Abstract
The plant alkaloid methyllycaconitine (MLA) is considered to be a selective antagonist of the α7 subtype of neuronal nicotinic acetylcholine receptor (nAChR). However, 50 nM MLA partially inhibited (by 16%) [3H]dopamine release from rat striatal synaptosomes stimulated with 10 μM nicotine. Other α7-selective antagonists had no effect. Similarly, MLA (50 nM) inhibited [3H]dopamine release evoked by the partial agonist (2-chloro-5-pyridyl)-9-azabicyclo[4.2.1]non-2-ene (UB-165) (0.2 μM) by 37%. In both cases, inhibition by MLA was surmountable with higher agonist concentrations, indicative of a competitive interaction. At least two subtypes of presynaptic nAChR can modulate dopamine release in the striatum, and these nAChR are distinguished by their differential sensitivity to α-conotoxin-MII (α-CTx-MII). MLA was not additive with a maximally effective concentration of α-CTx-MII (100 nM) in inhibiting [3H]dopamine release elicited by 10 μM nicotine or 0.2 μM UB-165, suggesting that both toxins act at the same site. This was confirmed in quantitative binding assays with 125I-α-CTx-MII, which displayed saturable specific binding to rat striatum and nucleus accumbens withBmax values of 9.8 and 16.5 fmol/mg of protein, and Kd values of 0.63 and 0.83 nM, respectively. MLA fully inhibited 125I-α-CTx-MII binding to striatum and nucleus accumbens with a Kivalue of 33 nM, consistent with the potency observed in the functional assays. We speculate that MLA and α-CTx-MII interact with a presynaptic nAChR of subunit composition α3/α6β2β3* on dopamine neurons. The use of MLA as an α7-selective antagonist should be exercised with caution, especially in studies of nAChR in basal ganglia.
Nicotinic acetylcholine receptors (nAChR) are modulators of neuronal function in the central nervous system (Role and Berg, 1996). One way in which nAChR can modify neuronal activity is by facilitating neurotransmitter release, and this may be accomplished by presynaptic or somatodendritic nAChR (Wonnacott, 1997). Dopamine release in rodent striatum may be modulated by presynaptic nAChR on dopamine terminals (Wonnacott, 1997; Zhou et al., 2001) as well as somatodendritic nAChR on dopamine neurons in the substantia nigra (Clarke et al., 1987;Reuben et al., 2000; Klink et al., 2001). The nigrostriatal system has merited attention because of its relevance to the motor stimulant and addictive properties of nicotine and because these nAChR represent potential therapeutic targets for the treatment of Parkinson's disease.
Neuronal nAChR are pentameric ligand-gated cation channels, comprised of one or more different subunits, from a portfolio of nine α (α2–α10) and three β (β2–β4) subunits expressed in the nervous system (Lukas et al., 1999; Elgoyhen et al., 2001). Determination of the subunit composition of native nAChR presents a major challenge. This endeavor is hampered by a lack of subtype-selective nicotinic ligands, the majority of those available are antagonists derived from natural products. The α-conotoxins comprise a family of peptide toxins elaborated by Conus sp. and directed against particular nAChR subtypes (McIntosh et al., 1999). From studies of functional nAChR expressed in Xenopusoocytes, α-conotoxin-MII (α-CTx-MII) was originally defined as a specific antagonist of nAChR composed of α3 and β2 subunits (Cartier et al., 1996; Harvey et al., 1997).125I-α-CTx-MII labels a unique population of nicotinic binding sites in mouse and monkey brain (Whiteaker et al., 2000; Quik et al., 2001). This toxin partially blocks [3H]dopamine release from rat striatal synaptosomes stimulated with nicotine (Kulak et al., 1997) or anatoxin-a (Kaiser et al., 1998), providing evidence for the heterogeneity of presynaptic nAChR on dopamine terminals, with one population containing an α3β2 interface. Studies with the novel partial agonist UB-165 led us to propose an α4β2* nAChR subtype as a candidate for the other nAChR population (Sharples et al., 2000). The contribution of the β2 subunit to both nAChR subunits is consistent with the localization of β2 nAChR subunit immunoreactivity in most dopaminergic terminals in the dorsal striatum (Jones et al., 2001) and the absence of nicotine-evoked dopamine release from striatal synaptosomes or slices prepared from β2 null mutant mice (Grady et al., 2001; Zhou et al., 2001). However, the subunit composition of striatal presynaptic nAChR is likely to be more complex than pairwise combinations of subunits and this is denoted by the asterisk (Lukas et al., 1999).
