key: cord-0012887-188qnh2w
authors: Ma, Ze-gang; Jiang, Nan; Huang, Yuan-bing; Ma, Xiao-kuang; Brek Eaton, Jason; Gao, Ming; Chang, Yong-chang; Lukas, Ronald J; Whiteaker, Paul; Neisewander, Janet; Wu, Jie
title: Cocaine potently blocks neuronal α(3)β(4) nicotinic acetylcholine receptors in SH-SY5Y cells
date: 2019-08-09
journal: Acta Pharmacol Sin
DOI: 10.1038/s41401-019-0276-y
sha: a4e1cd500608de9847b476c4de4eab7cb2863635
doc_id: 12887
cord_uid: 188qnh2w

Cocaine is one of the most abused illicit drugs worldwide. It is well known that the dopamine (DA) transporter is its major target; but cocaine also acts on other targets including nicotinic acetylcholine receptors (nAChRs). In this study, we investigated the effects of cocaine on a special subtype of neuronal nAChR, α(3)β(4)-nAChR expressed in native SH-SY5Y cells. α(3)β(4)-nAChR-mediated currents were recorded using whole-cell recordings. Drugs were applied using a computer-controlled U-tube drug perfusion system. We showed that bath application of nicotine induced inward currents in a concentration-dependent manner with an EC(50) value of 20 µM. Pre-treatment with cocaine concentration-dependently inhibited nicotine-induced current with an IC(50) of 1.5 μM. Kinetic analysis showed that cocaine accelerated α(3)β(4)-nAChR desensitization, which caused a reduction of the amplitude of nicotine-induced currents. Co-application of nicotine and cocaine (1.5 μM) depressed the maximum response on the nicotine concentration-response curve without changing the EC(50) value, suggesting a non-competitive mechanism. The cocaine-induced inhibition of nicotine response exhibited both voltage- and use-dependence, suggesting an open-channel blocking mechanism. Furthermore, intracellular application of GDP-βS (via recording electrode) did not affect cocaine-induced inhibition, suggesting that cocaine did not alter receptor internalization. Moreover, intracellular application of cocaine (30 µM) failed to alter the nicotine response. Finally, cocaine (1.5 μM) was unable to inhibit the nicotine-induced inward current in heterologous expressed α(6)/α(3)β(2)β(3)-nAChRs and α(4)β(2)-nAChRs expressed in human SH-EP1 cells. Collectively, our results suggest that cocaine is a potent blocker for native α(3)β(4)-nAChRs expressed in SH-SY5Y cells.

Cocaine addiction is a serious public health problem worldwide that affects normal brain function, leading to alterations in memory as well as behavioral and neuronal physiology. Although many approaches have been pursued to address cocaine addiction, cocaine remains one of the most abused illicit drugs worldwide, and there are currently no approved medications to treat cocaine addiction.

It is well known that cocaine increases intrasynaptic dopamine via its inhibition of dopamine transporters, producing a rewarding effect [1, 2] . However, cocaine also acts on other brain targets involved in reward, such as neuronal nicotinic acetylcholine receptors (nAChRs) [3] [4] [5] . Epidemiological studies suggest that smoking increases the intake of cocaine and that cocaine use, in turn, increases cigarette consumption [6] . However, little is known about the mechanisms that would support such interactions. In laboratory animals, stimulation of nAChRs by nicotine increases cocaine-induced locomotor sensitization [7] , while blockade with a nonselective nAChR antagonist such as mecamylamine reduces cocaine reinforcement in rats [8] . Additionally, pretreatment with nicotine reduces cocaine-conditioned place preference (CPP) established in rats, while mecamylamine slightly attenuates cocaine-CPP in rats [9] [10] [11] . The roles of the α 4 β 2 nAChRs in the modulation of cocaine self-administration are still controversial. It has been reported that α 4 β 2 nicotinic receptor desensitizing compounds can decrease the self-administration of cocaine and methamphetamine in rats [12] , while in the monkey cocaine self-administration model, varenicline, a partial agonist of α 4 β 2 nAChRs and a full agonist of an α 7 nAChRs, did not attenuate the reinforcing effects of cocaine [13] .

Although the interactions between cocaine and nAChRs have been established in Xenopus oocytes expressing system [3, 4] , the specific pharmacological effects of cocaine on native nAChR subtypes are still unclear. Behavioral experiments showed that local injection of a selective α 4 β 2 nAChR antagonist (dihydro-βerythroidine, DHβE) into the ventral tegmental area (VTA) prevents cocaine-induced locomotor activity [14] , while the α 7 -selective antagonist methyllycaconitine (MLA) did not alter cocaineinduced behavioral sensitization [15] . Recently, nAChRs containing the α 6 subunit (α 6 * nAChRs), but not those containing the α 4 subunit, were shown to play a role in the induction of cocaineconditioned reward [16] , and we have recently reported that cocaine blocks human α 6 * nAChRs expressed in human SH-EP1 cells [17] . The α 3 β 4 nAChR may also be an important target that mediates the effects of cocaine. Although most widely expressed in the peripheral nervous system, α 3 β 4 nAChRs are also highly expressed in some brain regions such as the medial habenula, interpeduncular nucleus, and fasciculus retroflexus and moderately expressed in the VTA, dorsolateral tegmentum and basolateral amygdala [18] [19] [20] [21] . Several studies have shown that α 3 β 4 nAChRs play a role in drug-induced reward and reinforcement [22] [23] [24] [25] [26] . A recent study demonstrated that inhibition of α 3 β 4 nAChRs by high-affinity α 3 β 4 -nAChR ligands attenuates cocaineinduced CPP and behavioral sensitization [27] , suggesting that α 3 β 4 nAChRs may participate in the rewarding effect of cocaine.

