©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
A New -Conotoxin Which Targets 32 Nicotinic Acetylcholine Receptors (*)

(Received for publication, November 13, 1995; and in revised form, January 8, 1996)

G. Edward Cartier (1) Doju Yoshikami (1) William R. Gray (1) Siqin Luo (1) Baldomero M. Olivera (1) J. Michael McIntosh (1) (2)(§)

From the  (1)Departments of Biology and (2)Psychiatry, University of Utah, Salt Lake City, Utah 84112

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
Discussion
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have isolated a 16-amino acid peptide from the venom of the marine snail Conus magus which potently blocks nicotinic acetylcholine receptors (nAChRs) composed of alpha3beta2 subunits. This peptide, named alpha-conotoxin MII, was identified by electrophysiologically screening venom fractions against cloned nicotinic receptors expressed in Xenopus oocytes. The peptide's structure, which has been confirmed by mass spectrometry and total chemical synthesis, differs significantly from those of all previously isolated alpha-conotoxins. Disulfide bridging, however, is conserved. The toxin blocks the response to acetylcholine in oocytes expressing alpha3beta2 nAChRs with an IC of 0.5 nM and is 2-4 orders of magnitude less potent on other nAChR subunit combinations. We have recently reported the isolation and characterization of alpha-conotoxin ImI, which selectively targets homomeric alpha7 neuronal nAChRs. Yet other alpha-conotoxins selectively block the muscle subtype of nAChR. Thus, it is increasingly apparent that alpha-conotoxins represent a significant resource for ligands with which to probe structure-function relationships of various nAChR subtypes.


INTRODUCTION

The muscle subtype of nicotinic acetylcholine receptor (nAChR) (^1)is one of the best understood ligand-gated channels due in part to the availability of a large number of protein and small molecule ligands which serve as specific probes for this channel. The nAChR is a heteropentameric ion channel complex and is a member of a superfamily that includes glycine, GABA(A), and 5-HT(3) receptors(1) . The mammalian nAChR has the subunit composition (alpha1)(2)beta1 in developing muscle, and the subunit is replaced by an subunit in mature muscle. In mammalian neurons the situation is much more complex with at least seven alpha subunits, designated alpha2-alpha7 and alpha9 (in chick there is also an alpha8 subunit), and three beta subunits, beta2-beta4. The alpha2, alpha3, and alpha4 subunits can each combine with beta2 or beta4 subunits to form functional channels when expressed in Xenopus oocytes, e.g. alpha2beta2, alpha3beta2, alpha2beta4, etc. In addition, alpha7 and alpha9 subunits can be expressed as functional homooligomers in this system. Studies employing either nucleotide probes or antibodies indicate that each of these alpha and beta subunits have a unique pattern of anatomical expression in the central nervous system(2) . However, the precise structural composition and functional role of the different neuronal subtypes of nicotinic receptors are less well understood. The development of subtype-specific ligands will greatly aid progress in this area.

Although a number of valuable nicotinic antagonists have been described, few are highly subtype-selective, particularly in the case of neuronal nAChRs. d-Tubocurarine, an alkaloid from the Chondrodendron tomentosum bush, used for centuries as an arrow poison to kill wild game, blocks both muscle and neuronal nAChRs (3) . In addition it binds to all neuronal nicotinic receptors with more or less similar affinities(4) . Likewise, dihydro-beta-erythroidine, the hydrogenated derivative of erythroidine, isolated from trees and shrubs of the genus Erythrina is a competitive antagonist at both muscle and neuronal nAChRs(4) . Lophotoxin, a small cyclic diterpene, is used by the soft shell coral Lophogorgia rigida to discourage its consumption by fish(5) . This toxin forms a covalent bond with Tyr of the alpha-subunit of Torpedo nAChRs, irreversibly blocking the binding of ACh to the receptor(6, 7) . Studies with nAChRs expressed in Xenopus oocytes reveal that this toxin blocks muscle nAChRs as well as alpha2beta2, alpha3beta2, and alpha4beta2 neuronal nAChRs(8) . Neosurugatoxin, a glycoside from the gastropod Babylonia japonica(9) , potently but nonselectively blocks alphaxbeta2 nAChRs expressed in oocytes, where x is 2, 3, or 4(8) . The synthetically derived small molecules trimethaphan and mecamylamine discriminate between ganglionic and neuromuscular nAChRs and are used clinically as ganglionic blocking agents(3) .