Midbrain dopamine neurons express most nAChR subunits (Klink et al., 2001), including a particularly high expression of mRNA corresponding to the α6 and β3 nAChR subunits (Le Novère et al., 1996). Patch-clamp recording and single cell polymerase chain reaction analysis of rat midbrain dopamine neurons, and comparison with data from transgenic mice deficient in a particular nAChR subunit, led to the tentative subunit compositions α4α6/α3α5(β2)2 and (α4)2α5(β2)2 for two major nAChR subtypes found on cell bodies of these neurons in the substantia nigra and ventral tegmentum (Klink et al., 2001). Typical fast desensitizing α7-type nAChR currents are also observed in some midbrain dopamine neurons (Pidoplichko et al., 1997; Klink et al., 2001), but α7* nAChR do not seem to mediate [3H]dopamine release from striatal synaptosomes as the α7-selective antagonists α-bungarotoxin (αBgt) and α-conotoxin-ImI (α-CTx-ImI) are without effect (Rapier et al., 1990; Kulak et al., 1997). However, we previously noted that 50 nM methyllycaconitine (MLA), a potent α7-selective antagonist, produced a partial inhibition of anatoxin-a-evoked [3H]dopamine release from striatal synaptosomes (Kaiser and Wonnacott, 2000), in agreement with the previous observation of Clarke and Reuben (1996). Klink et al. (2001) have also shown that low nanomolar concentrations of MLA inhibit a population of non-α7 nAChR currents, correlated with the tentative subunit composition α4α6α5(β2)2, in the cell bodies of mesencephalic dopamine neurons.
MLA is a hexacyclic norditerpenoid alkaloid, isolated fromDelphinium sp. (Wonnacott et al., 1993). MLA is a competitive nicotinic antagonist with approximately 100-, 1,000-, and 10,000-fold higher affinity for α7 nAChR, compared with α3β2, α4β2, and muscle nAChR, respectively (Alkondon et al., 1992; Drasdo et al., 1992; Wonnacott et al., 1993). The pharmacology and distribution of [3H]MLA binding sites in rodent brain tissue corresponds well with that of125I-αBgt binding sites (Davies et al., 1999;Whiteaker et al., 1999), and sensitivity to low nanomolar concentrations of MLA has been interpreted as evidence for α7 nAChR. However, in avian neuronal preparations discrepancies between MLA- and αBgt-sensitive responses have been reported (Yum et al., 1996; Yu and Role, 1998), raising the possibility that minority populations of neuronal nAChR with particular subunit combinations may show differential sensitivity to these two toxins.
In this study, we have characterized pharmacologically the MLA-sensitive portion of nAChR-mediated [3H]dopamine release from striatal synaptosomes. We show that nanomolar concentrations of MLA inhibit the same nAChR population as α-CTx-MII. Using125I-α-CTx-MII to label nAChR directly, we confirm that MLA potently interacts with this site. The relationship between presynaptic and somatodendritic nAChR on dopamine neurons, with respect to subunit composition, is discussed.
Materials and Methods
Sprague-Dawley rats were obtained from the University of Bath Animal House (Bath, UK) breeding colony and the Health Sciences Center (Denver, CO). [7,8-3H]Dopamine ([3H]dopamine, specific activity 1.78 TBq/mmol) and 125I autoradiographic microscales were purchased from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). α-CTx-MII was provided by Tocris Cookson (Bristol, UK). α-CTx-ImI, MLA, (−)-nicotine, αBgt, mecamylamine, pargyline, and nomifensine were obtained from Sigma Chemical (Poole, Dorset, UK). (±)-UB-165 was synthesized by Professor T. Gallagher (University of Bristol, Bristol, UK) as described previously (Wright et al., 1997). 125I-α-CTx-MII was synthesized by addition of an N-terminal tyrosine and subsequent iodination, as described by Whiteaker et al. (2000). All other chemicals used were of analytical grade and obtained from standard commercial sources.