The role of α 3 β 4 nAChRs in drug addiction behaviors is intriguing. However, little is known about the mechanisms that support the interactions between cocaine and α 3 β 4 nAChRs. In the present study, we used patch-clamp whole-cell recording to evaluate the acute effects of cocaine on native α 3 β 4 nAChRs expressed in SH-SY5Y cells, and we also explored the mechanisms underlying these pharmacological effects.

Cell culture Human SH-SY5Y neuroblastoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin and 2 mM glutamine. Previous studies demonstrated that native α 3 β 4 nAChRs were stably expressed in SH-SY5Y cells. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO 2 and then plated in 35-mm dishes for patchclamp recording.

Expression of human neuronal α 4 β 2 nAChRs in SH-EP1 human epithelial cells Heterologous expression of human α 4 β 2 nAChRs has been previously described [28, 29] . Briefly, human nAChR α 4 and β 2 subunits, subcloned into pcDNA3.1-zeocin or hygromycin vectors, respectively, were introduced into native nAChR-null SH-EP1 cells [30] to create the SH-EP1-hα 4 β 2 cell line. Cells were maintained as low-passage-number cultures in a medium augmented with 0.5 mg/mL zeocin and 0.4 mg/mL hygromycin and were passaged once per week by splitting cultures 1/10 just as they reached confluence to maintain the cells in a state of proliferative growth.

Expression of human neuronal α 6 /α 3 β 2 β 3 nAChRs in human SH-EP1 cells The human epithelial cell line expressing α 6 /α 3 chimera β 2 β 3 nAChRs (α 6 N/α 3 Cβ 2 β 3 nAChRs) was developed by Dr. Whiteaker's laboratory [31] , and its pharmacological response properties have been profiled recently [32] . α 6 /α 3 denotes a chimeric subunit composed of the extracellular, ligand-binding domain of the human α 6 subunit fused to the first transmembrane domain and followed by the sequence of the human α 3 nAChR subunit; this approach reproducibly increases expression compared to that of native α 6 subunits while retaining α 6 -like pharmacology. Consensus-sequence β 2 and β 3 human nAChR subunit clones were also used. Wild-type SH-EP1 cells were transfected with nAChR subunit clones using the cationic polymer Effectene (Qiagen, Valencia, CA, USA). These cells were maintained in DMEM (Invitrogen) with 10% horse serum (Invitrogen), 5% fetal bovine serum (HyClone, GE Healthcare Life Sciences, Pittsburgh, PA, USA), 1 mM sodium pyruvate, and 4 mM L-glutamine, supplemented with 0.25 mg/mL zeocin, 0.13 mg/mL hygromycin B, and G418 at 0.6 mg/mL, ensuring positive selection of transfectants. Low-passage-number (1−40 from our frozen stocks) cultures were used to ensure phenotypic stability. Cells were typically passaged once per week by splitting just-confluent cultures 1:20−1:40 to maintain cells in proliferative growth.

Patch-clamp whole-cell current recordings and data acquisition Standard whole-cell current recordings were performed as previously described, using an application system that enables fast application and removal of drugs [33, 34] . Briefly, SH-SY5Y cells and two types of transfected SH-EP1 cells were prepared in 35-mm culture dishes without polylysine coating, plated on the bottoms of the dishes, and later placed on the stage of an inverted microscope (Axiovert 200; Zeiss, Germany). Cells were perfused with standard external solution (2 mL/min) and bubbled with 95% O 2 /5% CO 2 . Glass microelectrodes with 3-5 MΩ resistance between the pipette and extracellular solution were used to form tight seals (>1 GΩ) on the surface of the cells, and standard wholecell current recording was initiated by applying suitable suction and then waiting 5 to 10 min for the exchange of the pipette solution and the cytosol. Subsequently, recorded cells were lifted up gently from the bottoms of the culture dishes, which improved both solution exchange and accurate evaluation of the differences in the kinetics of agonist-induced whole-cell currents. Before capacitance and resistance compensation, access resistance (Ra) was measured; experiments with Ra values of less than 20 MΩ were accepted. Pipette and whole-current capacitance were both minimized, and series resistance was compensated routinely to 80%. The recorded cells were voltage clamped at a holding potential of −60 mV in SH-SY5Y cells and −40 mV in two types of transfected SH-EP1 cells. Inward currents induced by nicotine were measured (200B amplifier; Molecular Devices, Sunnyvale, CA, USA). Current signals were typically filtered at 2 kHz, acquired at 5 kHz, and displayed and digitized online (Digidata 1440 A series A/D board; Molecular Devices, Sunnyvale, CA, USA). Data acquisition and analyses were performed using pClamp10.0 (Molecular Devices), and results were plotted using Prism 5.0 software (GraphPad Software, Inc., CA, USA). All experiments were performed at room temperature (22 ± 1°C).

