The Journal of Neuroscience, September 17, 2003, 23(24):8445-8452
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-Conotoxin PIA Is Selective for 6 Subunit-Containing Nicotinic Acetylcholine Receptors
Cheryl Dowell,1
Baldomero M. Olivera,1
James E. Garrett,4
Sarah T. Staheli,1
Maren Watkins,2
Alexander Kuryatov,5
Doju Yoshikami,1
Jon M. Lindstrom,5 and
J. Michael McIntosh1,3
Departments of 1Biology, 2Pathology, and 3Psychiatry, University of Utah, Salt Lake City, Utah 84112, 4Cognetix, Salt Lake City, Utah 84108, and 5Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104
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Abstract
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Until now, there have been no antagonists to discriminate between heteromeric nicotinic acetylcholine receptors (nAChRs) containing the very closely related 
6 and 3 subunits. nAChRs containing 3, 4, or 6 subunits in combination with 
2, occasionally 4, and sometimes 3 or 5 subunits, are thought to play important roles in cognitive function, pain perception, and the reinforcing properties of nicotine. We cloned a novel gene from the predatory marine snail Conus purpurascens. The predicted peptide, -conotoxin PIA, potently blocks the chimeric 6/323 subunit combination as expressed in oocytes but neither the muscle nor the major neuronal nAChR 42. Additionally, this toxin is the first described ligand to discriminate between nAChRs containing 6 and 3 subunits. Exploiting the unusual intron conservation of conotoxin genes may represent a more general approach for defining conotoxin ligand scaffolds to discriminate among closely related receptor populations.
Key words: nicotinic; -conotoxin; 6; 2; 3; Conus
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Introduction
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Nicotinic acetylcholine receptors (nAChRs) in the CNS are involved in a variety of normal physiological functions, including cognition, reward, motor activity, and analgesia. They are implicated in the pathophysiology and treatment of disease states, including chronic pain syndromes, Parkinson's, and Alzheimer's. Eleven neuronal nAChR subunits have been cloned in mammals, 2–7, 9 and 10, and 2–4 (Lips et al., 2002
). Different combinations of subunits produce distinct nAChR subtypes. Homologous subunits are organized around a central cation channel that is gated by the endogenous ligand acetylcholine and the tobacco plant toxin nicotine. subunits and most subunits associate to form heteropentamers, generally with a stoichiometry of two and three subunits. Neither 5 nor 3 subunits have been found to form nAChRs when expressed alone or in pairs with the other subunits. However, both will associate to form functional nAChRs when combined with at least one other and one other subunit (Ramirez-Latorre et al., 1996; Wang et al., 1996; Groot-Kormelink et al., 1998). The 5 and 3 subunits are therefore referred to as structural subunits, because they lack residues believed to be necessary to form a ligand-binding site. Instead, they are believed to assemble in a position analogous to that of the 1 subunit in muscle nAChRs.
The 2 subunit is widely expressed in the CNS and appears critical for nicotine reinforcement. 2-Containing nAChRs modulate nicotine-induced dopamine release in the striatum. This release is absent in 2 knock-out mice, which show attenuated self-administration of nicotine (Picciotto et al., 1998; Whiteaker et al., 2000). The 2 subunit can assemble with various subunits to form distinct subtypes of nAChRs. For example, midbrain dopaminergic nuclei (substantia nigra, ventral tegmental area) are critical to reinforcement and associative motor learning (Berke and Hyman, 2000). Dopaminergic neurons within these nuclei, in addition to 2, contain 4, 6, and 3 nicotinic subunits (Klink et al., 2001). Thus, there is a need for ligands that discriminate among subpopulations of 2 subunit-containing nAChRs. -Conotoxin MII, a peptide toxin isolated from the marine cone snail Conus magus, was initially thought to be selective for 32 nAChRs (Cartier et al., 1996). However, at the time of its characterization, no method for expressing 6-containing nAChRs was known. Subsequently, it has been shown that -conotoxin MII also blocks 62* nAChRs (Kuryatov et al., 2000).
