action for bupropion is that this drug inhibits the catecholamine reuptake in presynaptic neurons, modulating the concentrations of dopamine and norepinephrine in the synaptic cleft. However, the affinity of bupropion for the neurotransmitter transporter is only moderate, and there is not clear-cut evidence explaining the dual antidepressant and anti-nicotinic modes of action elicited by bupropion.</p><p>In addition to the current clinical uses of bupropion, this drug behaves pharmacologically as a noncompetitive antagonist (NCA)1 on several nicotinic acetylcholine receptors (AChRs) (3,4) [reviewed in (5)]. AChRs are the paradigm of the Cys-loop ligand-gated ion channel superfamily. This genetically-linked superfamily includes types A and C γ-aminobutyric acid, type 3 5-hydroxytryptamine (serotonin), and glycine receptors [reviewed in (6–9)]. The malfunctioning of these receptors has been considered as the origin of several neurological disorders [reviewed in (8,10)]. For example, the evidence showing a higher rate of smokers in depressed patients than in the general population supports a possible role of AChRs in depression mechanisms [reviewed in (11)]. In this regard, it has been reported that hypercholinergic neurotransmission, which is associated with depressed mood states, may be mediated through excessive neuronal AChR activation and that the therapeutic actions of many antidepressants may be mediated in part through inhibition of one or more AChRs [reviewed in (12)].</p><p>Previous studies have shown that tricyclic antidepressants (TCAs) behave as NCAs of both muscle-type (13) and neuronal-type AChRs (14). Their noncompetitive inhibitory mechanisms on different members of the Cys-loop ligand-gated ion channel superfamily have been elucidated (13,15) [reviewed in (5)]. And more recently, the TCA binding site in the desensitized Torpedo AChR has been characterized by [3H]2-azidoimipramine photolabeling and molecular dynamics (16). Nevertheless, the mechanisms of AChR inhibition elicited by bupropion are poorly understood. In this regard, we want to determine the interaction of bupropion with muscle-type AChRs in different conformational states. To this end, we will use binding and functional approaches including radioligand competition binding assays using well known NCAs such as [piperidyl-3, 4-3H(N)]-N-(1-(2 thienyl)cyclohexyl)-3,4-piperidine ([3H]TCP) and [3H]imipramine, Ca2+ influx and macroscopic current recordings, thermodynamic and kinetic measurements using column-immobilized Torpedo AChRs, and molecular docking and dynamics studies. These studies will aid in further drug development.</p><!><p>[Piperidyl-3, 4-3H(N)]-(N-(1-(2 thienyl)cyclohexyl)-3,4-piperidine) ([3H]TCP; 42–45 Ci/mmol), and [3H]imipramine hydrochloride (47.5 Ci/mmol) were obtained from PerkinElmer Life Sciences Products, Inc. (Boston, MA, USA), and stored in ethanol at −20°C. Carbamylcholine chloride (CCh), suberyldicholine dichloride, acetylcholine chloride (ACh), tetracaine hydrochloride, (±)-epibatidine, (±)-bupropion hydrochloride, sodium cholate, cholesterol, phosphatidylserine, sphingomyelin, phosphatidic acid, pepstatin, leupeptin, aprotinin, calpain I, calpain II, benzamidine, paramethylsulfonylfluoride (PMSF), and polyethylenimine were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Phencyclidine hydrochloride (PCP) was obtained through the National Institute on Drug Abuse (NIDA) (NIH, Baltimore, USA). α-Bungarotoxin (α-BTx) was obtained from Invitrogen Co. (Carlsbad, CA, USA). [1-(Dimethylamino) naphtalene-5-sulfonamido]ethyltrimethylammonium perchlorate (dansyltrimethylamine) was purchased from Pierce Chemical Co. (Rockford, IL, USA). Salts were of analytical grade.</p><!><p>AChR native membranes were prepared from frozen Torpedo californica electric organs obtained from Aquatic Research Consultants (San Pedro, CA, USA) by differential and sucrose density gradient centrifugation, as described previously (17). Total AChR membrane protein was determined by using the bicinchoninic acid protein assay (Pierce Chemical Co.). Specific activities of these membrane preparations were determined by the decrease in dansyltrimethylamine (6.6 µM) fluorescence produced by the titration of suberyldicholine into receptor suspensions (0.3 mg/mL) in the presence of 100 µM PCP and ranged from 1.0 to 1.2 nmol of suberyldicholine binding sites/mg total protein (0.5–0.6 nmol AChR/mg protein). The AChR membrane preparations were stored at −80°C in 12 % sucrose.</p><!><p>The CMAC-Torpedo AChR column was prepared by the immobilization of solubilized Torpedo membranes following a previously described protocol (18). Torpedo membranes were first homogenized in buffer A (50 mM Tris-HCl buffer, pH 7.4, containing 1 mM pepstatin A, 0.02 mM leupeptin, 1 mM aprotinin, 1 mM calpain I, 1 mM calpain II, 1 mM benzamidine, 2 mM MgCl2, 3 mM CaCl2, 5 mM KCl, and 0.2 mM PMSF), and subsequently solubilized in buffer A containing 2% (w/v) sodium cholate and 1 mL glycerol, in the presence of 100 nM cholesterol, 60 µM phosphatidylserine, 20 µM sphingomyelin, and 60 µM phosphatidic. Then, 200 mg of the Immobilized Artificial Monolayer (IAM) liquid chromatographic stationary phase (ID = 12 µm, 300 Å pore; Regis Chemical Co.) was suspended in the supernatant, and the mixture was rotated at room temperature (RT) for 1 h. The suspension was dialyzed against 1 L of 50 mM Tris-saline buffer, pH 7.4, containing 5 mM EDTA, 100 mM NaCl, 0.1 mM CaCl2 and 0.1 mM PMSF, for 1 day. The suspension was then centrifuged at 700 × g at 4°C and the pellet (Torpedo-IAM) was washed three times with 10 mM ammonium acetate buffer, pH 7.4. The stationary phase was packed into a HR 5/2 column (GE Healthcare, Piscataway, NJ) to yield a 150 mm × 5 mm (ID) chromatographic bed, the CMAC-Torpedo AChR column. Finally, the CMAC-Torpedo AChR column was attached to the chromatographic system Series 1100 Liquid Chromatography/Mass Selective Detector (Agilent Technologies, Palo Alto, CA, USA) equipped with a vacuum de-gasser (G 1322 A), a binary pump (1312 A), an autosampler (G1313 A) with a 20 µL injection loop, a mass selective detector (G1946 B) supplied with atmospheric pressure ionization electrospray and an on-line nitrogen generation system (Whatman, Haverhill, MA, USA). The chromatographic system was interfaced to a 250 MHz Kayak XA computer (Hewlett-Packard, Palo Alto, CA, USA) running ChemStation software (Rev B.10.00, Hewlett-Packard).