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NEUROPHARMACOLOGY

Effect of Novel -Conotoxins on Nicotine-Stimulated [³H]Dopamine Release from Rat Striatal Synaptosomes

Departments of Psychiatry (J.M.M.) and Biology (L.A., J.M.M.), University of Utah, Salt Lake City, Utah

Received May 14, 2004; accepted August 17, 2004.

	Abstract

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Nicotine's action on the midbrain dopaminergic neurons is mediated by nicotinic acetylcholine receptors (nAChRs) that are present on the cell bodies and the terminals of these neurons. Previously, it was suggested that one of the nAChR subtypes located on striatal dopaminergic terminals may be an 32 subtype, based on partial inhibition of nicotine-stimulated [³H]dopamine release by -conotoxin MII, a potent inhibitor of heterologously expressed 32 nAChRs. More recent studies indicated that -conotoxin MII also potently blocks 6-containing nAChRs. In the present study, we have examined the nAChR subtype(s) modulating [³H]dopamine release from striatal terminals by using novel -conotoxins that have 37- to 78-fold higher selectivity for 6-versus 3-containing nAChRs. All of the peptides partially (20-35%) inhibit nicotine-stimulated [³H]dopamine release with IC₅₀ values consistent with those obtained with heterologously expressed rat 6-containing nicotinic acetylcholine receptors. These results, together with previous studies by others, further support the idea that 6-containing nicotinic receptors modulate nicotine-stimulated dopamine release from rat striatal synaptosomes.

Nigrostriatal dopamine (DA) neurons, which originate from within the substantia nigra compacta and send projections to the dorsal striatum, are involved in voluntary motor control. The selective loss of these neurons has been implicated in the pathology of the motor deficits associated with Parkinson's disease. Nicotine's actions on these neurons, and subsequent dopamine release within the striatum, may partly underlie this alkaloid's potential therapeutic effects in patients with Parkinson's. Midbrain (DA) neurons possess nicotine binding sites (Clarke and Pert, 1985), and nicotine directly activates these neurons (Pidoplichko et al., 1997; Yin and French, 2000). Moreover, nicotine stimulates release of DA within the striatal target region by direct action on nicotinic receptors on the DAergic terminals (Rapier et al., 1990; Grady et al., 1992; Clarke and Reuben, 1996; Wonnacott, 1997). In situ hybridization and immunohistochemical studies have revealed the presence of a variety of nAChR subunit mRNAs and protein within midbrain DAergic neurons, including 3-7 and 2 and 3 (Charpantier et al., 1998; Elliott et al., 1998; Sorenson et al., 1998; Klink et al., 2001; Azam et al., 2002), that could potentially result in numerous distinct subtypes of nAChRs. Therefore, due to lack of subtype-specific ligands, it has been difficult to determine the exact subtypes of nAChRs mediating DA neurotransmission. Recently, it was shown that -conotoxin MII (-MII), a Conus toxin isolated from Conus magus that potently inhibits the 32 nAChR subtype (Cartier et al., 1996), inhibited approximately 30 to 40% of nicotine-stimulated [³H]DA release from striatal synaptosomes (Kulak et al., 1997; Kaiser et al., 1998; Grady et al., 2001). However, more recent data indicate that -MII also has high affinity for 6-containing receptors. Studies with mutant animals have shown that ¹²⁵I--MII binding is largely preserved in 3 knockout mice, but it is abolished in 6 knockout mice (Champtiaux et al., 2002; Whiteaker et al., 2002). These studies suggest that the primary target of -MII in DA neurons may be an 6-rather than 3-containing receptor.

Until now, no ligands that distinguish between 6^* and 3^* nAChRs (asterisk indicates the possible presence of other subunits) have been available. In the present study, we have characterized nicotine-modulated [³H]DA release from rat striatal synaptosomes using the novel -conotoxin PIA (-PIA), isolated from Conus purpurascens that has approximately 78-fold higher selectivity for 6-versus 3-containing receptors. In addition, three recently developed structural analogs of -MII, with 37-, 54-, and 75-fold selectivity for 6-versus 3-containing receptors, were also used. The results implicate the involvement of 6^* rather than 3^* receptors in nicotine-evoked dopamine release from DAergic terminals.