We therefore turned again to the cone snails for a more selective ligand. Conus is a large genus of predatory snails that feeds on fish, molluscs, and marine worms. Their venoms are complex, often containing hundreds of different peptides. Indeed, the venoms from over 500 species of snails represent a substantial combinatorial library selected over millions of years of evolution to target specific components of the nervous system. Perhaps the most conserved feature of cone snail venom is the -conotoxins. These are a series of structurally and functionally related peptides that target nAChRs. Each species of cone snail examined contains at least four distinct -conotoxins, suggesting that, within the genus, there are literally thousands of novel peptides that act on nAChRs. The diversity in these peptides is likely accounted for by the wide variety of prey types of Conus (five different phyla). Specialized genetic mechanisms appear to be used to generate peptide sequence diversity.
In this study, we describe the cloning and characterization of a novel peptide from the venom of Conus purpurascens, commonly known as the purple cone. The peptide has high affinity and selectivity for 6-containing nAChRs.
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Materials and Methods
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Identification and sequencing of genomic DNA clone-encoding -conotoxin PIA. DNA from C. purpurascens hepatopancreas was isolated using the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN). Frozen tissue was placed in 600 µl of lysis buffer, homogenized with a disposable microcentrifuge pestle, and digested overnight with 60 µg of proteinase K at 55°C. The remainder of the procedure followed the suggested protocol from the manufacturer for marine invertebrates.
The resulting genomic DNA was used as a template for PCR using oligonucleotides, which were tailed for cloning, corresponding to the 3' end of the intron preceding the toxin region of -conotoxin prepropeptides and the 3' untranslated region (UTR) sequence of the prepropeptides (Schoenfeld, 1999). The resulting PCR product was purified using the High Pure PCR Product Purification Kit (Roche Products, Indianapolis, IN) following the suggested protocol of the manufacturer. The eluted DNA fragment was annealed to plasmid (p)AMP1 vector, and the resulting product was transformed into competent DH5 cells with the CloneAmp pAMP system for rapid cloning of amplification products (Invitrogen, Grand Island, NY), following suggested protocols of the manufacturer.
Identification and sequencing of a cDNA clone-encoding -conotoxin PIA. cDNA was prepared by reverse transcription of RNA isolated from the C. purpurascens venom duct as described previously (Jacobsen et al., 1998). The resulting cDNA served as a template for PCR using oligonucleotides corresponding to the conserved signal sequence and the 3' UTR sequence of -conotoxin prepropeptides. The resulting PCR product was purified using the High Pure PCR Product Purification Kit (Roche Products) following the suggested protocol of the manufacturer. The eluted DNA fragment was ligated to HincII-linearized pTZ19U vector, and the resulting product was transformed into competent DH5 cells as described previously (Jacobsen et al., 1998). The nucleic acid sequences of the resulting clones were determined according to the standard protocol for Sequenase version 2.0 DNA sequencing kit, as described previously (Jimenez et al., 1997).
Chemical synthesis. Peptide was synthesized on an amide resin (0.45 mmol/gm; Applied Biosystems, Foster City, CA) using Fmoc (N-(9-fluorenyl)methoxycarboxyl) chemistry and standard side chain protection except on cysteine (Cys) residues. Cys residues were protected in pairs with either S-trityl on Cys2 and Cys8 or S-acetamidomethyl on Cys3 and Cys16. Amino acid derivatives were from Advanced Chemtech (Louisville, KY). The peptides were removed from the resin and precipitated, and a two-step oxidation protocol was used to selectively fold the peptides as described previously (Walker et al., 1999). Briefly, the first disulfide bridge was closed by dripping the peptide into an equal volume of 20 mM potassium ferricyanide, 0.1 M Tris, pH 7.5. The solution was allowed to react for 30 min, and the monocyclic peptide was purified by reverse-phase HPLC. Simultaneous removal of the S-acetamidomethyl groups and closure of the second disulfide bridge was performed by iodine oxidation. The monocyclic peptide and HPLC eluent was dripped into an equal volume of iodine (10 mM) in H2O:trifluoroacetic acid (TFA):acetonitrile (78:2:20 by volume) and allowed to react for 10 min. The reaction was terminated by the addition of ascorbic acid diluted 20-fold with 0.1% TFA and the bicyclic product that was purified by HPLC.