The standard external solution for SH-SY5Y and SH-EP1 cells contained (in mM) 120 NaCl, 5 KCl, 2 MgCl 2 , 2 CaCl 2 , 25 D-glucose, and 10 HEPES and had a pH of 7.4, adjusted with Tris base. The pipette solution for SH-SY5Y cells contained (in mM) 140 Kgluconate, 10 HEPES, 0.2 EGTA, 5 KCl, 2 MgCl 2 , 4 Na 2 ATP, 0.3 Na 3 GTP, and Na 2 -phosphocreatine and had a pH of 7.3. For traditional whole-cell recording in SH-EP1 cells, the pipette solution contained (in mM) 110 dibasic Tris phosphate, 28 Tris base, 11 EGTA, 2 MgCl 2 , 0.5 CaCl 2 , and 4 Na-ATP and had a pH of 7.3. In the experiments, nicotine was used as the test agonist and induced whole-cell current responses. Nicotine was quickly applied to recorded cells using a computer-controlled U-tube drug perfusion system and could fully surround the recorded cells within 30 ms, as in our previous reports [33] [34] [35] . The interval between drug applications (2 min) was optimized specifically to ensure the stability of nAChR responsiveness (e.g., no functional rundown).

Homology modeling and ligand docking The amino acid sequences of the human α 3 (NP_000734) and β 4 (NP_000741) nAChR subunits and the crystal structure of the human α 4 β 2 nAChR (PDBID: 5kxi; Chain A: α 4 ; Chain B: β 2 ; Chain C: β 2 ; Chain D: α 4 ; Chain E: β 2 ) were downloaded from the NCBI website. Using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/ clustalo/), the α 3 sequence was aligned with the α 4 sequence (Chain A) from the crystal structure, and the β 4 sequence was aligned with the β 2 sequence (Chain B) from the crystal structure. The unaligned regions of the α 3 and β 4 subunits (mainly the signal peptide and the M3-M4 intracellular loop) were removed. The homology models of all five chains were built with ICM Pro 1.7 (MolSoft, San Diego, CA, USA; Chain A: α 3 ; Chain B: β 4 ; Chain C: β 4 ; Chain D: α 3 ; Chain E: β 4 ). Five model chains were combined to form a pentameric structure. The receptor was further converted to an ICM object, and energy was minimized using MolMechanics function in ICM Pro. The cocaine molecule was built from the crystal structure, with a PDBID of 2pgz. The docking was performed using ICM Pro with a docking space manually adjusted to cover the transmembrane domain of the receptor. The topscored pose was used for presentation using Discovery Studio 4.0 Visualizer (BIOVIA, San Diego, CA, USA).

To analyze the effects of cocaine on the nAChR whole-cell current responses, we measured the peaks of inward currents. Data are presented as the means ± standard errors. Statistical analysis was performed using paired t-tests when evaluating data obtained from a single cell or Student's t-test (unpaired values) or one-way ANOVA followed by Student-Newman-Keuls test when comparing data obtained from different cells; all these tests were conducted using GraphPad 5.0 software. P values less than 0.05 were considered significant. Curve fitting for agonist and antagonist concentration-response data were performed by GraphPad 5.0 software (GraphPad Software, Inc., CA, USA) using the logistic equation to provide fits for maximal and minimal responses, EC 50 or IC 50 values, and Hill coefficients.

Nicotine produces a concentration-response curve in native α 3 β 4 nAChRs in SH-SY5Y cells, and cocaine inhibits α 3 β 4 nAChRmediated currents in a concentration-dependent manner Patch-clamp whole-cell current recording in voltage-clamp mode was performed, and bath application of nicotine induced an inward current at a holding potential (V H ) of −60 mV. In order to obtain the concentration-response relationship of nicotineinduced currents, whole-cell currents induced by different concentrations of nicotine were measured from the same recorded cell (Fig. 1a) . From the 6 cells tested, normalized (to the response to 1000 μM nicotine) whole-cell current peak amplitudes in response to nicotine (Fig. 1b) , when plotted as a function of agonist concentration, were sigmoid and well fitted by the logistic equation for a single-site model. Fits to the data yielded an EC 50 value and a Hill coefficient of 20 µM and 1.02 for nicotine, demonstrating that nicotine acts as an agonist of α 3 β 4 nAChRs in SH-SY5Y cells. Then, we determined whether cocaine affected α 3 β 4 nAChR function in SH-SY5Y cells using patch-clamp whole-cell recording combined with fast drug application by a Utube apparatus. At a V H of −60 mV, bath application of 20 µM nicotine (nicotine EC 50 concentration for α 3 β 4 nAChRs) induced an inward current, and the application of 20 µM nicotine after 30 s pretreatment with different concentrations (0.1-30 µM) of cocaine reduced α 3 β 4 nAChR-mediated currents (Fig. 1c) . According to the fitting results from the concentration-response curves (Fig. 1d) , the IC 50 value and Hill coefficient for cocaine-mediated suppression of whole-cell peak currents were 1.56 µM and 1.04, respectively.

