Introduction

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Although cocaine binds to a variety of nervous system sites, its potency for reinforcing effects are best correlated with binding to the dopamine transporter (Ritz et al., 1987). The reinforcing effects of cocaine are attenuated in dopamine transporter and dopamine receptor knockout mice (Xu et al., 1994; Giros et al., 1996). In general, these findings are consistent with an obligatory role of the dopaminergic system in mediating the reinforcing properties of cocaine (see, however, Rocha et al., 1998). Thus, the functional effects and structural basis for cocaine interactions with neurotransmitter transporters (for dopamine and serotonin in particular) have justifiably received considerable attention. In contrast, evaluation of the potential for and functional relevance of cocaine effects on other critical elements in synaptic function has lagged behind. Central and peripheral effects of cocaine independent of binding to the dopamine transporter probably contribute to its addictive potential and toxicity profile.

Although cocaine is an effective inhibitor of voltage-gated sodium channels (resulting in its local anesthetic activity) and the structural basis for this interaction has been well characterized (Ragsdale et al., 1994; Li et al., 1999), few studies have examined interactions between cocaine and ligand-gated ion channels (LGICs) of the nervous system. This fact is perhaps somewhat surprising, because much of our current understanding of the pore structure and gating kinetics of an entire family of structurally related LGIC subtypes has arisen from the use of quaternary local anesthetic derivatives as structural probes of the neuromuscular junction subtype of nicotinic acetylcholine receptor (nAChR). Cocaine shares key structural features with other local anesthetics (specifically an ionizable amine group and a hydrophobic or aromatic moiety) and, like other local anesthetics, noncompetitively inhibits muscle nAChR (Swanson and Albuquerque, 1987; Niu et al., 1995). However, little information is available about the potential for inhibition of nervous system nAChRs by the cocaine concentrations normally associated with abuse and/or toxicity.

Neuronal nAChRs are pentameric LGICs that function either as homomers (7-9) or as heteromeric combinations of  (2-4, 6) and  (2 and 4) subunits. Brain nAChR subtypes have been implicated in a variety of central nervous system (CNS) processes, including presynaptic regulation of neurotransmitter release (for review, see Wonnacott, 1997). Moreover, activation and/or subsequent desensitization of CNS nAChR subtypes is believed to underlie behavioral addiction to nicotine (for review, see Dani and Heinemann, 1996). Additionally, neuronal nAChR subtypes of the peripheral nervous system are critical elements for transmission in sympathetic and parasympathetic ganglia. A few recent studies have begun to characterize interactions between central effects of cocaine and nicotine (Lerner-Marmarosh et al., 1995; Damaj et al., 1999); however, a clear picture of the effects of cocaine on the diverse subtypes of neuronal nAChRs has yet to take shape. The goals of the present study are to assess the potential for subtype-specific effects of cocaine on neuronal nAChRs and evaluate the underlying structural determinants.

	Materials and Methods

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Chemicals. Acetylcholine stock solutions were made daily and diluted to the working concentration in oocyte saline solution. Cocaine hydrochloride was provided by the National Institute on Drug Abuse and concentrated stock solutions were stored frozen in aliquots. All other chemicals were purchased from Sigma (St. Louis, MO) unless otherwise noted.

Production of Chimeric cDNAs and Mutagenesis. Rat nAChR cDNA clones were provided by Drs. Steve Heinemann and Jim Boulter (Salk Institute, La Jolla, CA). The production and functional evaluation of chimeric  subunits has been described previously (Papke et al., 1993). For these chimeras (2₂₂₈/4 and 4₂₃₀/2), the numeric subscript refers to the number of N-terminal amino acids contributed by a given  subunit. Chimeric nicotinic receptor  subunit cDNAs were created using homologous restriction sites conserved between  subunit sequences. Specifically, a BstXI site located in a stretch of conserved sequence that codes for the extracellular loop region between M2 and M3 was used to create chimeric subunits that exchange sequence between 3 and 4 (3₂₆₇/4 and 4₂₆₇/3). Similarly, a homologous PstI site located immediately after sequence coding for the first of the two cysteines in the N-terminal extracellular domain (which form the cysteine loop characteristic of the nicotinic gene family) was used to create chimeric subunits which exchange sequence between 2 and 4 (2₁₂₈/4 and 4₁₂₈/2). In each case, the numeric subscript refers to the amino acid immediately preceding the site at which the coding sequence is exchanged.  Subunit amino acids are numbered according to the system adopted for the muscle  subunit, in which the vicinal cysteine residues characteristic of nicotinic receptor  subunits are numbered 192 and 193. Restriction fragments were isolated from an agarose gel using the Geneclean III kit (Bio 101,Vista, CA) and subsequently ligated by homologous end ligation. After bacterial transformation, chimeric DNAs were evaluated by restriction digest and subsequent DNA sequencing.