Preparation of nAChR subunit cRNA. As reported previously for the human 6 subunit (Kuryatov et al., 2000), attempts to express the rat nAChR 6 subunit with 2in Xenopus oocytes consistently failed; that is, no ACh-gated currents could be detected. To improve functional expression, we created a chimeric nAChR of the rat 6 and 3 subtypes. The chimeric nAChR consists of amino acids 1–237 of the rat 6 subunit protein linked to amino acids 233–499 of the rat 3 subunit protein. The chimeric junction is located at the paired arginine residues immediately preceding the M1 transmembrane segment of the 3 subunit. The resulting chimeric nAChR represents the extracellular ligand-binding domain of the 6 subunit linked to the membrane-embedded region of the 3 subunit where the membrane-spanning segments are located. The 6/3 cDNA was constructed by introduction of BspEI sites at the chimeric junction into the 6 and 3 cDNA sequences using mutagenic primers to introduce the restriction sites through silent codon changes. The 6 and 3 segments were generated by PCR of rat brain cDNA using primers in the 5' and 3' untranslated regions of the corresponding cDNAs along with the internal mutagenic primers. The PCR products were digested with BspEI and ligated to generate the chimeric construct. The final chimeric construct was cloned and completely sequenced to confirm the correct cDNA sequence. To further improve expression levels, all of the 5' and 3' untranslated regions of the nAChR cDNA were deleted, and the chimeric construct was cloned into the Xenopus expression vector pT7TS, placing Xenopus globin 5' and 3' UTR regions around the nAChR cDNA. The expression construct, pT7TS/r63, was transcribed with T7 RNA polymerase to generate sense-strand RNA for oocyte expression.
Electrophysiology and data analysis. Clones of nAChR subunits were used to produce cRNA for injection into Xenopus oocytes as described previously (McIntosh et al., 2000). A previously described chimeric -subunit consisting of the human extracellular domain of 6 and the remaining part of the human 3 subunit was expressed with other human nAChR subunits (Kuryatov et al., 2000). The rat 6/3 chimera (described above) was expressed with previously cloned rat subunits (Cartier et al., 1996). For most nAChR subunits, 5 ng of cRNA was injected into the oocytes. For the muscle nAChR, 0.5–2.5 ng was used. For 6/32, 6/34, and 64 nAChRs, 12.5–50 ng of cRNA was used to improve the amount of functional expression. A 30 µl cylindrical oocyte-recording chamber fabricated from Sylgard (Dow Corning, Midland, MI) was gravity-perfused with ND96A (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 1 µM atropine, 5 mM HEPES, pH 7.1–7.5) at a rate of 
2 per minute (Luo et al., 1998). All toxin solutions also contained 0.1 mg/ml of bovine serum albumin to reduce nonspecific adsorption of peptide. Toxin was pre-applied for 6 or 10 min (for concentrations of <10 nM). ACh-gated currents were obtained with a two-electrode voltage-clamp amplifier (OC-725B; Warner Instruments, Hamden, CT), and data were captured as described previously (Luo et al., 1998). The membrane potential of the oocytes was clamped at -70 mV. To apply a pulse of ACh to the oocytes, the perfusion fluid was switched to one containing ACh for 1 sec. This was done automatically at intervals of 1–5 min. The shortest time interval was chosen such that reproducible control responses were obtained with no observable desensitization. The concentration of ACh was 10 µM for trials with 11
and 100 µM for all other nAChRs. Toxin was bath-applied for 5–10 min (10 min at concentrations <10 nM), followed by a pulse of ACh. Thereafter, toxin was washed away, and subsequent ACh pulses were given every 1 min, unless otherwise indicated. All ACh pulses contain no toxin; for it was assumed that little if any bound toxin washed away in the brief time (it takes <2 sec for the responses to peak). In our recording chamber, the bolus of ACh does not project directly at the oocyte but rather enters tangentially and then swirls and mixes with the bath solution. The volume of entering ACh is such that the toxin concentration remains at a level >50% of that originally in the bath until the ACh response has peaked (<2 sec).
The average peak amplitude of three control responses just preceding exposure to toxin was used to normalize the amplitude of each test response to obtain a "percentage response" or "percentage block." Each data point of a dose–response curve represents the average value ± SE of measurements from at least three oocytes. Dose–response curves were fit to the following equation:
where nH is the Hill coefficient with Prism software (GraphPad Software, San Diego, CA) on an Apple Power Macintosh.