</p><p>A 10 µL sample of 10 µM bupropion was injected onto the CMAC-Torpedo AChR column, and bupropion was monitored in the positive ion mode using single ion monitoring at m/z = 240.5 [MW + H]+ ion with the capillary voltage at 3000 V, the nebulizer pressure at 35 psi, and the drying gas flow at 11 L/min at a temperature of 350°C.</p><!><p>Human embryonic kidney (HEK293) cells were transfected with mouse α1, β1, δ, and ε cDNA subunits using calcium phosphate precipitation at a subunit ratio of 2:1:1:1 for α1:β1:δ:ε, respectively, essentially as previously described (19,20). For transfections, cells at 40–50 % confluence were incubated for 8–12 h at 37°C with the calcium phosphate precipitate containing the cDNAs in Dulbecco's Modified Eagle Medium (DMEM) plus 10% (v/v) fetal bovine serum (FBS). A plasmid encoding green fluorescent protein (pGreen lantern was also included to allow identification of transfected cells under fluorescence optics. Cells were used for outside-out patch measurements 1 or 2 days after transfection.</p><p>The TE671 cell line is a human rhabdomyosarcoma cell line (obtained from American Type Culture Collection, USA) that endogenously expresses the human fetal muscle AChR (i.e., α1β1γδ). TE671 cells were cultured in a 1:1 mixture of DMEM and Ham's F-12 Nutrient Mixture (Seromed, Biochrom, Berlin, Germany), supplemented with 10% (v/v) FBS. DMEM/Ham's F-12 contains 1.2 g/L NaHCO3, 3.2 g/L sucrose, and stable glutamine, as previously described (21). The cells were incubated at 37°C, 5% CO2 and 95% relative humidity. For passaging, the cells were detached from the cell culture flask by washing with phosphate-buffered saline and brief incubation (3–5 min) with trypsine (0.5 mg/mL)/EDTA (0.2 mg/mL). The cells were passaged every 3 days.</p><!><p>Ca2+ influx was determined as previously described (21). Briefly, 5 × 104 TE671 cells per well were seeded 72 h prior to the experiment on black 96-well plates (Costar, New York, USA) and incubated at 37°C in a humidified atmosphere (5% CO2/95% air). 16–24 h before the experiment, the medium was changed to 1% BSA in HEPES-buffered salt solution (HBSS) (130 mM NaCl, 5.4 mM KCl, 2 mM CaCl2, 0.8 mM MgSO4, 0.9 mM NaH2PO4, 25 mM glucose, 20 mM HEPES, pH 7.4). On the day of the experiment, the medium was removed by flicking the plates and replaced with 100 µL HBSS/1%BSA containing 2 mM Fluo-4 (Molecular Probes, Eugene, Oregon, USA) in the presence of 2.5 mM probenecid (Sigma, Buchs, Switzerland). The cells were incubated at 37°C in a humidified atmosphere (5% CO2/95% air) for 1 h. Plates were flicked to remove excess of Fluo-4, washed twice with HBSS/1% bovine serum albumin (BSA), and finally refilled with 100 µL of HBSS containing different concentrations of bupropion, and pre-incubated for 5 min. Plates were then placed in the cell plate stage of the fluorescent imaging plate reader (FLIPR) (Molecular Devices, Sunnyvale, CA, USA). A baseline consisting of 5 measurements of 0.4 sec each was recorded. Epibatidine (1 µM) was then added from the agonist plate (placed in the agonist plate stage of the FLIPR) to the cell plate using the FLIPR 96-tip pipettor simultaneously to fluorescence recordings for a total length of 3 min. The laser excitation and emission wavelengths are 488 and 510 nm, at 1 W, and a CCD camera opening of 0.4 sec. In parallel experiments, 1 µM epibatidine with different concentrations of bupropion were co-injected by several seconds.</p><!><p>For outside-out recordings, the pipette solution contained 140 mM KCl, 5 mM EGTA, 5 mM MgCl2 and 10 mM HEPES, pH 7.3. Extracellular solution (ECS) contained 150 mM NaCl, 5.6 mM KCl, 1.8 mM CaCl2, 5 mM MgCl2 and 10 mM HEPES, pH 7.3. The patch was excised in this configuration and moved into position at outflow of a perfusion system. The perfusion system allows for a rapid (0.1–1 ms) exchange of the solution bathing patch. A series of applications of 300 µM ACh were applied to the patch, as described before (13,22). The duration of agonist pulse was 200 ms. We recorded the responses following different protocols: +/− protocol: the patch was exposed 2 min to bath solution containing different concentrations of bupropion before the application of the agonist-containing solution; −/+ protocol: pulse of agonist solution containing bupropion without preincubation; +/+ protocol: the patch was exposed 2 min to bath solution containing different concentrations of bupropion before the application of the agonist solution containing bupropion. All currents were referred to those recorded in the same cell in the absence of bupropion (−/− protocol). In these experiments, control currents were also recorded after each protocol to assess recovery of the original peak current.</p><p>Macroscopic currents were filtered at 5 kHz, digitized at 20 kHz and stored on the hard disk. Data analysis was performed using IgorPro software (WaveMetrics Inc., Lake Oswego, OR). The ensemble mean current was calculated for 5–10 individual current traces. Mean currents were fitted by a single exponential function: (1)I(t)=I0exp(−t/τd)+I∞