	Materials and Methods

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Materials. The chemicals were obtained from the following sources: (-)-Nicotine hydrogen tartarate, pargyline HCl, bovine serum albumin, ascorbic acid (Sigma-Aldrich, St. Louis, MO), [³H]dopamine (dihydroxyphenylethylamine 3,4 [7-³H]; 28-30 Ci/mmol) (PerkinElmer Life and Analytical Sciences, Boston, MA), Ecolume scintillation cocktail (MP Biomedicals, Irvine, CA). -Conotoxins were synthesized as described previously (Cartier et al., 1996; McIntosh et al., 2004).

Tissue Preparation. Adult male albino rats (Simonsen Laboratories, Gilroy, CA) were kept two per cage on a 12:12-h light/dark cycle, with food and water available ad libitum. For each experiment, four adult male rats between 60 and 90 days old were used. The rats were decapitated and brains quickly removed. This procedure was approved by the Institutional Animal Care and Use Committee and is consistent with Federal guidelines. Synaptosomes were prepared essentially as described by Kulak et al. (1997). Briefly, the striata were quickly dissected on ice and placed in ice-cold 0.32 M sucrose buffer, pH 7.4 to 7.5. The dissected striata were homogenized by 14 gentle up and down strokes, followed by centrifugation at 1000g for 10 min at 4°C. The supernatant was centrifuged at 12,000g for 20 min at 4°C. The resulting P2 pellet was resuspended in 2 ml of Krebs-HEPES buffer (superfusion buffer) with composition 128 mM NaCl, 2.4 mM KCl, 1.2 mM KH₂PO₄, 0.6 mM MgSO₄, 3.2 mM CaCl₂, 25 mM HEPES, and 10 mM glucose and supplemented with 1 mM ascorbic acid and 0.1 mM pargyline. The synaptosomes were incubated for 10 min at 37°C to equilibrate with the superfusion buffer, followed by another 10-min incubation with 0.12 µM [³H]dopamine (specific activity 28-30 Ci/mmol) at 37°C. The synaptosomes were centrifuged at 3500 rpm for 5 min to get rid of excess radiolabeled dopamine. The pellet was resuspended in 4 ml of superfusion buffer, and 1 ml was transferred into each of four conical tubes containing 3 ml of superfusion buffer and subsequently loaded into the superfusion chambers containing 13-mm-diameter A/E glass fiber filters (Gelman Instrument Co., Ann Arbor, MI). One tube (4-ml total volume) contained enough synaptosomes for six chambers of the superfusion apparatus.

Superfusion. The superfusion system had 12 identical channels and was set up as described in Kulak et al. (1997). Once synaptosomes were loaded into the superfusion apparatus, they were washed for 20 min with either superfusion buffer alone or buffer plus varying concentrations of the toxins, at a rate of 0.5 ml/min. -Conotoxin MII[E11A], due to its slow on-rate, was flowed on for 40 min. After the wash period, 2-min fractions were collected in 5-ml polypropylene vials containing 4 ml of Ecolume scintillation cocktail. At the end of the third 2-min fraction, a 1-min pulse of nicotine or nicotine plus toxin was applied, followed by five 2-min washes with superfusion buffer alone. For studies where -MII and -PIA or -MII and -MII[E11A] were coapplied, synaptosomes were perfused for 20 or 40 min, respectively, with buffer containing 100 nM -MII and 100 nM -PIA or 100 nM -MII and 10 nM -MII[E11A] and pulsed with nicotine as described above. At the end of the superfusion, filters containing the synaptosomes were taken out and placed directly in vials containing 4 ml of Ecolume to determine total [³H]DA uptake. Radioactivity collected in each fraction was quantitated by liquid scintillation spectroscopy, with Beckman Coulter 5801 and 9800 liquid scintillation counters, tritium efficiency approximately 50%.

Data Analysis. Throughout this article, tritium release is presumed to correspond directly to amounts of radiolabeled transmitter release, because it has been shown previously that tritium released by nAChR agonists is proportional to total radiolabeled transmitter released (Rapier et al., 1988).