Effects of cocaine on α 3 β 4 nAChR-mediated currents induced by different nicotine concentrations In order determine the mechanisms (competitive or noncompetitive) of how cocaine inhibits nicotine-induced responses, the nicotine concentration-response relationship curves in the presence and absence of 1.5 µM cocaine were compared. As shown in Fig. 2a , nicotine-induced inward currents increased with increasing nicotine concentrations, forming a sigmoid-shaped curve (open circle symbols, Fig. 2c ). In the presence of 1.5 µM cocaine, nicotine-induced currents were reduced compared to those for nicotine exposure alone (solid circle symbols, Fig. 2b) . Unpaired ttest analysis for each nicotine concentration pair with 1.5 µM cocaine showed highly significant differences between nicotine alone and nicotine plus cocaine at all tested nicotine concentrations, including 10 (inhibitory rate = 40.8% ± 12.6%), 100 (inhibitory rate = 40.8% ± 7.7%) and 1000 µM (inhibitory Fig. 2c , black circle symbols). The inhibitory rates of these different concentrations of cocaine against nicotineinduced currents were not significantly different (one-way ANOVA, F(2, 15) = 0.09, P = 0.91). For normalized nicotine concentrationresponse curves with and without cocaine, the EC 50 value was not significantly different between the nicotine group and the nicotine plus cocaine group (Fig. 2d) . In the presence of nicotine alone or nicotine plus 1.5 µM cocaine, the EC 50 values of the groups were 21.55 ± 4.39 µM and 22.79 ± 6.03 µM, respectively, which were not significantly different (unpaired t-test, t(16) = 0.165, P = 0.87). Collectively, the data show that cocaine reduces the maximal response of the nicotine concentration-response curve ( Fig. 2c ) but does not change the nicotine EC 50 value (Fig. 2d) , suggesting noncompetitive inhibition.

Kinetic analysis of cocaine-induced inhibition in α 3 β 4 nAChRmediated currents In further tests, we examined kinetic changes in α 3 β 4 nAChRmediated whole-cell current after acute exposure to 1.5 µM cocaine. As shown in Fig. 3 , cocaine did not alter the rising slope of the 20 µM nicotine-induced whole-cell current (Fig. 3a, b) but increased the decay rate (Fig. 3a, right traces) . Statistical analysis demonstrated that after 1.5 µM cocaine exposure, α 3 β 4 nAChR channel activation did not alter the rising slope of the current (from −0.60 ± 0.10 to −0.58 ± 0.12 pA/ms, paired t-test, P > 0.05, n = 8, Fig. 3b ), but channel inactivation was accelerated, as measured by the current decay constant (tau values from 628.4 ± 135.3 to 161.6 ± 22.1 ms, paired t-test, P < 0.05, n = 8, Fig. 3c) . These results suggest that acute cocaine application inhibits nicotine-induced currents by accelerating channel inactivation.

Use dependence of cocaine suppression of α 3 β 4 nAChR-mediated currents It has been reported that cocaine inhibits nAChRs through an open-channel block mechanism. Under patch-clamp recording conditions, one feature of open-channel block is use dependence. Thus, we examined whether cocaine-induced inhibition in α 3 β 4 nAChR-mediated current is use-dependent. To exclude the phenomenon of nAChR desensitization caused by nicotine itself, we applied 5 doses of nicotine alone for 1 s each at 30 s intervals, and we found that the second nicotine response was reduced (82.7% ± 5.2%) compared to the first one; however, the difference was not statistically significant (paired t-test, t(7) = 1.92, P = 0.09), and subsequent nicotine responses were stabilized at this level without further rundown (Fig. 4a top trace and 4d red curve) . Under the same conditions, repetitive applications of nicotine (20 µM) in the continuous presence of 1.5 µM cocaine led to gradual decreases in responses induced by nicotine (Fig. 4b) . In a variant of the same protocol, 1.5 µM cocaine was applied only for the 1 st and 5 th traces of nicotine application, whereas the standard extracellular solution took the place of the cocaine solution during the second, third and fourth traces. With this protocol, the recorded cell was exposed only two times (first and fifth) of nicotine (Fig. 4c) , assuming nAChRs were less frequently used compared to five doses of nicotine. Fig. 4d summarizes the nicotine current amplitude changes of the control, 5-exposure and 2-exposure protocols. Comparison of the amplitude ratios of the fifth and first current traces between these two protocols demonstrated that in the presence of 1.5 µM cocaine, the ratio was 61.2% ± 8.8% after repeated challenges to 5 nicotine exposures, while the ratio was 96.7% ± 13.7% after two nicotine exposures (unpaired t-test, t(15) = 6.42, P < 0.05, Fig. 4e ).

These results indicate that cocaine-mediated suppression of α 3 β 4 -nAChR function was use-dependent.