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Results
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Cloning of -conotoxin PIA
Conus venoms are extremely complex, with an estimated 200 peptides in the venom of a single Conus species. There are 500 species of cone snails; in each species, there appears to be on average one-half dozen -conotoxins targeted to nAChRs. Although the mature -conotoxin sequences diverge considerably, the organization of -conotoxin genes reveals considerable conservation across species. The intron immediately preceding the toxin sequence, the signal sequence within the exon, and the 3' untranslated region show a high degree of conservation. Proteolytic processing of the larger precursor protein leads to the formation of the mature -conotoxin. Conserved features of the -conotoxin gene structure were exploited to isolate a novel -conotoxin gene from Conus purpurascens. Two complementary approaches were used. In the first, direct amplification of the -conotoxin gene sequence from genomic DNA was performed. The coding region of the signal sequence and most of the propeptide region was separated from the toxin-coding region and 3' untranslated region by a single intron. Primers were designed to this conserved intron sequence and used in conjunction with primers to the conserved 3' untranslated region to PCR-amplify the toxin-coding portion of the -conotoxin gene. In the second approach, reverse-transcription PCR of venom duct RNA was performed. This was accomplished by designing oligonucleotide primers on the basis of conserved regions in the signal sequence and the 3' untranslated region. Both methods yielded clones encoding -conotoxin PIA. Figure 1A shows the results for amplification of genomic DNA. Clones of -conotoxin MII from Conus magus and -conotoxin GIC are shown for comparison in Figure 1B; as can be appreciated, the prepropeptide regions of the three peptides are highly conserved. This is in contrast to the mature toxin regions that are much more divergent, particularly in the C-terminal portions of the peptides.
Chemical synthesis of -conotoxin PIA
The -conotoxin gene sequences were conceptually translated to identify the conotoxin peptide. Because of the highly conserved nature of the cysteine residues in -conotoxins and conserved proteolytic processing sites, the mature toxin sequences can be reliably identified from the gene sequence. Complete chemical synthesis of the predicted mature toxin was undertaken. It is important to note that -conotoxin PIA has not yet been isolated from Conus purpurascens venom. Therefore, it is possible that there are post-translational modifications in the native peptide. Such modifications could influence the properties of the native peptide compared with the synthetic peptide described in this study.
To synthesize the peptide, two assumptions were made. First, it was assumed that the disulfide-bonding pattern of -conotoxin PIA was the same as that of all previously characterized -conotoxins, that is, Cys1 to Cys3 and Cys2 to Cys4. Cys residues were orthogonally protected to direct the formation of disulfide bonds in this configuration. The first and third cysteine residues were protected with acid-labile groups, which were removed first after a cleavage from the resin; ferricyanide was used to close the first disulfide bridge. Reverse-phase HPLC was used to purify the monocyclic peptide; subsequently, the acid-stable acetometomethyl groups were removed from the second and fourth cysteines by iodine oxidation, which also closed the second disulfide bridge. The fully folded peptide was purified again by HPLC. Laser desorption mass spectrometry (reflectron) of synthetic -conotoxin PIA was consistent with the amidated sequence (monoisotopic MH+: calculated, 1980.83; observed, 1980.71). Synthetic peptide was used in all subsequently described studies.
Toxin effects on heterologously expressed nAChRs
Injection of human 6 subunits into oocytes either alone or in combination with 2 or 3 subunits does not reliably produce functional nAChRs (Kuryatov et al., 2000; our observations). In these studies, we therefore used a previously reported chimera, wherein the large extracellular domain of 6 is joined to the transmembrane and intracellular portion of the closely related human 3 subunit (Kuryatov et al., 2000). Similarly in these studies, we failed to achieve expression of rat 6 when its cRNA was injected into oocytes either alone or in combination with 2 or 3. We therefore created and used a chimera of the rat 6 and 3 subunits analogous to the human chimera described above to improve functional expression.