where I0 and I∞ are the peak and the steady state current values, respectively, and τd is the decay time constant that measures the current decay due to desensitization. Current records were aligned with each other at the point where the currents had risen to 50% of its maximum level. Peak currents correspond to the value obtained by extrapolation of the decay current to this point. The ratio between the peak current or the desensitization rate in the presence (Ibupropion) and absence of the drug (Icont) was plotted as a function of bupropion concentration and the curve was fitted to the Hill equation: (2)Ibupropion/Icont=IC50nH/IC50nH+[bupropion]nH)

 where IC50 is the concentration of bupropion that produces 50% inhibition of the peak currents.</p><!><p>We studied the influence of bupropion on either [3H]TCP or [3H]imipramine maximal binding to the Torpedo AChR in the resting/α-bungarotoxin (α-BTx)-bound and desensitized/CCh-bound states. In this regard, AChR native membranes (0.3 µM) were suspended in binding saline (BS) buffer (50 mM Tris–HCl, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) with 7 nM [3H]TCP or 13 nM in the presence of 1 mM CCh (desensitized/CCh-bound state), or with 7 nM [3H]TCP or 44 nM [3H]imipramine in the presence of 2 µM α-BTx (resting/α-BTx-bound state), and preincubated for 30 min at RT. α-Bungarotoxin is a competitive antagonist that maintains the AChR in the resting (closed) state (23). Nonspecific binding was determined in the presence of 50 µM PCP (desensitized/CCh-bound state experiments) or 100 µM PCP (or alternatively 200 µM tetracaine) (resting/α-BTx-bound state experiments), as was used previously (24,25). The total volume was divided into aliquots, and increasing concentrations of bupropion were added to each tube and incubated for 2 h at RT. AChR-bound radioligand 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).</p><p>The concentration–response data were curve-fitted by nonlinear least squares analysis using the Prism software. The corresponding IC50 values were calculated using the following equation: (3)θ=1/[1+([L]/IC50)nH]
 where θ is the fractional amount of the radioligand bound in the presence of inhibitor at a concentration [L] compared to the amount of the radioligand bound in the absence of inhibitor (total binding). IC50 is the inhibitor concentration at which θ = 0.5 (50% bound), and nH is the Hill coefficient. The nH values were summarized in Table 2.</p><p>Taking into account that the AChR presents one binding site for TCP (25,26) and imipramine (13,16), the observed IC50 values from the competition experiments described above were transformed into inhibition constant (Ki) values using the Cheng–Prusoff relationship (27):(4)Ki=IC50/{1+([NCA]/KdNCA)}

 where [NCA] is the initial concentration of [3H]TCP or [3H]imipramine, and KdNCA is the dissociation constant for [3H]TCP [0.83 µM in the resting state (25) and 0.25 µM in the desensitized state (26)], and for [3H]imipramine in the desensitized and resting states [0.8 and 3.8 µM, respectively; (16)]. In addition, the free energy change (ΔG) of bupropion interacting with the Torpedo AChR was determined as [reviewed in (7)]: (5)ΔG=RTlnKi

 where R is the gas constant (8.314 J mol−1 K−1), and T is the temperature in Kelvin. The calculated Ki and ΔG values for bupropion were summarized in Table 2.</p><!><p>In the non-linear chromatography approach, concentration-dependent asymmetric chromatographic traces are observed due to slow adsorption/desorption rates. The mathematical approach used in this study to resolve these non-linear conditions was the Impulse Input Solution (28). The chromatographic data were analyzed using PeakFit v4.11 for Windows Software (SPSS Inc., Chicago, IL) following a previously reported protocol (29). The details of this approach and its application to the determination of the binding kinetics of non-competitive inhibitors to neuronal AChRs were presented earlier (29,30). Briefly, the resultant peaks were fitted to the Impulse Input Solution model by adjusting four variables, namely a0−a3. The a2 variable was directly used for the calculation of the dissociation rate constant (koff) according to this equation: (6)koff=1/(a2t0)

 where the dead time of the column, t0, is determined as the time required for the elution of water. The a3 value was used to calculate the association constant (Ka) for the formation of the ligand-receptor complex in equilibrium using this relationship: (7)Ka=a3/[bupropion]
 where [bupropion] is the concentration of bupropion. Both Ka and koff values can be used to further calculate the association rate constant, kon (kon = Ka · koff).</p><!><p>Chromatographic elutions of bupropion from the CMAC-Torpedo AChR column were carried out at the following temperatures: 10, 12, 16, 20, and 25°C. The elution procedure was conducted using a mobile phase composed of 10 mM ammonium acetate buffer (pH 7.4):methanol (85:15, v/v) delivered at a flow rate of 0.2 mL/min. For the first set of experiments, the CMAC-Torpedo AChR column was equilibrated by passing 125 nM α-BTx to the mobile phase through the column for 1 h. In addition, a new column was treated with a 10 µL injection of 1 mM epibatidine, and the temperature studies were repeated. All studies were carried out in triplicate.</p><p>For the temperature-dependence studies, van't Hoff plots were constructed according to the following linear regression equation [reviewed in (7)]:
(8)lnKa=(ΔS°/R)−(ΔH°/R)(1/T)

 where ΔS° and ΔH° are the standard entropy change and standard enthalpy change, respectively. Finally, the free energy change at 293K (ΔG20) was calculated using the Gibbs-Helmholtz equation [reviewed in (7)]: (9)ΔG20=ΔH°−TΔS°