Superfusion of Synaptosomes.
[3H]Dopamine release from rat striatal synaptosomes was measured as described previously (Kaiser et al., 1998). In brief, male Sprague-Dawley rats (250–350 g) were killed by cervical dislocation and decapitated, and striata (including dorsal striatum and nucleus accumbens; 180–240 mg wet tissue/rat) were rapidly dissected. P2 synaptosomes were obtained by homogenization followed by differential centrifugation and were loaded with [3H]dopamine (0.1 μM, 0.132 MBq/ml) for 15 min at 37°C. Synaptosomes were deposited on GF/B filter discs (Whatman, Maidstone, UK) in open chambers of a superfusion apparatus (model SF-12; Brandel, Gaithersburg, MD), with striata from two rats providing enough tissue for 12 superfusion chambers. Synaptosomes were superfused with Krebs-bicarbonate buffer of the following composition: 118 mM NaCl, 2.4 mM KCl, 2.4 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, and 10 mM glucose, pH 7.4, saturated with 95% O2, 5% CO2 and supplemented with 1 mM ascorbic acid, 8 μM pargyline, and 0.5 μM nomifensine to prevent dopamine degradation and reuptake, respectively. The flow rate was 0.5 ml/min.
After a 20-min washout period, the synaptosomes were superfused for a further 10 min in Krebs' buffer with or without antagonist (except in the case of αBgt, which was present in the perfusate for 1 h). The nicotinic agonists (−)-nicotine (10 or 100 μM) or (±)-UB-165 (0.2 or 1 μM) were then applied for 40 s, alone or in combination with antagonist, separated from the bulk buffer flow by 10-s air bubbles. Two-minute fractions were collected and counted in a TRI-CARB liquid scintillation analyzer (model 1600, counting efficiency 48%; Packard Instrument Company, Inc., Downers Grove, IL).
Radioactivity remaining in the synaptosomes at the end of the experiment was determined by counting the filters from the superfusion chambers. Total radioactivity present in synaptosomes at the time of agonist stimulation was calculated as the sum of subsequently released [3H]dopamine plus radioactivity remaining on the filters.
Superfusion Data Analysis.
Evoked tritium release above baseline was calculated as a percentage of the total radioactivity present in the synaptosomes immediately before stimulation. The baseline was derived, using SigmaPlot version 2.0 (Jandel Scientific, San Rafael, CA), by fitting the following double exponential decay equation to the data: y =ae−bx +ce−dx, where a andc are initial (at x = 0) release in each phase, b and d are the decay constants in each phase, and x is the fraction number.
Data are expressed as percentages of the corresponding controls, assayed in parallel in the absence of antagonist, and are the mean ± S.E.M. of several independent experiments, each consisting of two to three replicate chambers for each condition in each experiment. Statistical analyses were performed using the unpaired Student'st test, and one-way ANOVA with Tukey's post hoc test.
Quantitative Autoradiography of 125I-α-CTx-MII Binding.
125I-α-CTx-MII binding was assessed by quantitative autoradiography. The methods used were similar to those detailed in Whiteaker et al. (2000). Six male Sprague-Dawley rats (300–350 g) were killed by cervical dislocation, and the brains were removed from the skull and rapidly frozen by immersion in isopentane (−35°C, 30 s). The frozen brains were wrapped in aluminum foil, packed in ice, and stored at −70°C until sectioning. Tissue sections (20 μm in thickness) were prepared, using a Leica CM1850 cryostat/microtome refrigerated to −23°C, and thaw mounted onto subbed microscope slides (Richard Allen, Richland, MI). Slides were subbed by incubation with gelatin (1% w/v)/chromium aluminum sulfate (0.1% w/v) for 2 min at 37°C; drying overnight at 37°C, incubation at 37°C for 30 min in 0.1% (w/v) poly-l-lysine in 25 mM Tris, pH 8.0; and drying at 37°C overnight. Mounted sections were stored, desiccated, at −70°C until use. Twelve series of sections were collected from three brains for use in the saturation binding experiments, with 15 series being collected from the remaining brains for use in the inhibition binding experiments. Sections were collected from the front to the back of the striatum (approximately +2.2 mm to −1.8 mm relative to bregma).