Baseline release was determined as average of two fractions before and two fractions after the peak release. Average baseline was subtracted from the evoked release and the resulting values divided by the baseline to yield the evoked release as a percentage over baseline. For all data, except the release profiles in Fig. 1, percentage of release over baseline was normalized to average release with nicotine alone. IC₅₀ values were determined by nonlinear regression analysis using Prism (Graphpad Software Inc., San Diego, CA). All statistical analysis was performed with Prism. Toxin effects were analyzed by one-way analysis of variance, followed by Dunnett's post hoc for comparisons with nicotine control or Newman-Keuls or Bonferroni's (where indicated) for multiple pairwise comparisons.

View larger version (33K): [in this window] [in a new window]   Fig. 1. Representative profiles of nicotine-stimulated [3H]DA release from striatal synaptosomes. Striatal synaptosomes preloaded with [3H]DA were stimulated with a 1-min pulse of 3 µM nicotine (indicated by arrow). This concentration of nicotine stimulated a peak release of 80 to 100% above baseline (solid lines denoted as "control"). Simultaneous application of 1 µM -conotoxins (a saturating concentration) inhibited the peak release by approximately 20 to 30% (broken lines). The -conotoxins that were tested are -conotoxin MII (A), -conotoxin PIA (B), -conotoxin MII[H9A] (C), -conotoxin MII[L15A] (D), and -conotoxin MII[E11A] (E).

	Results

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Inhibition of Nicotine-Stimulated [³H]DA Release by -Conotoxin PIA and MII. A 1-min pulse of 3 µM nicotine (a submaximal concentration) resulted in [³H]DA release approximately 80 to 100% above baseline (Fig. 1). It has previously been shown that this nicotine-evoked release is completely Ca²⁺ dependent and is blocked by the nonselective nAChR antagonist mecamylamine at a concentration of 100 µM (Kulak et al., 1997). Both -PIA and -MII dose dependently inhibited nicotine-stimulated [³H]DA release with IC₅₀ values of 1.48 and 1.04 nM, respectively (Fig. 2; Table 1). However, this inhibition only reached significance at concentrations 1 nM for -MII and 10 nM for -PIA (Fig. 2). Maximum inhibition for both toxins was reached at 10 nM, because application of higher concentrations of either toxin did not produce significantly more inhibition than that seen with 10 nM. At the same toxin concentrations, block of nicotine-stimulated [³H]DA release was not significantly different between -MII and -PIA.

View larger version (22K): [in this window] [in a new window]   Fig. 2. -MII and -PIA inhibition of nicotine-stimulated [3H]DA release from striatal synaptosomes. The values are normalized to release obtained with 3 µM nicotine alone (100% release above baseline). Both toxins dose dependently inhibited nicotine-evoked [3H]DA release with IC50 values in the low nanomolar range. For -MII, inhibition was significantly different from nicotine control at concentrations of 1 nM and greater (***, p < 0.001; Dunnett's post hoc), whereas for -PIA, inhibition became significant at concentrations of 10 nM and greater (###, p < 0.001; Dunnett's post hoc). Values are mean ± S.E.M. from four to nine separate experiments.

To determine whether the similar inhibition of nicotine-stimulated [³H]DA release seen with -MII and -PIA is due to their action on the same site, the toxins were simultaneously applied in the presence of 3 µM nicotine. Coapplication of both toxins at a maximally effective concentration (100 nM) did not produce a greater inhibition than either one applied alone (Fig. 3), suggesting that both toxins exert their effects by action on the same nAChR site(s).

View larger version (34K): [in this window] [in a new window]   Fig. 3. Inhibition of nicotine-evoked [3H]DA release from striatal synaptosomes in the presence of both -MII and -PIA. Coapplication of maximally effective concentrations of -MII and -PIA (100 nM) did not produce a greater inhibition than that seen with 100 nM of each toxin alone. Percentage of [3H]DA release above baseline is normalized to release obtained with 3 µM nicotine alone (100% release above baseline). Values are mean ± S.E.M. from four separate experiments. ***, p < 0.001, compared with nicotine control; Student's t test.