The mature toxin sequence of -conotoxin PIA bears some resemblance to -conotoxins MII and GIC isolated from Conus magus and Conus geographus, respectively. The latter two peptides potently target neuronal subtypes in preference to the muscle subtype of nAChR (Table 1). We therefore examined the effect of -conotoxin PIA on heterologously expressed neuronal and muscle nAChRs in Xenopus oocytes. As shown in Figure 2, 100 nM -conotoxin PIA abolishes the ACh-induced response on human 6/323 nAChRs. In contrast, 10 µM -conotoxin PIA has no effect on the adult form of the human muscle nAChR, indicating a high degree of preference for the neuronal nAChR. The effect of -conotoxin PIA was reversed only after several minutes of toxin washout in the case of human 6/323 and human 6/3/2 nAChRs. In contrast, although high concentrations of -conotoxin PIA also blocked the human 32 nAChR, this effect was rapidly reversed after toxin washout (Fig. 3).
To assess the effect of the toxin on rodent nAChRs, concentration–response analysis was conducted on rat 6/32 (with and without 3), 32, 42, and 22 subunit combinations heterologously expressed in Xenopus oocytes. The results are shown in Figure 4 and Table 2. In contrast to previously isolated-conotoxins and other known nAChR antagonists, -conotoxin PIA shows selectivity for heteromeric nAChRs containing the extracellular 6 subunit sequence in comparison with 3, 4, and 2 subunits. We note that the 3 subunit, when coinjected with chimeric 6/3 and 2, substantially increased expression in oocytes. However, the 3 subunit did not significantly affect the IC50 of the -conotoxin PIA (Fig. 4, Table 2). 3 is also believed to be present in native 6-containing nAChRs, where it presumably acts as a structural subunit (Lena et al., 1999). Concentration–response analysis for -conotoxin PIA was also performed on human chimeric 6/323 nAChRs and gave similar results (IC50, 1.7 nM; 95% confidence interval; 1.1–2.8 nM) to rat chimeric 6/323 (IC50, 0.95 nM; 95% confidence interval; 0.72–1.3 nM)
-Conotoxin PIA was also tested on 4-containing nAChRs. Potency was comparatively low on 24, 34, and 44 nAChRs. Although the 64 subunit combination is present in the chick (Vailati et al., 1999), it has not been identified yet in mammalian systems. Nevertheless, we assessed the toxin affinity for this subtype. No currents were seen using rat 6/34 subunits when 5 ng of cRNA per subunit was injected; therefore, cRNA amounts were increased to 10–25 ng per subunit. -Conotoxin PIA potently blocked this combination for both rat and human subunits (Table 2). Larger amounts of cRNA (40 ng) were also needed to see expression of r64. Expression was small, usually leading to currents <5 nA, and occurred only in a minority of oocytes at 7 d after injection. -Conotoxin PIA blocked rat 64 and chimeric 6/34 with similar potencies (Fig. 4, Table 2). Thus, PIA selectively blocks 6/32 versus 32 nAChRs, and selectively blocks 6/34 versus 34 nAChRs, indicating the 6 versus 3 preference of this peptide. In contrast, -conotoxin MII does not distinguish well between 6/323 and 32 nAChRs. The IC50 was 0.40 nM (95% confidence interval; 0.29–0.54 nM) for 6/323, and the IC50 for 32 was 1.7 nM (95% confidence interval; 1.3–2.4 nM).
Toxin off-rates were examined for rat 6/323, 6/32, and 32 nAChRs. As in the case of the human nAChRs, the effect of -conotoxin PIA is much more slowly reversed during washout from 6/32 (with or without 3) versus 32 nAChRs (Fig. 5a). Thus, -conotoxin PIA distinguishes between these two nAChRs, both in potency and kinetics of unblock. In addition, the toxin also dissociates slowly from rat 4-containing subunit combinations (which under the described conditions may lead to a small underestimation of its potency at these subtypes) (Fig. 5b). However, for both r34 and r6/34, preincubation with -conotoxin PIA for 20 min did not significantly change the IC50 compared with that obtained with 10 min preincubation (data not shown).
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Figure 5. Off-rate kinetics of -conotoxin PIA. a, -Conotoxin PIA was bath-applied for 5 min to oocytes heterologously expressing the indicated rat2-containing nAChRs. The concentration of toxin was 10 nM for6/32, 100 nM for6/323, and 10µM for32.kofffor 6/323 = 0.17 min-1. koff for 6/32 nAChRss was similar (0.19 min-1). In contrast, the block of 32 nAChRs by -conotoxin PIA was fully reversed within 1 min of toxin washout. b,-Conotoxin PIA was applied as described above to rat4-containing nAChRs. The toxin concentration was 1 µM for 64, 6/34, and 34, and 10 µM for r24 and 44. koff for 34 was 0.44 min-1. Recovery from block for 64 and 6/34 was comparatively slow, with only 25 and 30% recovery, respectively, after 10 min.