Before incubation with 125I-α-CTx-MII, sections were incubated in binding buffer [144 mM NaCl, 1.5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 20 mM HEPES, and 0.1% (w/v) bovine serum albumin, pH 7.5] containing PMSF (1 mM; to inactivate endogenous serine proteases) at 22°C for 15 min. For all 125I-α-CTx-MII binding reactions, the standard binding buffer was also supplemented with 5 mM EDTA, 5 mM EGTA, and 10 μg/ml each of aprotinin, leupeptin trifluoroacetate, and pepstatin A to protect the ligand from endogenous proteases.
Saturation binding experiments were performed by incubation of six series of sections with concentrations of125I-α-CTx-MII varying between 0.05 and 2.5 nM to determine total binding at each concentration for 2 h at 22°C. Nonspecific binding [in the presence of 10 mM (−)-nicotine] was determined in a series of adjacent sections, and receptor-specific binding was determined as the difference between total and nonspecific binding at each concentration. The ability of MLA to inhibit125I-α-CTx-MII binding was determined by coincubation of sections with 0.5 nM125I-α-CTx-MII and varying concentrations of MLA (1–1000 nM) for 2 h at 22°C. After incubation with125I-α-CTx-MII, the slides were washed as follows: 60 s in binding buffer (twice at 22°C), 10 s in 0.1× binding buffer (twice at 0°C), and twice at 0°C for 10 s in 5 mM HEPES, pH 7.5. Sections were initially dried with a stream of air then by overnight storage at 22°C under vacuum.
Mounted, desiccated sections were apposed to film (3 days, Hyperfilm β-Max film; Amersham Biosciences UK). To allow quantitation, each film was also exposed to radioactive microscales. After the films had been exposed they were developed and signal intensity in selected brain regions was measured by digital image analysis. Films were illuminated using a Northern Light light box, and autoradiographic images of the sections and standards were captured using a charge-coupled device imager camera. Signal intensity was determined using NIH Image 1.61. Six independent measurements from different tissue sections were made for nucleus accumbens and striatum, under each incubation condition, for each rat. The optical density measurements for each brain area were averaged, and the mean optical density was used to calculate the degree of labeling by reference to the relevant standard curve.
Analysis of Autoradiography Experiments.
Results for saturation binding experiments were calculated using the Hill equationB = BmaxLn /(Ln +Kdn), whereB is the binding at the free ligand concentrationL, Bmax is the maximum number of binding sites, Kd is the equilibrium binding constant, and n is the Hill coefficient. Values of Bmax,Kd, and n were calculated using the nonlinear least-squares fitting algorithm of SigmaPlot version 5.0 (Jandel Scientific). Results for inhibition of125I-α-CTx-MII binding were calculated using a one-site fit: B =B0/(1 +I/IC50), where B is ligand bound at inhibitor concentration I,Bo is the binding in the absence of inhibitor, and IC50 is the concentration of inhibitor required to reduce binding to 50% ofBo. Values forKi (inhibition binding constant) were derived by the method of Cheng and Prusoff (1973):Ki = IC50/(1 +L/Kd).
Results
MLA Partially Inhibits Nicotine-Evoked [3H]Dopamine Release from Striatal Synaptosomes.
Nicotine-evoked [3H]dopamine release from rat striatal synaptosomes was monitored in the presence and absence of a variety of nicotinic antagonists. Nicotine (10 μM; delivered as a 40-s pulse) elicited a peak of radioactivity above basal release, and this was almost totally abolished in the presence of the general nAChR antagonist mecamylamine (10 μM; Fig.1A). The residual release was equivalent to the nonspecific release observed when agonist was replaced with buffer. In the presence of MLA (50 nM), a small decrease in the amount of nicotine-evoked [3H]dopamine release was consistently observed (Fig. 1, A and B).