Inhibition of Nicotine-Stimulated [³H]DA Release by -MII and -PIA in the Presence of Varying Concentrations of Nicotine. A number of studies have suggested that [³H]DA release from striatal terminals is modulated by at least two different nAChR subtypes (Kulak et al., 1997; Sharples et al., 2000; Grady et al., 2002), which have different affinities for nicotine (Kuryatov et al., 2000; Champtiaux et al., 2003). To determine whether different proportion of -MII and -PIA sensitive nAChRs are activated at different nicotine concentrations, 10 nM -MII and -PIA were tested with nicotine concentrations in the range 300 nM to 10 µM. Both toxins were used at a concentration of 10 nM because electrophysiological studies in our laboratory had indicated that for -PIA, this concentration would selectively block a larger proportion of 6-rather than 3-containing receptors (Dowell et al., 2003) (also see Table 1). At 10 nM, -PIA significantly inhibited nicotine-stimulated release at all concentrations of nicotine tested (approximately 20%; p < 0.001), and this inhibition was similar across all nicotine concentrations (Fig. 4A). Similarly, -MII significantly inhibited nicotine-stimulated [³H]DA release at all concentrations of nicotine (p < 0.001; Fig. 4B). However, -MII displayed a trend toward greater inhibition of nicotine-stimulated [³H]DA release at 300 nM and 1 µM nicotine compared with 3 and 10 µM nicotine, although this difference did not reach significance (p = 0.165; Fig. 4B). Between -MII and -PIA, there was significantly more inhibition of nicotine-stimulated [³H]DA release with -MII at 300 nM and 1 µM nicotine compared with -PIA at the same nicotine concentrations (Fig. 4).

View larger version (62K): [in this window] [in a new window]   Fig. 4. -MII and -PIA inhibition of nicotine-stimulated [3H]DA release in presence of varying concentration of nicotine. Percentage of [3H]DA release above baseline is normalized to release in presence of the corresponding nicotine concentration alone (control). A, -PIA (10 nM) significantly inhibited nicotine-stimulated [3H]DA at all nicotine concentrations as compared with corresponding nicotine control (***, p < 0.001; Dunnett's post hoc), and this inhibition was similar across all nicotine concentrations. B, -MII (10 nM) significantly inhibited nicotine-stimulated [3H]DA release at all nicotine concentrations compared with corresponding nicotine control (***, p < 0.001; Dunnett's post hoc), although this inhibition was slightly, although insignificantly, greater at 300 nM and 1 µM nicotine. However, at 300 nM and 1 µM nicotine, -MII significantly inhibited more [3H]DA release than -PIA at the same nicotine concentrations. Absolute values for nicotine control (percent above baseline): A: 300 nM, 61.7 ± 8.2; 1 µM, 78.1 ± 8.4; 3 µM, 101.3 ± 7.6; 10 µM, 118.9 ± 12.3; and B: 300 nM, 70.9 ± 11.7; 1 µM, 94.1 ± 7.1; 3 µM, 101.7 ± 11.9; 10 µM, 122.9 ± 17.2 (#, p < 0.05; ##, p < 0.01; Bonferroni's selected pairs of columns post hoc). Values are mean ± S.E.M. from three to four separate experiments.