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Discussion
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Toxins represent powerful tools for distinguishing among various subtypes of ligand- and ion-gated channels. In particular, polypeptide toxins from land and sea snakes have proven invaluable for characterizing nAChRs. Certain alkaloids such as methyllycaconitine, isolated from the seeds of the larkspur plant, are also quite useful. More recently, small peptides from the venomous gastropod Conus have been shown to discriminate between various forms of nAChRs. Previous studies of -conotoxin MII indicated its ability to discriminate among 32 versus other nAChR subtypes. However, at the time of initial characterization of -conotoxin MII, the pharmacology of 6-containing nAChRs was unknown. The 6 subunit is structurally closely related to 3. This study indicates that -conotoxin MII discriminates poorly between 32 nAChRs and 62* nAChRs. 62* nAChRs appear central to important physiological and possibly pathological conditions. 62 nAChRs appear to be involved in the modulation of dopamine release (Champtiaux et al., 2002) and are significantly reduced or eliminated in primate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine models of Parkinson's disease (Quik et al., 2001). We therefore wished to investigate whether other species of Conus snails produce toxins that have the ability to distinguish 62* from other 2-containing nAChRs. We used conserved features of the Conus genome to probe for novel peptide ligands.
Conus purpurascens, also known as the purple cone, is moderately common in shallow water from the Gulf of California to Peru. It is often found in tide pools and on rocky ledges where it hunts fish (Keen, 1972; Walls, 1979). We used both genomic cloning and cDNA libraries in C. purpurascens to isolate a gene encoding a novel -conotoxin. On the basis of the clone, we synthesized the predicted mature peptide toxin. It should be noted that the native toxin has not been isolated yet from the venom of Conus purpurascens. Post-translational modifications of the native peptide, if present, may confer different properties than those indicated for the synthetic peptide described in this study. The predicted structure is a 16 amino acid peptide with two disulfide bonds. The disulfide bonds were folded in the arrangement of Cys1–Cys3, Cys2–Cys4, because this conformation is present in all previously characterized venom-isolated -conotoxins.
Because the 6 subunit did not express with 2 or 2 plus 3, we used a chimeric 6/3 subunit to allow expression. The putative structural subunit, 3, did not substantially affect either toxin potency or off-rate kinetics. That is, the block by -conotoxin PIA of 6/323 nAChRs was not significantly different from that of 6/32 nAChRs. Both rat 6 with 4 and the rat chimeric 6/3 with 4 gave similar IC50s with -conotoxin PIA. This indicates that, as expected, the toxin binds to determinants on the extracellular portion of the nAChR and that the remaining 3 portion of the chimeric 6/3 subunit does not substantially affect peptide binding. Also note that the IC50 for -conotoxin MII for chimeric rat 6/323 determined in this study (0.4 nM) is in reasonable agreement with the affinity of -conotoxin MII (1.3 nM) for rat 62* nAChRs immunoprecipitated from the striatum (Zoli et al., 2002). This again suggests that the 6/3 chimera as expressed in Xenopus oocytes provides a good approximation of the native 6 extracellular binding site. In null mutant mice, [125I]-conotoxin MII binding is abolished in 6-/- mice, yet primarily retained in 3-/- mice (Champtiaux et al., 2002; Whiteaker et al., 2002). Given the similar affinity of -conotoxin MII for 6/32 and 32 nAChRs found in this study, we interpret the results with radiolabeled MII to indicate that the predominant CNS binding site of MII is an 62* nAChR.