</p><!><p>Amino acid sequences in the M2 transmembrane segments of the AChR ion channel are highly conserved between different species and subunits. However, the absolute numbering of amino acid residues varies greatly between subunits, thus, the residues in M2 of AChR subunits are referred to here using the prime nomenclature (1' to 20'), corresponding to residues Met243 to Glu262 in the Torpedo AChR α-subunit. As binding targets for modeling we used a structural model of the pore region of AChR based on the cryo-electron microscopy structure of the Torpedo AChR determined at ~4 Å resolution (PDB ID 2BG9) (32,33). A model of the mouse muscle AChR subtype was constructed using homology/comparative modeling method with Torpedo 2BG9 model used as a template.</p><p>Computational simulations were performed using the same protocol as recently reported (16). In the first step, R- and S-bupropion molecules were prepared using HyperChem 6.0 (HyperCube Inc., Gainesville, FL). Sketched molecules were optimized using the semiempirical method AM1 (Polak-Ribiere algorithm to a gradient lower than 0.1 kcal/Å/mol) and then transferred for the subsequent step of ligand docking. The Molegro Virtual Docker (MVD 2008.2.4.0 Molegro ApS Aarhus, Denmark) was used for docking simulations of flexible ligands into the rigid target AChR model. In this step the complete structures of target receptors were used. The docking space was limited and centered on the middle of the ion channel and extended enough to ensure covering of the whole channel domain for sampling simulations (docking space was defined as a sphere of 21 Å in diameter). The actual docking simulations were performed using the following settings: numbers of runs = 100; maximal number of interactions = 10,000; maximal number of poses = 10), and the pose representing the lowest value of the scoring function (MolDockScore) for each ligand was selected for further simulations. In the last step, molecular dynamics were performed using the Yasara 6.10.18 package (Yasara Biosciences, Graz, Austria). Complexes of Torpedo or mouse muscle AChR ion channel models with bupropion isomers were edited to provide coordinates for the membrane domain only. For each subunit, M1-M4 transmembrane helices plus connecting loops were left in the system, all other parts of the protein were removed. These models were further inserted into periodic boxes (88Å×88Å×68Å), and solvated with water molecules using the Yasara default algorithm. Although fixing constrains for backbone atoms were assigned, all side-chain atoms were left free to move during the simulations. In all further simulations the AMBER99 force field for both protein and ligand structures was used (Yasara BioSciences) with the cutoff 7.86 Å and particle-mesh Ewald longrange function for electrostatic interactions. The initial complexes were pre-optimized with steepest descent method followed by 500 steps of simulated annealing. Finally, the actual 1.0 ns molecular dynamics was performed using the following parameters: temperature = 298 K; multiple timesteps = 1 fs for intramolecular and 2 fs for intermolecular forces; PressureControl – waterprobe (0.99 g/mL) ensemble. Snapshots of the simulations were saved every 5 ps.</p><p>The trajectories of molecular dynamics simulations were characterized by calculation of the emulated binding energy defined here as the difference between the energy of the complete complex system and a summation of energy of the ligand and the energy of the hydrated protein alone. All these calculations were performed with the same AMBER force field settings.</p><!><p>The change in fluorescence in TE671 cells after epibatidine stimulation is caused by an increase in intracellular Ca2+. The major source of Ca2+ is the extracellular buffer. Omission of Ca2+ from the extracellular solution does not produce the signal elicited by agonist stimulation (21). Epibatidine-induced α1β1γδ AChR activation is blocked by pre-incubation (~5 min) with bupropion with an inhibitory potency (IC50) of 20.5 ± 3.9 µM (Table 1). Although the receptors are initially in the resting/activatable state (21) during pre-incubation with bupropion, the subsequent addition of the agonist will make them open and desensitize, and therefore the final effects of bupropion will be a combination of the effects at each state. In addition, the co-injection of epibatidine and bupropion produced an IC50 = 10.5 ± 2.1 µM (Table 1). This value is identical to that obtained by 86Rb+ efflux experiments using the same human muscle AChR (4). Considering that this protocol is performed in the time regime of seconds, the observed blocking effect will be a combination of the effects elicited by bupropion on the open and desensitized ion channels. The fact that the nH values are close to unity (Table 1) indicates that both bupropion blocking processes are produced in a non-cooperative manner. In turn, this suggests that there is only one binding site for bupropion on each conformational state or that there are binding sites with similar affinity.</p><!><p>To dissect the effects of bupropion on different conformational states, we studied its effects on outside-out patches rapidly perfused with 300 µM ACh. To determine the state-dependence of bupropion action we used different perfusion protocols (see Experimental Procedures). In control data, the current reaches the peak after 0.1–1 ms and then decays with a time constant (τd) of about 15–30 ms due to desensitization (Fig. 1). This τd corresponds to a desensitization rate of 37 ± 8 s−1, given that at saturating agonist concentrations 1/τd is a good estimation of the desensitized rate. When bupropion is present only in the ACh activation solution (−/+ protocol), a concentration-dependent increase in the decay rate (1/τd) is observed (Fig. 1A). At 200 µM bupropion, the decay rate increases to 294 ± 10 s−1. A plot of the normalized decay rate as a function of bupropion concentrations yields an IC50 value of 40.1 ± 5.1 µM and a nH of 0.99 ± 0.11 (Table 1). No significant changes on the peak currents are observed under this protocol application. At all bupropion concentrations, decays are well fitted by a single exponential function. This observation discards a fast open-channel block mechanism, since a two-component decay time course should have been observed instead (34,35). However, the observed monoexponential decay might also result from channel block with a slow unblocking rate, as described before for TCAs acting on muscle AChRs (13). Distinguishing between slow channel block and increased desensitization is not possible from these experiments.