Preparation of RNA and Oocyte Injection. After linearization and purification of cloned cDNAs, RNA transcripts were generated using the mMessage mMachine in vitro RNA transcription kit (Ambion, Austin, TX). Resultant RNA transcripts were evaluated by UV spectroscopy and denaturing agarose gel electrophoresis (visualized with ethidium bromide). RNAs were diluted to a concentration of 600 ng/µl and stored frozen in ribonuclease-free water at 80°C.

Electrophysiology. Two-electrode voltage-clamp recordings were made at room temperature in oocyte saline solution supplemented with 1.8 mM CaCl₂ and 1 µM atropine. All recordings were made using a Turbo Tec 01C amplifier (Adams & List, Westbury, NY) at a holding potential of 50 mV unless otherwise noted. Recording electrodes were filled with 3 M KCl and typically had resistances in the range of 0.5 to 3 M.

G°

	Results

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Cocaine Inhibition of nAChR Subtypes. Because the oocyte expression system permits regulated expression of nAChRs of known subunit composition, it is possible to associate pharmacological effects with specific subunit types. The effect of application of 10 µM cocaine with 30 µM ACh on a number of nAChR subunit combinations is shown in Fig. 1A, whereas inhibition curves for the various subunit combinations are shown in Fig. 1B (IC₅₀ values are summarized in Table 1). The relative efficacy of the 30 µM agonist concentration in the absence of cocaine ranged from 41 to 58% of the maximal current (I_max) for all of the wild-type receptor combinations tested with the exception of 22 and 24 (12 and 18%, respectively; Table 2). Because of the lower potency for activation of 22 and 24 receptors, the effects of cocaine were also tested in combination with an agonist concentration near the EC₅₀ value for these receptor subtypes (130 µM ACh). No significant difference in cocaine IC₅₀ value was detected for either subunit combination (data not shown).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Cocaine inhibition of nAChR subtypes. A, responses of neuronal nAChRs to 30 µM ACh in the absence and presence (middle trace) of 10 µM cocaine. ACh was applied for 15 s in each case, and the bar above the first trace (upper left) shows the timing of the application. In this figure and subsequent figures, the trace at left is the response to a control application of ACh alone, the middle trace is the response to application of ACh with cocaine, and the trace at right is the response to a second control application of ACh representing recovery from inhibition. Each application is separated by approximately 4 min. B, concentration dependence of cocaine inhibition of nAChR. Each data point represents the mean response of at least four oocytes to the coapplication of 30 µM ACh with varying concentrations of cocaine relative to 30 µM ACh alone. C, G° and G° values associated with cocaine binding to nAChR subunit combinations. Boxed values indicate G° values for comparisons of 2- versus 4-containing receptors, and circles indicate G° values for 4- versus 3- and 2-containing receptors.

Contribution of  Subunit to Inhibition by Cocaine. Individual nicotinic receptor subunits share a conserved membrane topology composed of an N-terminal extracellular segment followed by four hydrophobic transmembrane domains. Comparison of the 2 and 4 subunit amino acid sequences reveals a number of differences in the N-terminal putative extracellular domains of the two subunits (Fig. 2A). In contrast, only a single amino acid differs between the pore-lining second transmembrane (M2) domains of the two subunits. To distinguish between these two potential regions of cocaine interaction (N terminus versus membrane-spanning domains), the effects of cocaine were tested on receptors incorporating chimeric  subunits in which all sequence N-terminal to M1 is exchanged between 2 and 4 (Fig. 2B). Functional effects of these exchanges on the kinetics of inhibition by neuronal bungarotoxin have been evaluated previously (Papke et al., 1993).

View larger version (52K): [in this window] [in a new window]   Fig. 2.   Cocaine inhibition of  subunit chimeras. A, sequence alignment of nAChR  subunits. Identical amino acids are shaded black. Conservative differences are in gray. The four putative transmembrane domains are boxed. *, 13' position. The position of the break point in the chimeras is shown. B, schematic depicting structure of  subunit chimeras. In this figure and in Fig. 6, the thicker bars represent putative transmembrane regions. C, responses of neuronal nAChRs to an application of 30 µM ACh in the absence and presence (smaller amplitude trace) of 10 µM cocaine. Note the kinetic differences in the control responses. The length of the agonist application is shown by the bar above the traces. D, the mean response of nAChR subtypes to the coapplication of 30 µM ACh with 10 µM cocaine. Each column represents the mean (±S.E.) of at least four oocytes.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Cocaine inhibition of 13'  subunit mutant nAChRs. A, responses of 13' mutant receptors to 30 µM ACh in the absence and presence (middle trace) of 10 µM cocaine. Each application is separated by approximately 4 min and the bar above the first trace shows the timing of the agonist application. B, concentration dependence of cocaine inhibition of nAChR. Each data point represents the mean response (±S.E.) of at least four oocytes to the coapplication of 30 µM ACh with varying concentrations of cocaine and is normalized to a 30 µM ACh control response.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Effects of mutation of  subunit 13' residue on activation by acetylcholine. A, concentration-response data for acetylcholine activation of 34 and 3413'V receptors expressed relative to a control application of 30 µM ACh. B, concentration-response data for acetylcholine activation of 32 and 3213'F receptors expressed relative to the ACh maximum. Each data point represents the mean (±S.E.) of three to eight responses.