There is a high degree of identity between the extracellular portion of human 6 and 3, and between rat 6 and 3 nAChR subunits. Despite these similarities, -conotoxin PIA has an 75-fold lower IC50 for 6/323 nAChRs compared with 32 nAChRs. In the extracellular regions, there is also considerable homology between rat 6, 4, and 2 subunits. Nevertheless, -conotoxin PIA has an IC50 >10 µM for the rat 42 and 22 subunit combinations heterologously expressed in oocytes. -Conotoxin PIA also potently blocks 6/34 versus 24, 34, and 44 nAChRs, also demonstrating that toxin selectivity determinants are located on the extracellular portion of the 6 subunit. 62* nAChRs are located in the striatum, where they are believed to modulate dopamine release (Zoli et al., 2002). We note that -conotoxin PIA potently blocks nicotine-stimulated dopamine release in rat striatal synaptosomes with low nanomolar potency (Azam et al., 2002a).
Structure–function studies of the 3 nAChR subunit and -conotoxin MII indicate that lysine 185 and isoleucine 188 of 3 are critical amino acids for toxin binding (Harvey et al., 1997). These are conserved between 3 and 6 subunits. Because -conotoxin PIA, unlike -conotoxin MII, is able to distinguish 6 from 3, it appears that at least some of the nAChR-binding determinants for the two toxins are distinct. Table 3 shows residues that are present in 6 but not identical or homologous to residues in aligned positions of 2, 3, and 4 subunits. These residues account for 10% of the total amino acids in the extracellular region. Only one of the identified residues differs between rat and human 6. This conserved nature of the 3 and 6 subunits suggests that a nAChR mutagenesis approach would be useful to establish the residues in 6 responsible for high-affinity -conotoxin PIA binding.
-Conotoxins PIA and MII have identical spacing of Cys residues, disulfide connectivity, and the SNPV (serine, asparagine, proline, valine) sequence in the first peptide loop. Therefore, it follows that differences in either the N terminal or loop 2 sequences account for the differences in selectivity between 6 and 3 subunits. In addition, whereas MII has some affinity for muscle and 42 subtypes (IC50, 430 nM and 3.7 µM, respectively) (Cartier, 2002), PIA has no measurable effects on these subtypes at 10 µM (Figs. 2, 4).
Differences between -conotoxin MII and -conotoxin PIA also result in differences in off-time kinetics between the two toxins. Both toxins show a relatively long off-time for 6/323 nAChRs (this study; our unpublished observations). In contrast, whereas -conotoxin MII shows a relatively long off-time for 32 nAChRs (Cartier et al., 1996), block by -conotoxin PIA reverses quickly (Fig. 5). Thus, block by -conotoxin PIA of 6/323 versus 32 nAChRs may be distinguished in two ways. The first is the 75-fold lower IC50 for 6/323 nAChRs; the second is the prolonged koff relative to 32. Off-rate kinetics for -conotoxin PIA was dependent on the subunit. The koff for -PIA was substantially longer for 64 than 6/32. Likewise, the koff for 34 was longer than that of 32 (Fig. 5). This suggests that -conotoxin PIA has binding determinants on the subunit, as demonstrated previously for -conotoxin MII (Harvey et al., 1997). In addition, it indicates that the subunit influences both the toxin on-rate and off-rate. The range of kinetic values for -conotoxin PIA is consistent with that of -conotoxins reported previously (Cartier et al., 1996; McIntosh et al., 2002). The IC50 of -conotoxin PIA was also related to the subunit. For both 3 and 6 subunits, the presence of a 2 versus 4 subunit led to a lower IC50 (Table 2).
To our knowledge, this is the first demonstration that 6 versus non-6-containing nAChRs may be pharmacologically distinguished by an antagonist. This has implications for several pathophysiological states. The 6 subunit is highly expressed in CNS regions including the nigrostriatal pathway, which is pathologically affected in Parkinson's disease, vision-related nuclei, the locus ceruleus implicated in attention, and ventral tegimental area that modulates reward and addictive behavior (Le Novère et al., 1996, 1999; Vailati et al., 1999; Klink et al., 2001; Azam et al., 2002b; Champtiaux et al., 2002).
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Footnotes
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Received May 5, 2003;
revised July 22, 2003;
accepted July 24, 2003.
This work was supported by National Institutes of Health Grants MH53631 (J.M.M.), NS11323 (J.M.L.), and GM48677 (B.M.O., D.Y.).
Correspondence should be addressed to J. Michael McIntosh, Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112. E-mail: mcintosh{at}biology.utah.edu.
Copyright © 2003 Society for Neuroscience 0270-6474/03/238445-08$15.00/0
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Abstract
Introduction