</p><p>To determine if bupropion affects AChR activation when acting through the resting/activatable state, we exposed the patch to bupropion for 2 min, and then activated the receptors with an ACh solution free of the drug (+/− protocol). Pre-application of the drug decreases the peak current in a dose-dependent manner. No significantly changes in the decay rate were observed in the presence of different concentrations of bupropion with respect to control currents. After washing out the drug with bath solution the peak current can be completely recovered (95 ± 2 %). Thus, the effects of bupropion are very different when it acts on the open state, where it increases the decay rate, or on the resting/activatable state, where it decreases the peak current. The IC50 calculated for the inhibition of the peak current is 0.40 ± 0.04 µM and the nH is 1.90 ± 0.30 (Table 1). Thus, the inhibitory potency is much higher when the drug acts from the resting/activatable state than from the open state. The decrease in the peak current produced by bupropion acting at the resting/activatable state may arise from block of resting channels or from an increase of the desensitization of resting channels.</p><p>To determine if the effects of bupropion on both states are additive, the patch was continuously exposed to the drug (+/+ protocol). For this experiment we used the drug concentrations corresponding to the IC50 values for the actions at each state. Thus, the patch was pre-incubated with 0.4 µM bupropion and then activated with a solution containing 300 µM ACh plus 40 µM bupropion. The decrease of the peak current measured in the +/+ protocol is quantitatively similar to that measured using the +/− protocol (48.4 ± 3.4 %). Analogously, the decrease of the decay rate determined under the +/+ protocol is similar to that achieved under the −/+ protocol (49.5 ± 2.8 %). In conclusion, the continuous exposure of the receptor to the drug produces the same inhibition as the sum of the individual effects at the resting/activatable and open states. Full recovery of the peak current and of the decay time constant (93 ± 6 %) are achieved after washing the drug, indicating that the inhibition is reversible (Fig. 1C).</p><p>In conclusion, macroscopic current recordings show that bupropion inhibits AChR by two mechanisms mediated by binding to specific conformational states. More specifically, when bupropion binds to the resting/activatable state, channel opening is impaired, whereas when it binds to the open state, desensitization rate from the open state is increased or alternatively, slow channel block occurs.</p><!><p>We have previously characterized the binding sites for PCP/TCP (24,25,36) and imipramine (13,16) in Torpedo AChRs. Thus, we want to determine the location of the bupropion binding site relative to these NCA loci. To this end, we determined the influence of bupropion on either [3H]TCP (Fig. 2) or [3H]imipramine (Fig. 3) maximal binding to Torpedo AChRs in different conformational states. Bupropion inhibits ~100% the specific binding of [3H]TCP (Fig. 2) and [3H]imipramine (Fig. 3) to either the desensitized/CCh-bound or resting/α-BTx-bound state. The obtained Kis (Table 2) indicate that bupropion binds to the Torpedo AChR in either the resting/α-BTx-bound or desensitized/CCh-bound state with practically the same affinity as TCAs (13,16). Comparing the Ki values in different conformational states, we can indicate that bupropion binds to the PCP and TCA binding sites with ~2-fold higher affinity in the desensitized/CCh-bound compared to that in the resting/α-BTx-bound state. The same binding affinity difference (~1.5-fold) was obtained by using frontal affinity chromatography (data not shown), confirming the radioligand binding results.</p><p>The fact that the calculated nH values are close to unity (Table 2) indicates that bupropion inhibits [3H]TCP and [3H]imipramine binding in a non-cooperative manner. These data suggest that bupropion interacts with just one binding site, and that bupropion probably inhibits [3H]TCP and [3H]imipramine binding in a steric fashion. Schild-plot analyses of imiprimine-induced [3H]TCP binding inhibition support this mechanism of competition (37).</p><!><p>Figure 4A shows the expected asymmetric traces for bupropion when it is chromatographed on the CMAC-Torpedo AChR column. The data were used to determine the koff and Ka values according to equations 6 and 7, respectively, and thus, to further calculate the kon values for bupropion when it binds to the Torpedo AChR in different conformational states. These dynamic parameters might help to explain the observed ~2-fold higher affinity of bupropion for the desensitized AChR compared to the resting AChR (see Table 2). However, the results indicate that the dissociation rate constant (koff) of bupropion was practically the same when the column was exposed to either epibatidine (the AChR is mainly in the desensitized state) or α-BTx (the AChR is mainly in the resting state) (see Table 3). This is also reflected by similar ΔG values for the resting and desensitized states obtained by dynamic measurements (approximately −31 kJ/mol for both states; see Table 3), contrary to a small difference in the ΔG values obtained by radioligand binding (−33 and −30 kJ/mol for the desensitized and resting states, respectively; see Table 2). In this regard, mechanisms other than ligand dynamics might explain the observed difference in the affinity of bupropion when the receptor is in different conformational states.