Voltage dependence of cocaine inhibition in α 3 β 4 nAChRmediated currents

Another feature of open-channel block is voltage dependence. Thus, we examined whether cocaine showed different inhibitory rates at two different holding potentials. The effects of changes in the holding potential (V H ) on cocaine inhibition were tested in the α 3 β 4 nAChRs. As shown in Fig. 5a , the application of 1.5 µM cocaine at a standard V H of −60 mV resulted in approximately 50% inhibition of the peak inward current induced by nicotine. The application of the same concentration of cocaine at a V H of −20 mV decreased the observed inhibition to approximately 5% of the control value (Fig. 5b) . Statistical analysis demonstrated that after 1.5 µM cocaine exposure at a V H of −60 mV, the responses to nicotine were 55.7% ± 10.9% of the responses induced by nicotine alone (paired t-test, t(7) = 2.81, P = 0.02, n = 8). When the V H was raised to −20 mV, the responses to nicotine after 1.5 µM cocaine exposure were 92.5% ± 21.1% of the responses induced by nicotine alone (paired t-test, t(7) = 0.35, P = 0.74, n = 8). The responses to nicotine at V H of −20 mV were 35.9% ± 8.5% (n = 8) of the responses induced by nicotine alone at V H of −60 mV (Fig. 5c) . These results demonstrated the voltage dependence of the inhibition of α 3 β 4 nAChRs by cocaine.

Cocaine inhibition of the α 3 β 4 nAChR-mediated current did not occur via an intracellular mechanism In order to test the hypothesis that cocaine inhibition of α 3 β 4 nAChR-mediated current is mediated via intracellular mechanisms, cocaine (30 µM) was added to the pipette solution and applied to cells intracellularly throughout whole-cell recordings. Under these conditions, bath application of 20 µM nicotine induced stable responses. After conversion to whole-cell recording mode followed by a waiting period of more than 20 min, which allowed full loading of cocaine into the cell, there were no declines in responses to nicotine. However, these responses decreased when nicotine and cocaine (1.5 µM) were coapplied extracellularly to the same recorded cell. In these same recorded cells, bath application of cocaine (30 µM) completely blocked nicotine-induced inward currents (Fig. 6a) . Statistical analysis also demonstrated that after intracellular application of 30 µM cocaine, bath application of 1.5 µM cocaine also inhibited nicotine-induced responses by 43.52% ± 10.48% (paired t-test, t(6) = 3.49, P = 0.01; Fig. 6b ). These Effects of receptor internalization on cocaine-induced inhibition of α 3 β 4 -nAChR function Since endocytosis or exocytosis of several transmembrane receptors occurs on the order of seconds to minutes through processes modulated by agonists and antagonists, we evaluated the effects of cocaine in SH-SY5Y cells preloaded with 600 µM GDP-βS (added to the pipette solution) for 20 min following conversion to whole-cell recording configuration, which has been reported to prevent AMPA receptor internalization [36] . Preloading cells with GDP-βS for 20 min had no effect on nicotineinduced currents or on cocaine-induced suppression of α 3 β 4 nAChR function (Fig. 7) . Statistical analysis showed that acute cocaine (1.5 µM) application still significantly inhibited nicotineinduced currents after intracellular application of 600 µM GDP-βS (one-way ANOVA followed by the Student-Newman-Keuls test, F(2, 26) = 4.79, P = 0.016). These results support the hypothesis that the modulatory effect of cocaine on α 3 β 4 nAChRs does not occur via receptor internalization.

Effects of cocaine on α 4 β 2 -containing and α 6 -containing nAChRs The effects of 1.5 µM cocaine on the function of native α 3 β 4 nAChRs in SH-SY5Y cells and on α 4 β 2 or α 6 /α 3 β 2 β 3 nAChRs heterologously expressed in SH-EP1 cells were evaluated. The EC 50 concentrations of nicotine for α 6 /α 3 β 2 β 3 (1 µM) [32] , α 4 β 2 (3 µM) [37] , and α 3 β 4 (20 µM, see Fig. 1 ) were used. As shown in Fig. 8a , at a V H of −40 mV (approximately the resting potential of SH-EP1 cells), 1.5 µM cocaine was unable to inhibit nicotine-induced inward currents mediated by α 6 /α 3 β 2 β 3 (Fig. 8ai) or α 4 β 2 (Fig. 8aii ) nAChRs in SH-EP1 cells. However, the same concentration of cocaine blocked approximately 50% of nicotine-induced inward current mediated by α 3 β 4 nAChRs in SH-SY5Y cells (Fig. 8aiii) at a V H of −60 mV. Statistical analysis also demonstrated that 1.5 µM cocaine significantly inhibited nicotine-induced inward currents mediated by α 3 β 4 nAChRs in SH-SY5Y cells (paired t-test, t(7) = 3.16, P = 0.02) but had no effect on α 6 (paired t-test, t(19) = 0.12, P = 0.91) and α 4 β 2 nAChRs (paired t-test, t(7) = 0.42, P = 0.68) in SH-EP1 cells (Fig. 8b) .