The concentration-response relationship for the effect of MLA on [3H]dopamine release evoked by 10 μM nicotine (Fig. 1B) showed a significant (p < 0.01) inhibition of the response between 30 and 100 nM MLA. Higher concentrations of MLA were not examined because MLA becomes nonselective above 100 nM and would interact with various non-α7 subtypes of nAChR (Drasdo et al., 1992; Wonnacott et al., 1993). At 10 nM MLA, a concentration frequently used to inhibit α7 nAChR, there was a small but nonsignificant reduction in the response to 10 μM nicotine.
MLA (50 nM) was compared with other α7-selective nAChR antagonists for inhibition of nicotine-evoked [3H]dopamine release from rat striatal synaptosomes (Fig. 1C). Nicotine at a maximally effective (100 μM) and a submaximal concentration (10 μM; Fig. 1C, inset) were compared. At the higher concentration, nicotine elicited approximately 50% more [3H]dopamine release than 10 μM nicotine; in both cases the responses were decreased to the level of a buffer control in the presence of mecamylamine (10 μM). MLA significantly inhibited [3H]dopamine release evoked by 10 μM nicotine, by 16.3 ± 5.5% (n = 4,p < 0.05) but had no significant effect on release evoked by the higher concentration of nicotine (Fig. 1C). However, maximally effective concentrations of the α7-selective nAChR antagonists αBgt (40 nM) and α-CTx-ImI (1 μM; Pereira et al., 1996) did not diminish [3H]dopamine release from striatal synaptosomes stimulated with either concentration of nicotine.
The inhibition by MLA, but not by the other α7 nAChR antagonists, suggested that MLA was interacting with a non-α7 nAChR. To explore this possibility, other nAChR antagonists were used to clarify the subtype of nAChR involved. In the presence of 100 nM α-CTx-MII, a concentration that has been shown to selectively and maximally inhibit α3β2* nAChR (Cartier et al., 1996; Kaiser et al., 1998), [3H]dopamine release evoked by 10 μM nicotine was decreased by 35.5 ± 4.9% (n = 3,p < 0.01; Fig. 2). MLA also partially inhibited nicotine-evoked [3H]dopamine release, consistent with previous experiments (discussed above), but when coapplied with α-CTx-MII no additional inhibition of [3H]dopamine release was observed (26.2 ± 3.2% inhibition, n = 3,p < 0.01; Fig. 2).
The antagonist DHβE has a broad specificity for neuronal nAChR, but is more selective for non-α7-containing nAChR at low concentrations (1 μM; Harvey et al., 1996). At 1 μM, DHβE produced a substantial inhibition (62.7 ± 6.7%) of [3H]dopamine release evoked by 10 μM nicotine. Neither α-CTx-MII (100 nM) nor MLA (50 nM) in combination with DHβE (1 μM) showed any additivity with respect to the extent of inhibition observed (Fig. 2).
Subtype-selective agonists that activate only one of the nAChR subtypes present on striatal dopamine terminals would provide another approach to characterizing the action of MLA on striatal synaptosomes. We recently characterized the novel synthetic agonist UB-165 (Sharples et al., 2000), and showed it to be a partial agonist in releasing [3H]dopamine from striatal synaptosomes because it predominately activated the α-CTx-MII-sensitive nAChR subtype. From the effects of MLA on nicotine-evoked [3H]dopamine release (Figs. 1 and 2), we predicted that UB-165-evoked [3H]dopamine release should also be sensitive to low nanomolar concentrations of MLA. We compared two concentrations of UB-165: a maximally effective concentration (1 μM) and one that approximates to its EC50 value (0.2 μM; Sharples et al., 2000).