</p><!><p>In previous studies, an increase in the temperature produced a reduction in the chromatographic retention of dextromethorphan and levomethorphan (31). The data could then be analyzed using the van't Hoff plot to calculate the changes in enthalpy (ΔH°) and entropy (ΔS°) associated with the interactions of the ligands with the immobilized AChR. In this paper, non-linear chromatography was used to calculate the Ka constants to finally construct the van't Hoff plots. In this regard, temperature dependent changes in bupropion retention on immobilized Torpedo AChRs exposed to α-BTx (resting state) were obtained. Since the resulting van't Hoff plot was linear (Fig. 5), the thermodynamic parameters ΔH° and ΔS° were calculated from the slopes and intercepts of the van't Hoff plots, respectively, according to eq. 8, whereas ΔG20 was calculated according to eq. 9 (Table 4). Bupropion retention on immobilized Torpedo AChRs exposed to epibatidine (desensitized state) also produced the expected temperature dependence and thus, a linear van't Hoff plot (Fig. 5), allowing the calculation of the respective thermodynamic parameters (Table 4). The linearity of van't Hoff plots indicates an invariant retention mechanism over the temperature range studied (31,38).</p><p>The entropic contributions (i.e., −TΔS° values) for the AChR-ligand interactions were calculated using the ΔS° values from Table 4 at 293K. The results indicate that the entropic contributions are higher (~ −25 and −26 kJ/mol) than the enthalpic contributions (ΔH° ~ −5 and −6 kJ/mol; see Table 4), for the resting and desensitized states, respectively. This indicates that the bupropion-AChR interactions in both conformational states were mainly entropy-driven [reviewed in (7)]. In this regard, bupropion may induce local or global conformational changes or solvent reorganization in the binding pocket [reviewed in (7)]. Negative ΔH° values also suggest the existence of attractive forces (e.g., van der Waals, hydrogen bond, and electrostatic interactions) forming the stable complex. The calculated ΔG20 values (Table 4) coincide very well with that determined by either radioligand (see Table 2) or chromatographic (see Table 3) experiments. The thermodynamic parameters are similar in both the resting and desensitized states, suggesting that mechanisms other than thermodynamic differences might explain the observed ~2-fold higher affinity of bupropion for the desensitized state compared to the resting state.</p><!><p>During molecular modeling simulations, R- and S-bupropion isomers were docked to models of Torpedo (Fig. 7A,B) and mouse muscle AChR ion channels (Fig. 7C), respectively. Molegro Virtual Docker generated a series of docking poses and ranked them using energy-based criterion using the embedded scoring function in MolDockScore. Based on this ranking, the lowest energy pose of the ligand–receptor complex was selected and presented in Fig. 7A,B (Torpedo model) and Fig. 7C (mouse muscle model), respectively. The MolDockScore values for best ranked complexes are presented in Table 5. These data only serve to compare the interaction of bupropion isomers with each AChR model (Torpedo and muscle mice AChRs, respectively), and are not expected to be considered reliable in absolute terms. In other words, these values cannot be compared to those obtained by other experimental procedures.</p><p>Molecular modeling results also indicate that either R- or S-bupropion in the neutral state, interacts within the middle portion of the channel in the cavity formed between valine (position 13') and serine (position 6') rings. Interestingly, the same docking site for both isomers in the protonated state was found (data not shown). A very similar mode of binding was observed in previous simulations where bupropion was docked into the α3β4 ion channel (29,39). Analyses of the complexes show that the docked molecule interacts only with all five M2 helices, provided by each subunit. Comparison of Molegro scoring function values (see Table 5) suggest that both bupropion enantiomers present lower energies (a difference of approximately −10 kJ/mol) of interaction with the mouse muscle AChR ion channel when compared to the Torpedo model.</p><p>Best scored complexes obtained by docking were edited and limited to membrane domain of the receptor and then subjected to molecular dynamics simulations as described in the Experimental Procedure section. To verify whether the ligand configuration is stable within the binding site, simulations were performed keeping the backbone of the protein frozen, while the ligand and all side-chains were left free to move. Although the simulations lasted for 1.0 ns, the systems reached equilibrium after 0.2–0.25 ns as monitored by the trajectory of total energy (data not shown). During dynamics simulations, the molecules did not significantly change the position comparing to the starting pose, indicating that that simulated ligand–receptor complex is stable. In fact, the molecules interacted exclusively with nearby pore-lining residues at overlapping sites between the serine (position 6') and valine (position 13') rings, without interacting with the external or intracellular mouths of the ion channel. Table 5 includes the emulated binding energy values averaged during the 1.0 ns trajectory of molecular dynamics for each complex. Although these emulated measurements cannot be considered absolute values, they show that the calculated binding energy values fluctuate significantly during the dynamics simulations, producing standard deviations of ~10% the averaged value. In this respect, there is no significant difference between the emulated binding energy values for bupropion in the neutral state interacting with either the Torpedo AChR or the mouse muscle AChR. This lack of difference between both AChR types was also obtained using bupropion in the protonated state. Based on these results we did not pursue additional studies to demonstrate the difference between both isomers on muscle-type AChRs. However, further experiments need to be performed to demonstrate whether this is also true on neuronal-type AChRs such as the α3β4 AChR where bupropion also binds (4).