Contribution of  Subunit to Inhibition by Cocaine. The N-terminal domain of nicotinic receptor  subunits includes at least three stretches of amino acids previously demonstrated to contribute to agonist binding (loops A-C; for review, see Changeux and Edelstein, 1998). Thus, it is possible that cocaine inhibits 42 receptors by a competitive mechanism involving amino acids in the N terminus of the 4 subunit. Alternatively, cocaine may inhibit 42 receptors by a noncompetitive mechanism involving binding to either extracellular or pore sites (including the N and C termini, transmembrane domains, and extracellular loop region between M2 and M3).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Cocaine inhibition of 42 receptors as a function of agonist concentration. Acetylcholine (10 µM-1 mM) was applied in the absence and presence of 30 µM cocaine. Each data point is expressed relative to the ACh maximum and represents the mean (±S.E.) of three to six responses.

View larger version (70K): [in this window] [in a new window]   Fig. 6.   Cocaine inhibition of  subunit chimeras. A, sequence alignment of nAChR  subunits. Identical amino acids are shaded black. Conservative differences are in gray. The three principle loops believed to participate in agonist binding and the four putative transmembrane domains are denoted. The conserved restriction sites for PstI and BstXI used in constructing the 2/4 and 3/4 subunit chimeras are also shown. Numbering is relative to the vicinal cysteines (192 and 193) indicated by the asterisk. B, schematic depicting structure of  subunit chimeras. Chimeric subunits were created using homologous restriction sites shared between either 2 and 4 (PstI) or 3 and 4 (BstXI). C, responses of  subunit chimeric receptors to 30 µM ACh in the absence and presence (middle trace) of 30 µM cocaine. The bar above the first trace shows the timing of the agonist application. D, the mean response of nAChR subtypes to the coapplication of ACh with cocaine. Each column represents the mean relative response (±S.E.) of four or more oocytes to the application of 30 µM ACh with 30 µM cocaine.

Voltage Dependence of Cocaine Inhibition. As a second approach to localizing determinants of cocaine affinity, we sought to evaluate the disposition of cocaine binding sites with respect to the membrane electric field. The effects of changes in holding potential on inhibition were tested for the 34, 32_13'F, and 42 subunit combinations (Fig. 7). These subunit combinations were chosen to ensure that only a single class of binding site was evaluated in each case. For example, in the case of 34 receptors, any effects of holding potential on inhibition are likely associated primarily with a site on the 4 subunit because 32 receptors show little inhibition by cocaine at the concentration used in these experiments (10 µM; Fig. 1). Similarly, in the case of 42 receptors, it is likely that any observed effects of holding potential are primarily associated with the 4 subunit because the 2 subunit shows only low cocaine affinity.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Voltage dependence of cocaine inhibition of 34 and 42 nAChRs. Acetylcholine (30 µM) was applied in the absence and presence of either 10 µM (34) or 20 µM (42) cocaine after a brief equilibration period at the specified holding potential. Each data point represents the mean (±S.E.) response of four or more cells.

	Discussion

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Although the addictive properties of cocaine are likely mediated via interactions with neurotransmitter transporters, our data indicate that cocaine can also inhibit members of the nicotinic family of nervous system LGICs. Serum concentrations of cocaine can rise to a peak near 1 µM within 30 min after nasal insufflation (Jeffcoat et al., 1989) and brain concentrations are likely to be higher because of the lipophilic nature of the drug (Nayak et al., 1976). Our data indicate that cocaine inhibits particular nAChR subtypes with an affinity comparable with that displayed for the open/inactivated state of voltage-gated sodium channels (~10 µM; Wright et al., 1997), suggesting the possibility of neuronal nAChR-mediated cocaine effects. Cocaine potency shows considerable variability across nAChR subtypes, with IC₅₀ values ranging between 2 µM (44) and 60 µM (32). Although inhibition of the 34 subunit combination is voltage-dependent and affected by mutation of the 13' residue of the pore-lining M2 domain, inhibition of the 42 subtype shows only very weak voltage dependence and is probably associated with N-terminal sequence immediately preceding M1 (Fig. 8).