[3H]Dopamine release evoked by 0.2 and 1 μM UB-165 was 20 and 29%, respectively, of that elicited by 10 μM nicotine (Fig. 3). Mecamylamine (10 μM) reduced the responses of both concentrations of UB-165 to a similar level, approximately 7% of the nicotine response. α-CTx-MII (100 nM) and MLA (50 nM) inhibited [3H]dopamine release evoked by 0.2 μM UB-165 to a similar extent (38.9 ± 3.1 and 37.1 ± 3.9% inhibition, respectively), compared with 53.9 ± 6.0 and 61.3 ± 4.4% inhibition by DHβE (1 μM) and mecamylamine (10 μM), respectively (Fig. 3). Coapplication of α-CTx-MII and MLA produced no additive effect. At the higher agonist concentration (1 μM UB-165), α-CTx-MII and DHβE continued to inhibit the response (by 40.0 ± 7.2 and 57.0 ± 7.1%, respectively) but MLA no longer exerted any significant effect.
Inhibition by MLA of 125I-α-CTx-MII Binding.
The effects of MLA on nicotinic agonist-evoked [3H]dopamine release suggest that it acts competitively at the same nAChR subtype as α-CTx-MII. To address this proposition, 125I-α-CTx-MII was used to label nicotinic sites in rat striatum and nucleus accumbens, and the ability of MLA to displace this specific binding was investigated.125I-α-CTx-MII displayed saturable specific binding to striatum and nucleus accumbens (Fig.4A), withBmax values of 9.8 ± 1.3 and 16.5 ± 4.6 fmol/mg of protein andKd values of 0.63 ± 0.19 and 0.83 ± 0.55 nM, respectively. Competition assays with serial dilutions of MLA over the range 1 nM to 1 μM were carried out using 0.5 nM 125I-α-CTx-MII, a concentration approximating its Kd. MLA was able to fully displace specific binding of the radioligand to both striatum and nucleus accumbens (Fig. 4B), with Kivalues of 32.9 ± 12.9 and 34.6 ± 13.8 nM, respectively.
Discussion
The present study demonstrates that the presumed α7-selective compound MLA may antagonize other nAChR subtypes found at rat striatal dopaminergic nerve terminals, at concentrations frequently used to selectively block α7 nAChR. The ability of 50 nM MLA to partially inhibit nicotinic agonist-evoked [3H]dopamine release was consistently observed at low agonist concentrations, but was surmountable at higher agonist concentrations, suggesting a competitive mode of action. Because none of the other α7-selective compounds examined (αBgt and α-CTx-ImI) had any effect on nicotine-evoked [3H]dopamine release from striatal synaptosomes, this strongly argues that MLA is not acting through an α7 nAChR. Moreover, the lack of additivity with α-CTx-MII suggests that both antagonists act at the α-CTx-MII-sensitive nAChR subtype. Confirmation of this interaction was provided by the ability of low nanomolar concentrations of MLA to displace 125I-α-CTx-MII binding to rat striatum and nucleus accumbens.
The absence of a discernible α7 nAChR-mediated component in nicotine-evoked [3H]dopamine release from striatal synaptosomes is well documented (Rapier et al., 1990; Kulak et al., 1997) and substantiated by the lack of effect of αBgt and α-CTx-ImI in the present study (Fig. 1C). In contrast, we have shown previously that these agents, as well as MLA, antagonize nicotine-evoked [3H]dopamine release from striatal slices, interpreted as evidence for the involvement of an indirect α7 nAChR-mediated component in the slice (Kaiser and Wonnacott, 2000). In the present study, [3H]dopamine release from striatal synaptosomes was examined to focus on presynaptic nAChR localized on the dopamine terminals.
The inhibition by 50 nM MLA of [3H]dopamine release evoked by either nicotine or UB-165 was surmountable (Figs. 1C and 3), consistent with a competitive interaction. Similarly, Clarke and Reuben (1996) observed a surmountable inhibition by MLA of nicotine-evoked [3H]dopamine release from striatal synaptosomes. These authors demonstrated complete inhibition by MLA with an IC50 value of 38 nM, whereas nicotine-evoked [3H]noradrenaline release was notably less sensitive to MLA (IC50 = 1 μM). Full dose-response curves were not determined in the present study because the current appreciation of the heterogeneity of nAChR subtypes governing nicotine-evoked [3H]dopamine release compromises the analysis of such profiles. MLA (50 nM) also partially inhibited (by 37%) [3H]dopamine release from striatal synaptosomes stimulated with 1 μM anatoxin-a, but had no effect when the agonist concentration was increased to 25 μM (Kaiser and Wonnacott, 2000). Thus, MLA potently and competitively inhibits a portion of striatal [3H]dopamine release elicited by a number of nicotinic agonists acting at presynaptic nAChR.