Numerous protein toxins which act at muscle nAChRs have been isolated from a variety of snake venoms and proven highly useful for studying nAChRs. Two toxins from the Taiwanese banded krait, Bungarus multicinctus, have been particularly well characterized. The major nicotinic antagonist in this venom, alpha-bungarotoxin, in addition to blocking the muscle receptor, potently blocks alpha7 subunit-containing neuronal nAChRs(10) . Methyllycaconitine, an alkaloid toxin from the seeds of Delphinium brownii has markedly greater affinity for the I-alpha-bungarotoxin binding site in brain versus that in muscle demonstrating that these receptor subtypes can be pharmacologically distinguished(11) . A minor component of Bungarus venom, kappa-bungarotoxin (also known as neuronal bungarotoxin, toxin F, or Bgt 3.1) preferentially blocks alpha3beta2 receptors(8) , although the presence of venom purification contaminants has led to inconsistent findings(8, 12) . Unfortunately, due to limited availability of venom, this potent toxin is commercially unavailable at the present time.

A growing number of nicotinic antagonists have been isolated from the venom of the carnivorous marine snail Conus and are known as alpha-conotoxins. In contrast to snake alpha-toxins (60-80 amino acids), alpha-conotoxins are much smaller (12-25 amino acids), a feature which has allowed them to be readily chemically synthesized (13) . alpha-Conotoxins, which target the muscle nAChR are enjoying increasing use due to their recently discovered ability to differentiate between the two acetylcholine binding sites on the receptor. In the mouse muscle-derived BC(3)H-1 cell line alpha-conotoxins MI, GI, and SIA (respectively from Conus magus, Conus geographus, and Conus striatus) selectively bind to the ACh binding site at the alpha/ interface with more than 10^4-fold greater affinity than the site at the alpha/ interface(14, 15) . With Torpedo nAChR, the situation is reversed. alpha-Conotoxins MI and GI bind at the alpha/ interface with approximately 2 orders of magnitude greater affinity than the alpha/ interface(15, 16) . Like alpha-conotoxins MI, GI, and SIA, alpha-conotoxin EI, from Conus ermineus, prefers the alpha/ to the alpha/ interface of receptors in BC(3)H-1 cells, but with only 30-fold difference. In contrast to these other alpha-conotoxins, with Torpedo receptors, alpha-conotoxin EI preferentially binds the alpha/ versus the alpha/ interface by a 400-fold difference in affinity and is the only ligand known to possess this selectivity(17) . Thus, these alpha-conotoxins can serve as specific probes to investigate structure-function relationships of nAChRs (18) .

There are approximately 500 species of Conus. Each of these predatory gastropods hunt prey from one of five different phyla, and all of these prey have cholinergic synapses(19) . Thus, there is a potentially very wide diversity of nAChRs for conotoxins to target, and it is likely therefore that there are a comparably wide spectrum of cholinergically active peptides in the venom of Conus. We are seeking to exploit this situation to develop a bank of peptides which act on specific subtypes of neuronal nicotinic receptors. By use of a bioassay involving intracranial injections into mice to guide purification, we previously isolated alpha-conotoxin ImI which, unlike other alpha-conotoxins, selectively targets alpha7, and to a lesser degree alpha9, nAChRs(20, 21) . In the present study we used a much more specific screening assay to purify a novel nicotinic antagonist from C. magus venom. Voltage-clamped Xenopus oocytes expressing alpha3beta2 nAChRs were used in the assay to isolate alpha-conotoxin MII. We report the structural characterization and nAChR subtype selectivity of this peptide.