Effects of -Conotoxin MII Analogs -MII[H9A], -MII[L15A], and -MII[E11A] on Nicotine-Stimulated [³H]DA Release. Although -conotoxin MII does not distinguish well between 3- and 6-containing receptors (Kuryatov et al., 2000; Dowell et al., 2003; McIntosh et al., 2004), substitution of Ala for His9 shifts the selectivity of -MII toward 6-containing receptors, with an approximately 75-fold higher preference for 6-versus 3-containing nAChRs (Table 1). Similarly, replacement of Leu15 with Ala and Glu11 with Ala increases the selectivity of -MII for 6-containing nAChRs by approximately 37- and 54-fold, respectively. Double Ala substituted -MII analogs further increase selectivity (McIntosh et al., 2004). However, rapid off-rate kinetics combined with an apparent binding preference for the resting state of the nAChR make these analogs unsuitable for experiments with prolonged agonist application (our unpublished observations). The three singly substituted -MII analogs were examined for their effects on nicotine-stimulated [³H]DA release to further assess the role of 6-containing receptors in modulation of this release. All analogs displayed dose-dependent inhibition of nicotine-stimulated [³H]DA release, with IC₅₀ values of 1.25 nM for -MII[H9A], 0.84 nM for -MII[L15A], and 0.025 nM for -MII[E11A] (Fig. 5; Table 1). -MII [H9A] and -MII[L15A] significantly inhibited release at concentrations 10 nM (p < 0.01; Fig. 5), whereas -MII[E11A] was the most potent of the analogs, significantly inhibiting [³H]DA release at concentrations 0.1 nM (p < 0.001; Fig. 5). For -MII[H9A] and -MII[L15A], application of 100 nM and 1 µM did not produce any greater inhibition than seen with 10 nM, whereas for -MII[E11A], inhibition at 0.1 nM was not statistically different from that at higher concentrations (Fig. 5). -MII[E11A] displayed a trend toward larger maximal inhibition than the other toxins; however, this difference did not reach statistical significance. -MII[E11A] blocks 42 nAChRs with an IC₅₀ > 10,000 nM and therefore any larger block by -MII[E11A] is unlikely to be due to blockade of this receptor subtype. Additionally, coapplication of -MII with -MII[E11A] did not produce a statistically greater inhibition that either one alone (32 ± 7.9% inhibition with 100 nM -MII, 31.4 ± 11.8% with 10 nM -MII[E11A], and 42 ± 13% inhibition with 100 nM -MII plus 10 nM -MII[E11A]), suggesting action on the same site.

View larger version (28K): [in this window] [in a new window]   Fig. 5. -MII[H9A], -MII[L15A], and -MII[E11A] inhibition of nicotine-stimulated [3H]DA release from striatal synaptosomes. Percentage of [3H]DA release above baseline is normalized to release obtained with 3 µM nicotine alone (100% release above baseline). All three analogs dose dependently inhibited nicotine stimulated [3H]DA release with IC50 values in the picomolar to low nanomolar range. For -MII[H9A] and -MII[L15A], inhibition was significant at concentrations of 10 nM and greater (**, p < 0.01, Dunnett's post hoc), whereas -MII[E11A] showed the highest potency, with significant inhibition obtained at 0.1 nM and greater (###, p < 0.001; Dunnett's post hoc). Values are mean ± S.E.M. from three to four separate experiments.

	Discussion

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In the present study, we have used novel -conotoxins to assess the subtypes of nAChRs involved in nicotine-stimulated DA release in rat striatum. All of the toxins partially inhibit nicotine-stimulated [³H]DA release, consistent with there being at least two populations of nAChRs that modulate DA release (Sharples et al., 2000; Grady et al., 2002). The IC₅₀ values of the toxins are similar to IC₅₀ values of 6-containing nAChRs heterologously expressed in oocytes, suggesting that the nAChRs located on striatal DAergic terminals most likely contain an 6 rather than an 3 subunit (Table 1). The selectivity of these conotoxin peptides have allowed us to perform the first functional characterization of 6^* versus 3^* nAChRs in rat striatal synaptosomes.

Previously, it had been suggested that [³H]DA release from striatal terminals was modulated in part by an 32 nAChR subtype (Kulak et al., 1997; Kaiser et al., 1998; Sharples et al., 2000). This conclusion was based, in part, on inhibition of nicotine-stimulated [³H]DA release by -conotoxin MII. -conotoxin MII was previously shown to block the 32 subunit combination with 2 to 4 orders of magnitude lower IC₅₀ than other subunit combinations, including 22, 24, 34, 42, and 44 (Cartier et al., 1996). However, -MII was subsequently shown to also have low nanomolar affinity for 6-containing receptors (Vailati et al., 1999; Kuryatov et al., 2000; Vailati et al., 2000; Barabino et al., 2001). Recent studies with mutant mice suggest that -conotoxin MII binds with high affinity to 6^* nAChRs in the central nervous system (Champtiaux et al., 2002; Whiteaker et al., 2002).