MLA seems to interact with α-CTx-MII-sensitive nAChR because inhibition by the two toxins of nicotine- or UB-165-evoked [3H]dopamine release is not additive when they are applied together (Figs. 2 and 3). This inference is supported by the ability of MLA to potently displace125I-α-CTx-MII binding.125I-α-CTx-MII labeled a small population of specific sites in rat striatum and nucleus accumbens, with a subnanomolar binding affinity. The density of sites (10 and 16 fmol/mg of protein, respectively) agrees well with numbers of specific binding sites reported for the corresponding brain regions in mouse (Whiteaker et al., 2000) and monkey (Quik et al., 2001), assuming 1 mg of protein corresponds to 10 mg of tissue. In the monkey caudate putamen, more than 95% of specific 125I-α-CTx-MII binding sites were lost in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned animals. Together with the correlation with dopamine transporters, this strongly supports the localization of 125I-α-CTx-MII binding sites to dopaminergic terminals in these brain regions (Quik et al., 2001). αBgt (1 μM) failed to inhibit125I-α-CTx-MII binding to mouse striatum, and conversely α-CTx-MII, at concentrations up to 1 μM, did not compete for 125I-αBgt binding to mouse brain membranes (Whiteaker et al., 2000). Thus, the ability of MLA to fully displace125I-α-CTx-MII binding (Fig. 4) with aKi of ∼33 nM reflects a non-α7 nAChR activity for this ligand.
The observations in the present study parallel the findings of Klink et al. (2001) for nAChR responses recorded electrophysiologically from dopamine cell bodies in the rat midbrain. These authors showed that in most (75%) dopamine neurons, acetylcholine or nicotine evoked slow whole cell currents that were partially blocked by low nanomolar concentrations of MLA and α-CTx-MII in a nonadditive manner.
This raises the question of the subunit composition of nAChR subtype(s) with which MLA potently interacts. The majority of rat midbrain dopamine neurons express the α3, α4, α5, α6, β2, and β3 subunits (Klink et al., 2001). α-CTx-MII was originally defined as a selective antagonist of nAChR composed of α3 and β2 subunits, with >100-fold lower potency at other pairwise nAChR subunit combinations or α7 nAChR expressed in Xenopus oocytes (Cartier et al., 1996; Harvey et al., 1997; Kaiser et al., 1998). Because binding of CTx-MII to one α3β2 interface would be sufficient for functional inhibition, the toxin specificity was extended to include α3β2-containing (or α3β2*) nAChR (Kulak et al., 1997; Kaiser et al., 1998). However, the α3 subunit shares high sequence identity with the α6 subunit (Le Novère and Changeux, 1995) that is highly expressed in dopamine neurons (Le Novère et al., 1996;Goldner et al., 1997; Klink et al., 2001). Deletion of α6 subunit expression by in vivo administration of antisense oligonucleotides decreased nicotine-induced effects on locomotor activity (Le Novère et al., 1999), consistent with a role in motor functions executed by the striatum. The α6 subunit is reluctant to form functional nAChRs in pairwise combination with a β subunit, supporting its participation in more complex subunit assemblies, and the efficient expression of α6 in combination with a variety of mammalian subunits has been demonstrated in heterologous systems (Kuryatov et al., 2000). Immunoisolated α6-containing nAChR from chick retina display moderately high affinity for α-CTx-MII (Ki = 66 nM), but relatively low affinity for MLA (1.3 μM; Vailati et al., 1999).