</p><!><p>Bupropion clinically acts as an antidepressant and it is now also marketed as an aid to smoking cessation (2). Pharmacologically, bupropion also behaves as a NCA on several AChR types [reviewed in (5)]. In this regard, this study is an attempt to characterize the interactions of bupropion with different muscle-type AChRs in distinct conformational states and to determine its molecular mechanisms of inhibition. To this end, radioligand competition binding experiments, Ca2+ influx and macroscopic current recordings, thermodynamic and kinetic measurements, and molecular docking and dynamics studies were performed.</p><!><p>In a first attempt to determine the effect of bupropion on epibatidine-activated Ca2+ influx in TE671 cells, pre-incubation and co-injection protocols were used. The inhibitory potency of bupropion was in the 10–20 µM concentration range as was previously determined by 86Rb+ efflux experiments using the same cell type (4). Since the Ca2+ influx approach could not distinguish the effect of bupropion on a particular conformational state, we recorded macroscopic currents of agonist-activated receptors rapidly pre-(mainly in the resting state) or co-incubated (mainly in the open state) with bupropion (Fig. 1).</p><p>Macroscopic currents show that the interaction of bupropion with the open state decreases the decay time constant of currents activated by 300 µM ACh. At this ACh concentration, the decay rate equals the rate of desensitization from the double liganded open state (40). Thus, our results reveal that from the open state bupropion increases the desensitization rate. The current decay was adequately fitted using a single exponential function, which discards the possibility of a fast open-channel blockade mechanism. However, the occurrence of slow channel blockade, which has been described for other NCAs (41), maybe result in similar changes in macroscopic currents. If bupropion blocked the AChR and its unblocking process were quite slow, distinguishing between this process and the increase in the desensitization rate would be difficult (13).</p><p>Macroscopic current recordings also show a decrease in the peak current when bupropion interacts with the resting/activatable channel. Thus, together with the acceleration of decay rate from the open state, bupropion inhibits the opening of channels by binding to the resting/activatable state. The latter effect might be due to either an increase in desensitization of the resting/activatable state or a direct blockade of unliganded channels. A similar effect is observed with TCAs (13). The potency of inhibition is greater when bupropion acts on the resting/activatable state than on the open state. A similar finding was reported for the inhibition of different Cys-loop receptors by TCAs (15,42).</p><p>The two different effects detected for each different conformational state, i.e. reduced peak current and increased decay rate, are additive when the receptor is exposed continuously to the drug. Thus, the mechanism by which bupropion inhibits AChR function is selective for each conformational state.</p><!><p>The results from the radioligand competition binding (see Table 2) and frontal chromatographic (data not shown) experiments indicate that bupropion binds with higher affinity to the desensitized/agonist-bound Torpedo AChR compared to the resting/α-BTx-bound AChR. Unfortunately, neither the dynamic nor the thermodynamic studies could explain the observed state-dependant ligand affinity (see Table 3).</p><p>The radioligand competition experiments also indicate that bupropion inhibits the binding of both [3H]imipramine and [3H]TCP (the structural and functional analog of PCP) to both desensitized/CCh-bound and resting/α-BTx-bound AChRs with nH values close to unity (Table 2). Hill coefficients close to unity indicate a non-cooperative interaction between bupropion and PCP or imipramine, respectively, suggesting that bupropion interacts with only one binding site on each AChR conformational state. Although this evidence suggests a steric mode of competition between bupropion and PCP or imipramine, we cannot predict whether the location of the bupropion binding site is the same or distinct in each conformational state.