The cocaine molecule docks with the channel domain of the homology model of the human α 3 β 4 nAChR with subunit stoichiometry of (α 3 )2(β 4 )3

To explore the possible binding site of cocaine on α 3 β 4 nAChRs, we performed homology modeling of human α 3 β 4 nAChR with the subunit stoichiometry of (α 3 )2(β 4 )3 using the crystal structure of human α 4 β 2 as the template and ligand docking to the transmembrane channel domain docked to the channel domain of the α 3 β 4 homology model. As shown in Fig. 9 , the highestscored position of the docked cocaine is located in the center of the channel pore, below the channel gate but above the channel selectivity filter, and completely occludes the ion-conducting pore. The location of the docked cocaine in the pore domain is very similar to the location of the GABA A receptor channel blocker picrotoxin in the pore [38] . Thus, our docking result is consistent with a channel-blocking effect of cocaine on α 3 β 4 nAChRs.

In the present study, we found that cocaine potently blocks α 3 β 4 -nAChR function. Using natively expressed α 3 β 4 nAChRs in SH-SY5Y cells, we demonstrated that bath application of cocaine inhibits α 3 β 4 nAChR-mediated whole-cell currents in a concentrationdependent manner with an IC 50 value of 1.5 µM. Kinetic analysis showed that cocaine accelerated α 3 β 4 nAChR desensitization, which caused a reduction in the amplitude of nicotine-induced currents. Cocaine inhibited α 3 β 4 nAChRs in a noncompetitive manner. Cocaine inhibition of α 3 β 4 nAChRs was also usedependent and voltage-dependent. In addition, cocaine-induced inhibition is mediated neither via intracellular sites nor through changes in receptor internalization. Finally, potent cocaine inhibition occurs specifically in α 3 β 4 nAChRs but not in α 4 β 2 -or α 7 nAChRs. These studies provide experimental evidence that cocaine directly inhibits native α 3 β 4 nAChR function.

Cocaine inhibits native α 3 β 4 -nAChR function in SH-S5Y5 cells Previous research found that cocaine inhibits heterologously expressed nAChR function in Xenopus oocytes. For example, Damaj et al. reported that cocaine inhibits heterologously expressed nAChR-mediated currents in a concentrationdependent manner with an IC 50 range of 4.4-6.9 µM for α 4 β 2 nAChRs and 22-42.3 µM for α 3 β 2 nAChRs [3] . They also found that the cocaine-induced inhibition of nAChRs functions through a noncompetitive mechanism. Francis et al. systematically compared cocaine-induced inhibition in different subtypes of nAChRs expressed in oocytes and found that α 4 β 4 nAChRs exhibited the highest sensitivity, with an IC 50 of 2 µM, while α 3 β 2 showed the lowest sensitivity, with an IC 50 of 60 µM. They also found that cocaine inhibits α 3 β 4 nAChRs with an IC 50 of 6 µM [4] . By using transfected α 3 β 4 nAChRs in HEK 293 cells, Krivoshein et al. compared the possible mechanisms of cocaine-and MK801induced inhibition of α 3 β 4 nAChRs and found that both tested compounds exhibited noncompetitive inhibition under closed and open-channel conditions [39] . In addition to these expressed nAChRs, Acevedo-Rodriguez et al. found that cocaine also inhibits native (β 2 -containing) nAChRs in midbrain dopaminergic neurons with an IC 50 of 19.8 µM [5] . Collectively, these previous data suggest that cocaine serves as a low-potency antagonist of different subtypes of nAChRs. Thus far, however, whether cocaine modulates native α 3 β 4 -nAChR function is unknown. The present study provides experimental evidence that cocaine blocks native α 3 β 4 nAChR function in a concentrationdependent manner with an IC 50 value of 1.5 µM. This concentration is even lower than the IC 50 for cocaine-induced inhibition in dopamine transporters, indicating that native α 3 β 4 nAChR is highly sensitive to cocaine.

Mechanisms of cocaine-induced inhibition of native α 3 β 4 nAChRs Krivoshein et al. reported the mechanisms of cocaine-induced inhibition in transfected α 3 β 4 nAChRs in HEK 293 cells [39] . We believe that the present study exhibits some differences from Dr. Krivoshein's report. First, to explore the possible mechanisms of cocaine-induced inhibition in native α 3 β 4 nAChRs, we compared nicotine concentration-response curves with or without 1.5 µM Fig. 9 Cocaine molecule docked to the channel domain of the homology model of the human α 3 β 4 nAChR with the subunit stoichiometry of (α 3 )2(β 4 )3. a Top view (from the extracellular side) of the channel-lining domain (with only M2 domains for clarity). The docked cocaine is located in the center of the pore and has a potential hydrogen bond with α 3 Ser247 (Chain A). b Side view of the channel-lining domain, with one α 3 subunit (Chain D) removed from the front for clarity. The docked cocaine was located immediately above the selectivity filter (β 4 Glu241) up to the level at the 6′ position (α 3 Ser247, β 4 Ser248) of the second transmembrane domain (M2) with two potential π-anion interactions with two β 4 Glu241 residues of Chains C and D. The docked cocaine molecule was below the putative channel gate at the 9′ position (α 3 Leu250, β 4 Leu251)