View larger version (32K): [in this window] [in a new window]   Fig. 8.   Putative membrane topology of nAChR subunits and regions involved in determining cocaine affinity. In these studies, a stretch of sequence immediately preceding and inclusive of M1 and M2 of the 4 subunit (top, gray) is shown to contribute to determination of cocaine affinity. Based on the degree of sequence conservation in the membrane spanning domains of the  subunits, a smaller region preceding M1 (top, black) probably contains the cocaine binding site in 4. A second site associated with the presence of a phenylalanine residue (F) at the 13' position of the pore-lining M2 segment of the 4 subunit (bottom) also can determine cocaine affinity. The lower affinity 2 subunit has a valine residue (V) at this position.

The present study also identified  subunit residues important in the processes of receptor desensitization (Fig. 2) and activation (Fig. 4). Although the observed effects on receptor desensitization are associated with the N-terminal portion of the  subunit, the most significant changes in receptor activation properties are associated with mutation of the 13' residue of the 4 subunit. The processes of desensitization and binding/gating do not seem to be determined by common sequence elements because mutation of the 13' site does not yield appreciable differences in response time course (Fig. 3). A number of previous studies have illustrated effects of mutation of pore residues on channel gating. Most notably, the 9' position has also been implicated in the gating/binding process (Revah et al., 1991; Filatov and White, 1995; Labarca et al., 1995; Kearney et al., 1996). The observation that mutation of the 13' position has differential effects on receptor activation across the 2 and 4 subunits emphasizes the idea that sequence homologues in the pore region are not necessarily functional homologues.

Inhibition of 34 Receptors May Involve Open-Channel Block. Receptors incorporating the 3 subunit with chimeric subunits that pair N-terminal 2 sequence with 4 sequence C-terminal to M1 show sensitivity to cocaine inhibition comparable with wild-type 34 receptors whereas the reciprocal chimeric receptors (N-terminal 4 sequence with C-terminal 2 sequence) are inhibited by cocaine with a potency similar to wild-type 32 receptors. This result localizes the site of high-affinity cocaine binding to the C-terminal portion of the 4 subunit beyond M1. Within this region, exchange of the 13' site between 2 and 4 has reciprocal effects on the concentration dependence of cocaine inhibition, implicating the 13' position as a determinant of cocaine binding affinity in 34 receptors. Consistent with binding to a site in the ion channel pore, cocaine inhibition of 34 receptors exhibits voltage dependence. Given this observation and the dependence on sequence in the M2 region, cocaine may act as a conventional open-channel blocker on the 34 subtype of nAChR. However, the observation that cocaine has a lower potency at 32_13'F receptors compared with wild-type 34 receptors or chimeric 32₂₂₈/4 receptors may indicate that other sequence elements not contained within the M2 domain can contribute to the disposition of the cocaine binding site. Residues in the adjacent membrane-spanning domains or connecting loops could contribute directly to a cocaine-binding site or otherwise affect the exposure of the 13' residue to the channel pore. Additionally, because the functional role of the 13' position in receptor activation does not seem to be maintained across classes of  subunit (Fig. 4), exposure of this residue may differ during gating of the two receptor subtypes.

Potential Allosteric Inhibition of 42 Receptors. The  subunit chimeras localize a determinant of cocaine affinity to a region that includes a portion of the N-terminal extracellular segment with M1 and M2. On the basis of sequence identity alone, contributions from a large number of residues in this region, particularly residues in the membrane-spanning domains, can be eliminated (Fig. 6A). By concentrating on residues that differ between 4 and the other  subunits, a likely determinant of cocaine affinity can be further localized to a relatively nonconserved stretch of 50 amino acids immediately preceding M1. A region containing the vicinal cysteine pair thought to be a component of the acetylcholine binding site (loop C) is also contained within this stretch of amino acids, suggesting that cocaine may act in the vicinity of the agonist binding site. Consistent with a previous study (Lerner-Marmarosh et al., 1995), our results indicate that cocaine is not competitive with ACh over a 100-fold range in ACh concentration (30 µM-3 mM; Fig. 5). Therefore, cocaine probably binds to an allosteric site on 42 receptors and inhibits activation in a noncompetitive fashion.

Possible Functional Relevance of Cocaine Effects on nAChRs. Nicotinic receptors containing the 4 and 2 subunits are the predominant form of high-affinity nicotine receptors in the brain and the activation and/or subsequent desensitization of this subunit combination by nicotine is believed to be responsible for the addictive properties of the drug (Peng et al., 1994; Dani and Heinemann, 1996; Picciotto et al., 1998). Thus, this receptor type could provide a site of interaction for cocaine and nicotine effects. Synergistic effects between nicotine and the noncompetitive inhibitor mecamylamine on receptor turnover have been described previously (Peng et al., 1994).