</p><p>The location of the PCP binding site depends on the conformational state of the AChR ion channel [reviewed in (5)]. For instance, photoaffinity labeling studies using [3H]ethidium diazide, which binds with high affinity to the PCP locus, helped to determine the structural components of this site in the desensitized state (43). The results indicated that [3H]ethidium diazide mainly labeled residues at and close to the leucine ring (position 9') from the α1-M2 transmembrane segment. In addition, new photoaffinity labeling results using the hydrophobic probe 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine suggest that the PCP binding site is located between the threonine (position 2') and serine (position 6') rings, closer to the cytoplasmic end of the desensitized ion channel (44). Our photoaffinity labeling studies using [3H]2-azidoimipramine supports the idea that TCAs bind to the PCP locus in the desensitized ion channel (16). On the other hand, site-directed mutagenesis studies determined that the PCP binding site in the open ion channel includes residues located between the serine (position 6') and leucine (position 9') rings (45). Finally, we suggested that the PCP binding site in the resting state is located closer to the external mouth than that in the desensitized and open states (24,25,36). We also speculated that the aromatic tertiary amino group from the PCP (or TCP) molecule might interact with acidic residues (i.e., α1-Glu262) located at position 20' (e.g., the outer or extracellular ring) (25,36) [reviewed in (5)]. Considering our previous findings, we suggest that the secondary amino group from the bupropion molecule can also be responsible for the binding to one of the two Glu262 residues by charge interactions, when the receptor is in the resting/α-BTx-bound state.</p><p>Proposed mechanisms of bupropion binding were additionally studied by molecular modeling. Docking simulations followed by molecular dynamics of R- and S-bupropion were performed on two different variants of the AChR membrane domain, Torpedo (Fig. 6A,B) and mouse muscle (Fig. 6C) AChRs. The results from the docking simulations indicate no enantioselective interactions: both enantiomers form ligand–receptor complexes characterized by similar values of scoring function (Table 5). Although there is no experimental evidence of bupropion enantioselectivity on AChRs, the (2R,3R)-hydroxy bupropion isomer inhibits the muscle-type AChR with 3.7-fold higher potency than the (2S,3S)-hydroxy bupropion isomer (46), indicating the possibility of bupropion enantioselectivity. In order to determine bupropion enantioselectivity in AChRs, bupropion isomers will be used in further functional and binding experiments.</p><p>The analysis of the obtained molecular complexes clearly indicates that bupropion in either the neutral (see Fig. 7) or protonated form (data not shown) binds to the middle portion of the channel between the serine (position 6') and valine rings (position 13'). Exactly the same locus was observed for PCP and imipramine in the Torpedo AChR ion channel (16). However, a distinction was observed in the α3β4 ion channel: bupropion in the protonated state interacted with the polar region of the intermediate ring (position 1'), whereas the neutral form was positioned between the valine/phenylalanine (position 15') and serine (position 8') rings (29). The pocket formed by the cleft between the phenyl ring provided by β4-Phe and the isopropyl moiety from α3-Val interacts with the hydrophobic portion of bupropion, whereas hydrogen bonds are formed between polar residues at the serine ring and the polar region of bupropion. Considering this difference, distinct bupropion binding site locations may exist on each AChR ion channel. Nevertheless, we have to take into consideration that the α3β4 AChR ion channel was constructed by homology with the model of the 23 mer peptide imitating the M2 sequence of the Torpedo AChR δ subunit (29), whereas the current model was based on the cryo-electron microscopy structure of the Torpedo AChR determined at ~4 Å resolution (PDB ID 2BG9) (32,33).</p><p>The evidence obtained by molecular docking on the Torpedo AChR supports the competition experiments (see Table 2), indicating that there is a binding site for antidepressants that overlaps the PCP binding site in the Torpedo AChR ion channel. The thermodynamic parameters indicate that bupropion interacts with the Torpedo AChR ion channel either in the resting or in the desensitize state mainly by an entropy-driven process (see Table 4), forming a network of van der Waals, hydrogen bond, and polar interactions.</p><p>Considering the above results, we envision a dynamic process where bupropion binds first to the resting AChR, probably close to the mouth of the ion channel, decreasing the probability of ion channel opening. The remnant fraction of open ion channels is subsequently decreased by accelerating the desensitization process, and less probable by an open-channel blocking mechanism. Bupropion inhibits AChR function by interacting with a binding domain shared by tricyclic antidepressants and by PCP that is located between the serine and valine rings. This drug-receptor interaction is mediated mainly by an entropy-driven process.</p><!><p>This research was supported by grants from the Science Foundation Arizona and Stardust Foundation and the Office of Research and Sponsored Programs, Midwestern University (to H.R.), by grants from ANPCyT, CONICET, UNS, Loreal-UNESCO, and Fundación F. Fiorini (to C.B), by the FOCUS research subsidy from the Foundation for Polish Science (to K.J.). This research was also supported in part by the Intramural Research Program of the NIH, National Institute on Aging.</p><p>Abbreviations: NCA, noncompetitive antagonist; AChR, nicotinic acetylcholine receptor; PCP, phencyclidine; [3H]TCP, [piperidyl-3, 4-3H(N)]-(N-(1-(2 thienyl)cyclohexyl)-3,4-piperidine; CCh, carbamylcholine; ACh, acetylcholine; α-BTx, α-bungarotoxin; RT, room temperature; BS buffer, binding saline buffer (50 mM Tris–HCl, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4); NCA, noncompetitive antagonist; Ki, inhibition constant; Kd, dissociation constant; Ka, association constant; koff, dissociation rate constant; kon, association rate constant; IC50, ligand concentration that produces 50% inhibition (of binding or of agonist activation); nH, Hill coefficient; EC50, agonist concentration that produces 50% AChR activation; TCA, tricyclic antidepressants; DMEM, Dulbecco's Modified Eagle Medium; FBS, fetal bovine serum; FLIPR, fluorescent imaging plate reader.</p>