The findings in the present study strongly suggest that -MII-sensitive sites on rat striatal DAergic terminals contain an 6 rather than an 3 subunit. Although -MII displays similar potency in blocking rat 3- and 6-containing receptors in oocytes (Dowell et al., 2003; McIntosh et al., 2004), the results with the other toxins that have relatively lower potency for receptors containing the 3 subunit indicate the presence of an 6^* nAChR subtype on striatal DAergic terminals. The high affinity of -MII for both 3 and 6 subunits may be explained by the high homology (80%) in the extracellular region (ligand binding site) between 3 and 6 subunits, as well as conservation of residues between 3 and 6 that confer selectivity to -MII (Harvey et al., 1997). The residues within the toxins conferring the high selectivity for 6^* and lower selectivity for 3^* nAChRs are not currently known, although amino acids within the second disulfide loop are strong candidates. -MII and -PIA have identical amino acid sequence in the first loop, but their amino acid sequences are highly divergent in the second loop (Dowell et al., 2003). In addition, the three -MII analogs that display high selectivity for 6^* nAChRs have the alanine substitutions in their second loops (McIntosh et al., 2004).

Consistent with the current findings, recent immunoprecipitation studies have shown the presence of the 6 subunit in approximately 20% of [³H]epibatidine-labeled receptors in the rat striatum, with the 3 subunit present in only 3% of the receptors. Moreover, there was an almost complete disappearance of nAChRs containing the 6 subunit in 6-hydroxydopamine-lesioned striata, with no change in the 3 subunit (Zoli et al., 2002), indicating that the 6-containing sites are located primarily or exclusively on the DAergic terminals. The percentage of immunoprecipitated 6-containing [³H]epibatidine sites closely resembles the maximum inhibition of nicotine-stimulated [³H]DA release by the toxins in the present study. In addition to both 2 and 3, approximately 40% of 6-containing striatal nAChRs also contain the 4 subunit (Zoli et al., 2002; Champtiaux et al., 2003). However, none of the toxins in our present study may be able to distinguish between 62^* and 642^* nAChR subtypes. Toxin block of 642^* nAChRs would likely be dependent on the position of the 6 subunit. If the 6 subunit is present at a ligand binding interface, the -conotoxins used in this study would be expected to block the receptor, due to their high affinity for the 6/2 subunit interface (Dowell et al., 2003; McIntosh et al., 2004). If the 6 subunit is located at an 62 interface in both subunit combinations, then the binding of the toxin to that interface would be sufficient for complete block, based on the assumption that two molecules of nicotine are required to activate the nAChR (Lester and Dani, 1995). The binding of the -conotoxins to the 42 interface is unlikely, because all of the toxins, with the exception of -MII, do not block heterologously expressed 42 nAChRs at concentrations used in the present study (Cartier, 2003; McIntosh et al., 2004). Results from other studies suggest that the large -conotoxin-insensitive component of nicotine-evoked [³H]DA release is most likely mediated by 42^* nAChRs (Sharples et al., 2000; Zoli et al., 2002; Champtiaux et al., 2003).