cocaine. In our nicotine concentration-response curve, 1.5 µM cocaine reduced the maximal nicotine concentration-induced current without changing the nicotine EC 50 value, suggesting noncompetitive inhibition. This finding is consistent with the cocaine-induced inhibition observed in other subtypes of nAChRs [4] . Second, we demonstrated that cocaine-induced inhibition was dependent on use and voltage. This suggests that its suppression of nicotine-induced currents involves an agonist-induced transition of nAChRs to conformations having a higher affinity for the antagonist and allowing it free access to the binding site(s) [40] . It is reasonable to conclude that this transition is to an open-channel state. Third, through kinetic analysis, we found that cocaine accelerated α 3 β 4 nAChR desensitization during nicotine exposure, again suggesting that it blocked open channels. Finally, we applied either cocaine or GDP-βS intracellularly through the recording electrode and found that these manipulations did not affect α 3 β 4 nAChR function. Together, these findings suggest that cocaine has access to α 3 β 4 nAChRs in the resting state but principally acts as a noncompetitive antagonist, with a presumed binding site within the channel pore.

To visualize the potential mechanism whereby cocaine blocks the channel, we performed applied homology modeling and simulated ligand docking to the transmembrane domain of α 3 β 4 nAChR with the stoichiometry and arrangement of α 3 β 4 β 4 α 3 β 4 (Fig. 9a , extracellular view counterclockwise). The resulting topscored docking pose of cocaine was located in the center of the channel pore. The docked cocaine was located immediately above the selectivity filter (β 4 Glu241/α 3 Glu240) at the −1′ position, contacting residues at the 2′ (β 4 Thr244/α 3 Thr243), 5′ (β 4 Ile247/α 3 Ile246), and 6′ (β 4 Thr248/α 3 Thr247) positions (Fig. 9b) . This site is consistent with the location of a typical channel blocker in the pore, below the channel gate and immediately above the narrowest part of an open-channel selectivity filter.

The potential clinical significance of cocaine's suppression of α 3 β 4 -nAChR function Cocaine inhibits natural nAChR-mediated currents in VTA DA neurons and consequently increases the ratio of phasic to tonic DA release, thus potentially enhancing its reinforcing effects [5] . On the other hand, blocking nAChRs may mediate cocaine craving and addiction [41, 42] . Although α 3 β 4 nAChRs are most highly expressed in the medial habenula, interpeduncular nucleus and fasciculus retroflexus, they are expressed at low levels in the mesolimbic reward circuitry [43] and are involved in the rewarding effects of cocaine and possibly other psychostimulants [22] [23] [24] 44] . This idea is supported by recent studies showing that α 3 β 4selective nAChR ligands AT-1001 and AT-1012, which do not induce a place preference or aversion in mice per se, significantly and dose-dependently suppress the acquisition of CPP induced by a low dose of cocaine [27] .

Cocaine addiction is a serious social, medical, and economic problem worldwide. Countless attempts have been made to find compounds that will displace abused drugs, such as cocaine, from their sites of action in the nervous system. Thus far, no satisfactory drugs exist for the treatment of cocaine reward and dependence. Therefore, the finding of cocaine-directed inhibition of native α 3 β 4 nAChRs is highly significant for understanding the targets and mechanisms of cocaine and potential therapeutics for its abuse. Additionally, the high selectivity of cocaine for α 3 β 4 nAChRs might contribute to the cardiovascular effects of cocaine. It is well known that the α 3 β 4 subunit combination is prevalent in the nAChRs of autonomic ganglia neurons. nAChRs with the α 3 β 4 combination are also called "ganglia-type nAChRs". The inhibition of α 3 β 4 nAChRs by a low dose of cocaine may result in the modulation of cholinergic pathways in the neurons of autonomic ganglia, thereby inducing the possible adverse cardiovascular effects of cocaine. Furthermore, recent studies showed that α 3 β 4 nAChRs are predominantly expressed in human chromaffin cells, where they play a role in exocytosis [45] . This may be one of the mechanisms involved in the cardiovascular effects of cocaine. Therefore, the finding of cocaine-directed inhibition of native α 3 β 4 nAChRs is highly significant for understanding the targets and mechanisms of cocaine and potential therapeutics for its abuse.

In conclusion, using patch-clamp whole-cell recordings in cultured SH-SY5Y cells, we demonstrate that cocaine potently inhibits α 3 β 4 nAChR-mediated whole-cell currents with an IC 50 value of 1.5 µM. Cocaine-induced inhibition of the α 3 β 4 nAChRmediated current occurs in a noncompetitive manner through an open-channel block mechanism. This inhibition is not mediated by the alteration of α 3 β 4 nAChR internalization. In addition, the same concentration (1.5 µM) of cocaine fails to alter α 4 β 2 and α 6 * nAChR function. Based on these experimental results, we conclude that cocaine is a potent blocker of native α 3 β 4 nAChRs in SH-SY5Y cells.