MATERIALS AND METHODS

Peptide Isolation and Sequencing

Venom Extraction

Crude venom from dissected ducts of C. magus was collected in the Philippines, lyophilized, and stored at -70 °C until used. All reagents were precooled to, and extraction procedures were conducted at, 4 °C. Fifteen ml of 0.1% trifluoroacetic acid was added to 500 mg of lyophilized venom, and the mixture was vortexed for 20 min. This mixture was centrifuged at 17,000 times g for 20 min. The supernatant was transferred to a separate tube, and another 15 ml of 0.1% trifluoroacetic acid was added to the pellet which was then sonicated with a Sonifier (Branson Instruments) at setting #4, vortexed for 10 min, and centrifuged as above. The supernatants were combined and filtered through a Whatman GF/C filter (Whatman, Ltd, Maidstone, UK), and then placed in two Centriprep 30 microconcentrators (Amicon, Beverly, MA) which have a 30,000 molecular weight cut-off. The Centripreps were centrifuged at 1500 times g until the retentate in each was reduced to 5 ml (45 min). The filtrate was removed and 10 ml of 0.1% trifluoroacetic acid was added to the retentate of each Centriprep. The Centripreps were again centrifuged at 1500 times g for 120 min. The addition of trifluoroacetic acid reduced the viscosity of the retentate and improved the recovery of filtrate. Filtrates were combined and used for further purification of alpha-conotoxin MII.

RPLC Purification

All RPLC columns were from Rainin Instruments. Crude venom extract was fractionated on a semipreparative Vydac C18 column (10 mm times 25 cm, 5 µm particle size, 330 Å pore size) equipped with a guard module (catalog number 83-223-65). All other chromatographic purifications involved an analytical Brownlee C8 column (4.6 mm times 22 cm, RP300 packing, 7 µm particle size, 300 Å pore size) with a guard cartridge which also had RP300 packing material. Synthetic peptide was purified on a preparative Vydac C18 column (22 mm times 25 cm, 10 µm particle size, 330 Å pore size). For all chromatograms buffer A was 0.1% trifluoroacetic acid and buffer B was 0.1% trifluoroacetic acid, 60% acetonitrile. Trifluoroacetic acid (sequencing grade) was from Aldrich; acetonitrile (UV grade for semipreparative and analytical RPLC, non-spectro grade for preparative RPLC) was from Baxter.

Pyridylethylation and Purification of Modified Peptide

Peptide from the final purification was stored in the RPLC buffer in which it eluted. A 287-µl solution of this purified peptide (250 pmol) was combined with 14.4 µl (20:1 v/v) of 0.5 M Tris base which raised the pH to a value between 7 and 8 as measured with pH paper. Seventy-five µl of 50 mM dithiothreitol was added (final concentration 10 mM); the reaction vessel was flushed with argon, and the reaction incubated at 65 °C for 15 min. The solution was allowed to cool; 15 µl of 20% 4-vinyl pyridine in ethanol was added, and the solution was reacted for a further 25 min at room temperature in the dark. The solution was diluted 3-fold with 0.1% trifluoroacetic acid, and the alkylated peptide was loaded on the Brownlee column. After washing the column with 20% buffer B to allow the baseline to return to 10% of the initial reading, the peptide was eluted with the gradient described in Fig. 1, panel B.


Figure 1: Purification of alpha-conotoxin MII by RPLC. Panel A, filtrate of venom extract (38.2 ml) was loaded onto a semipreparative Vydac C18 column with 0% buffer B and subsequently eluted using a gradient system. The gradient was 0-15% buffer B/15 min, then 15-39% buffer B/72 min, then 39-65% buffer B/15 min, then 65-100% buffer B/5 min and held at 100% buffer B for 2 min. Flow rate was 5 ml/min. Panel B, fractions indicated by the arrow in Panel A were diluted with 2 volumes of 0.1% trifluoroacetic acid and repurified on an analytical Brownlee C8 column. The gradient was 20-50% buffer B/60 min at a flow rate of 1 ml/min. Panel C, the right half of a center cut of the absorbance peak indicated by the arrow in Panel B was diluted with 2 volumes of 0.1% trifluoroacetic acid and re-chromatographed as described in panel B to obtain the final purified product. A 5-ml sample loading loop was used in all chromatography. Buffer A, 0.1% trifluoroacetic acid; and buffer B, 0.1% trifluoroacetic acid, 60% acetonitrile. Absorbance was monitored at 220 nm.



Sequence Analysis

Sequencing was performed with Edman chemistry on an Applied Biosystems 477A Protein Sequencer at the Protein/DNA Core Facility at the University of Utah Cancer Center. Mass spectrometry was performed as described previously(17) .