-MII and -PIA both showed similar inhibition of nicotine-stimulated [³H]DA release across different concentrations of nicotine. Although this may be indicative of noncompetitive antagonism, it is more likely attributable to the slow off-rate kinetics of these toxins. Since the receptors have been preincubated with the toxins for at least 20 min, almost all the toxin molecules would be expected to be bound to the receptor when the nicotine is applied. However, at 300 nM and 1 µM nicotine, -MII displayed significantly greater inhibition of nicotine-stimulated [³H]DA release than PIA. This suggests possible presence of a high-affinity nAChR subtype that is blocked by -MII, but not -PIA. Possible candidates for this nAChR are 42 and/or 642 subtypes, which have EC₅₀ values of 340 nM and 1.3 µM for nicotine, respectively (Kuryatov et al., 2000). Both subtypes seem to be present on striatal DAergic terminals (Sharples et al., 2000; Zoli et al., 2002; Champtiaux et al., 2003). Although native 42^* nAChRs display very low affinity to -MII, as shown by immunoprecipitation and competition binding studies of native receptors (Zoli et al., 2002), it is possible that native 42^* receptors are more sensitive to functional blockade by this toxin. -MII has been shown to block heterologously expressed 42 nAChRs with an IC₅₀ value of 470 nM (Cartier, 2003), whereas -PIA does not show any block of this receptor subtype even at micromolar concentrations (Dowell et al., 2003). Additionally, the presence of the 5 subunit in a significant proportion of 42^* nAChRs on DAergic terminals (Zoli et al., 2002) could affect the sensitivity of the receptor for -MII. Immunoprecipitation studies indicate that some 62^* nAChRs on DAergic terminals also contain the putative structural 3 subunit (Zoli et al., 2002). In addition, knockout studies in mice suggest that most -MII sensitive sites in the striatum (presumably located exclusively on DAergic terminals) contain the 3 subunit (Cui et al., 2003). Until recently, we have had difficulty expressing functional receptors in Xenopus oocytes with only the chimeric 6/3 and 2 subunits and not the 3 subunit. However, using the 2 subunit in the high-expressing vector pGEMHE (Parker et al., 1998) with only the 6/3 subunit routinely yielded functional receptors, albeit at low levels. Both PIA and MII[E11A] block 6/32 versus 6/323 nAChRs with approximately equal IC₅₀ values (Dowell et al., 2003; McIntosh et al., 2004), suggesting that the presence of the 3 subunit has little influence on the potency of these toxins. Finally, each of the toxins tested in this study also has high affinity for 64 nAChRs (Barabino et al., 2001; Dowell et al., 2003; McIntosh et al., 2004). However, it is unlikely that the 4 subunit is involved in nicotine-stimulated dopamine release in the striatum for several reasons. Both in situ hybridization (Winzer-Serhan and Leslie, 1997; Azam et al., 2002) and single-cell reverse transcription-polymerase chain reaction studies (Klink et al., 2001) have indicated an absence of the 4 subunit mRNA in substantia nigra compacta DAergic neurons. In addition, immunoprecipitation studies indicate a lack of the 4 subunit protein on striatal DAergic terminals (Zoli et al., 2002). Finally, the IC₅₀ values of block of nicotine-stimulated [³H]DA release by -conotoxins are consistent with 62^* rather than 64^* nAChRs (Dowell et al., 2003; McIntosh et al., 2004).

One caveat of comparing results obtained from heterologously expressed nAChRs to properties of nAChRs in the native system is that heterologously expressed receptors may not have the same post-translational modifications as the native mammalian nAChRs and therefore may not display the same affinity for a given ligand. However, the toxins used in the present study have been tested in competition binding studies with ¹²⁵I--MII using mouse striatal homogenates, and they exhibited K_i values similar to IC₅₀ values obtained for heterologously expressed 6/323 nAChRs (Table 1).

In conclusion, data from the present study implicate 62^* nAChRs as one of the modulators of nicotine stimulated striatal DA release. Subpopulations of 62^* nAChRs containing 4 and/or 3 also seem to be present (Zoli et al., 2002; Champtiaux et al., 2003; Cui et al., 2003). However, at present, there are no ligands to further functionally discriminate among these subunit combinations.

	Footnotes

 
This work was supported by National Institute of Health Grant MH53631 (to J.M.M.) and Ruth L. Kirschstein National Research Service Award Postdoctoral Fellowship DA016835 (to L.A.). Portions of this work have been presented previously in Azam L, Dowell C, and McIntosh JM (2002) Characterization of nicotine-mediated dopamine release from striatal synaptosomes using a novel -conotoxin. Program 242.9. 2002 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2002. Online.

doi:10.1124/jpet.104.071456.

ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; DA, dopamine; -MII, -conotoxin MII; -PIA, -conotoxin PIA.

Address correspondence to: Dr. J. Michael McIntosh, 257 South 1400 East, Salt Lake City, UT 84112. E-mail mcintosh{at}biology.utah.edu

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