Expression of the β3 subunit is also limited to a few brain regions, notably catecholaminergic areas where it colocalizes with α6 nAChR subunit expression in the rat brain (Le Novère et al., 1996). Functional nAChRs comprised of rat α3, β2, and β3 subunits and expressed in Xenopus oocytes retain high sensitivity to α-CTx-MII (Luo et al., 2000), whereas transgenic mice lacking expression of the β3 nAChR subunit are deficient in specific binding sites for 125I-α-CTx-MII in the striatum (Booker et al., 1999). However, chicken immunoisolated β3-containing nAChR that are devoid of α6 subunits do not exhibit high affinity for α-CTx-MII or MLA (Vailati et al., 2000). Thus, the β3 subunit may not confer sensitivity to α-CTx-MII or MLA to nAChR but is crucial for the formation, targeting or stability of α-CTx-MII-sensitive nAChR in basal ganglia in vivo. Klink et al. (2001) propose that the β3 subunit is targeted to terminal regions of dopaminergic neurons and does not participate in somatodendritic nAChR; thus, nAChR at these two locations could exhibit pharmacological (Reuben et al., 2000) and biophysical differences. Indeed, Klink et al. (2001) reported that 1 nM MLA was maximally effective in blocking cell body responses, which is more potent than predicted from the neurochemical measurements of [3H]dopamine release summarized above, or itsKi for interaction with125I-α-CTx-MII binding sites (Fig. 4). Although this discrepancy may reflect methodological differences, it could also arise from distinct nAChR subtypes at cell body and terminal locations.
These considerations lead to the proposition that α-CTx-MII- and MLA-sensitive presynaptic nAChRs mediating striatal [3H]dopamine release are comprised of α3 and/or α6 subunits together with β2 and β3 subunits. The finding that 125I-α-CTx-MII binding to basal ganglia is absent in α6 null mutant mice (Champtiaux et al., 2002), but persists in α3 null mutant mice (Whiteaker et al., 2002), argues for the involvement of the α6 subunit, rather than the α3 subunit, in this nAChR subtype. Klink et al. (2001) proposed the subunit composition α4α6α5(β2)2 for α-CTx-MII- and MLA-sensitive somatodendritic nAChRs. Inclusion of the α4 subunit was based on the absence of responses in midbrain slices from α4 null mutant mice. The ability of nicotinic agonists to elicit dopamine release from striatal synaptosomes prepared from these transgenic animals has not been reported, so no evidence is available for the participation of the α4 subunit in the α-CTx-MII- and MLA-sensitive presynaptic nAChR.
One implication of this and related studies is that sensitivity to nanomolar concentrations of MLA should not be considered diagnostic of α7 nAChR, at least in studies of rodent basal ganglia. This is particularly pertinent for in vivo studies, where drug concentrations reaching the nAChR are not known. However, the caveat that must be placed on the nAChR subtype selectivity of MLA may also be exploited for the differentiation of minority subtypes of nAChR, by comparison of sensitivities to MLA and α-CTx-MII.
Acknowledgments
We are grateful to Tim Gallagher (University of Bristol) for the provision of UB-165.
Footnotes
-
This study was supported financially by the Biological and Biotechnological Sciences Research Council Project Grant 86/B11785 (to S.W.), National Institute on Drug Abuse Grant DA-12242 (to P.W., M.M., and J.M.M.), National Institute of Mental Health Grant MH-53631 (to J.M.M.), and a Biological and Biotechnological Sciences Research Council Cooperative Award in Science and Engineering Studentship in conjunction with GlaxoSmithKline (to A.J.M.). A.C.C. is supported, in part, by the National Institute on Drug Abuse Research Scientist Award DA-00197.
- Abbreviations:
- nAChR
- nicotinic acetylcholine receptor(s)
- α-CTx-MII
- α-conotoxin-MII
- αBgt
- α-bungarotoxin
- α-CTx-ImI
- α-conotoxin-ImI
- MLA
- methyllycaconitine
- ANOVA
- analysis of variance
- DHβE
- dihydro-β-erythroidine
- UB-165
- (2-chloro-5-pyridyl)-9-azabicyclo[4.2.1]non-2-ene
- Received February 15, 2002.
- Accepted March 14, 2002.
- The American Society for Pharmacology and Experimental Therapeutics