Different effects of cocaine and nomifensine on dopamine uptake in the caudate-putamen and nucleus accumbens

Cocaine receptors on dopamine transporters are related to self-administration of cocaine

Pharmacological characterization of nicotine's interaction with cocaine and cocaine analogs

Subtype-selective inhibition of neuronal nicotinic acetylcholine receptors by cocaine is determined by the alpha4 and beta4 subunits

Cocaine inhibition of nicotinic acetylcholine receptors influences dopamine release

Influence of cocaine use on cigarette smoking

Psychostimulant-induced behavioral sensitization depends on nicotinic receptor activation

Effects of nicotinic and NMDA receptor channel blockers on intravenous cocaine and nicotine selfadministration in mice

Molecular mechanism for a gateway drug: epigenetic changes initiated by nicotine prime gene expression by cocaine

Differences between nicotine and cocaineinduced conditioned place preferences

Nicotine receptor inactivation decreases sensitivity to cocaine

alpha4beta2 Nicotinic receptor desensitizing compounds can decrease self-administration of cocaine and methamphetamine in rats

Effects of varenicline on the reinforcing and discriminative stimulus effects of cocaine in rhesus monkeys

Contribution of dihydro-beta-erythroidine sensitive nicotinic acetylcholine receptors in the ventral tegmental area to cocaine-induced behavioral sensitization in rats

Inhibition of both alpha7* and beta2* nicotinic acetylcholine receptors is necessary to prevent development of sensitization to cocaine-elicited increases in extracellular dopamine levels in the ventral striatum

Differential roles of alpha6beta2* and alpha4beta2* neuronal nicotinic receptors in nicotine-and cocaine-conditioned reward in mice

Cocaine directly inhibits alpha6-containing nicotinic acetylcholine receptors in human SH-EP1 cells and mouse VTA DA neurons

Molecular and physiological diversity of nicotinic acetylcholine receptors in the midbrain dopaminergic nuclei

Measuring nicotinic receptors with characteristics of alpha4beta2, alpha3beta2 and alpha3beta4 subtypes in rat tissues by autoradiography

Alpha3beta4 subunitcontaining nicotinic receptors dominate function in rat medial habenula neurons

Activation of nicotinic acetylcholine receptors increases the frequency of spontaneous GABAergic IPSCs in rat basolateral amygdala neurons

Alpha-5/alpha-3 nicotinic receptor subunit alleles increase risk for heavy smoking

Genome-wide and candidate gene association study of cigarette smoking behaviors

The CHRNA5-CHRNA3-CHRNB4 nicotinic receptor subunit gene cluster affects risk for nicotine dependence in African-Americans and in European-Americans

The CHRNA5/A3/B4 gene cluster variability as an important determinant of early alcohol and tobacco initiation in young adults

A variant associated with nicotine dependence, lung cancer and peripheral arterial disease

High affinity alpha3beta4 nicotinic acetylcholine receptor ligands AT-1001 and AT-1012 attenuate cocaineinduced conditioned place preference and behavioral sensitization in mice

Characterization of human alpha 4 beta 2-nicotinic acetylcholine receptors stably and heterologously expressed in native nicotinic receptor-null SH-EP1 human epithelial cells

beta-Amyloid directly inhibits human alpha4beta2-nicotinic acetylcholine receptors heterologously expressed in human SH-EP1 cells

Characterization of nicotinic acetylcholine receptors expressed by cells of the SH-SY5Y human neuroblastoma clonal line

Structureactivity studies of 7-heteroaryl-3-azabicyclo[3.3.1]non-6-enes: a novel class of highly potent nicotinic receptor ligands

Pharmacological and functional comparisons of alpha6/alpha3beta2beta3-nAChRs and alpha4beta2-nAChRs heterologously expressed in the human epithelial SH-EP1 cell line

Electrophysiological, pharmacological, and molecular evidence for alpha7-nicotinic acetylcholine receptors in rat midbrain dopamine neurons

Functional properties of homomeric, human alpha 7-nicotinic acetylcholine receptors heterologously expressed in the SH-EP1 human epithelial cell line

Distinctive nicotinic acetylcholine receptor functional phenotypes of rat ventral tegmental area dopaminergic neurons

Role of AMPA receptor cycling in synaptic transmission and plasticity

Roles of nicotinic acetylcholine receptor beta subunits in function of human alpha4-containing nicotinic receptors

Structural model for gamma-aminobutyric acid receptor noncompetitive antagonist binding: widely diverse structures fit the same site

Mechanism-based approach to the successful prevention of cocaine inhibition of the neuronal (alpha 3 beta 4) nicotinic acetylcholine receptor

Kinetic and pharmacological characterization of desensitizing and non-desensitizing glutamate-gated chloride channels in cockroach neurons

The nicotinic acetylcholine receptor partial agonist varenicline and the treatment of drug dependence: a review

The role of acetylcholine in cocaine addiction

Structural and functional diversity of native brain neuronal nicotinic receptors

Comparison of the reinforcing properties of nicotine and cigarette smoke extract in rats

Human nicotinic receptors in chromaffin cells: characterization and pharmacology

This work was supported by grants from Barrow Neuroscience Foundation and NIDA (DA11064). Authors thank Kellen Vu for his assistance with editing the English for this manuscript.

Conflict of interest: The authors declare that they have no conflict of interest.