Peptide Synthesis

Linear Peptide

All amino acid derivatives were purchased from Bachem (Torrance, CA). The linear peptide chain was built on Rink amide resin by Fmoc (N-(9-fluorenyl)methoxycarbonyl) procedures with 2-(1H-benzotriole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate coupling, using an ABI model 430A peptide synthesizer. Side chain protection of non-Cys residues was in the form of t-butyl (Glu, Ser) and trityl (Asn, His). Orthogonal protection was used on cysteines: Cys^3 and Cys were protected as the stable Cys(S-acetamidomethyl), while Cys^2 and Cys^8 were protected as the acid-labile Cys(S-trityl). After assembly of the resin-bound peptide, the terminal Fmoc group was removed in situ by treatment with 20% piperidine in N-methylpyrrolidone. Linear peptide amide was cleaved from 93 mg of resin by treatment with 1 ml of trifluoroacetic acid/H(2)O/ethanedithiol/phenol/thioanisole (90/5/2.5/7.5/5 by volume) for 1.5 h at 20 °C. Released peptide was precipitated by filtering the reaction mixture into methyl-t-butyl ether which had been cooled to -10 °C. This procedure simultaneously cleaved peptide from the resin and deprotected the Cys(S-trityl) and the non-Cys residue side chains, but not the Cys(S-acetamidomethyl) residues. The cleavage reaction vessel was rinsed with 100% trifluoroacetic acid, and this rinse was also filtered into the methyl-t-butyl ether solution. The precipitate was washed two additional times with chilled methyl-t-butyl ether, and the supernatants were discarded. Pelleted peptide was dissolved by the addition of approximately 10 ml of 0.1% trifluoroacetic acid in 60% acetonitrile, with gentle swirling (to avoid foaming). The linear peptide solution was diluted with 190 ml of 0.1% trifluoroacetic acid and purified by RPLC on the preparative C18 Vydac column with a 10-60% buffer B gradient over 50 min. Flow rate was 20 ml/min. This gradient was also used for all subsequent preparative RPLC purifications of the synthetic peptide.

Peptide Cyclization

To form a disulfide bridge between Cys^2 and Cys^8 (i.e. the first and third cysteines), the major peptide fraction from the preparative RPLC (see above) was diluted to 1 liter with H(2)O and solid Tris base was added to increase the pH to 7.6. The solution was placed in a 4-liter flask and gently swirled at room temperature for 38 h at which time the reaction was judged to be complete by analytical RPLC. The pH of the solution was decreased to a value of 2-3 (measured with pH paper) by the addition of 4 ml of trifluoroacetic acid. The monocyclic peptide was then purified by RPLC and collected in a volume of 45 ml. Removal of the S-acetamidomethyl groups and closure of the second disulfide bridge (Cys^3-Cys, i.e. the second and fourth cysteines) was carried out simultaneously by iodine oxidation. The 45 ml of RPLC eluent containing the monocyclic peptide was dripped into a rapidly stirred 50-ml solution of 20 mM iodine in H(2)0/trifluoroacetic acid/acetonitrile/MeOH (50:20:20:10 by volume) over 5.5 min at room temperature. The reaction was allowed to proceed for another 15 min and terminated by the addition of ascorbic acid. The solution was diluted to 1 liter and the bicyclic peptide purified by RPLC.

Electrophysiology

cRNA Preparation

cDNA clones encoding nAChR subunits were provided by S. Heinemann and D. Johnson (Salk Institute, San Diego, CA). cRNA was transcribed using either RiboMAX large scale RNA production systems (Promega, Madison, WI) or an RNA transcription kit (Stratagene, La Jolla, CA). Diguanosine triphosphate (Sigma) was used for synthesis of capped cRNA transcripts according to the protocol of the manufacturer. Plasmid constructs of mouse and rat nAChR subunits were as described: alpha1, beta1, , (22) ; alpha2(23) ; alpha3(24) ; alpha4(25) ; alpha7(^2); alpha9(26) ; beta2(27) ; and beta4(28) .

cRNA Injection

cRNA was injected with a Drummond 10-µl microdispenser (Drummond Scientific, Broomall, PA) essentially as described by Goldin(29) . It was fitted with micropipettes pulled from glass capillaries provided for the microdispenser. The pipette tips were broken to an OD of 22-25 µm and back-filled with paraffin before mounting on the microdispenser. cRNA was drawn into the micropipette and 50 nl, containing 5 ng of cRNA of each subunit, was injected into each oocyte. In the case of muscle subunits, 0.5-2.5 ng of each subunit was injected.

Oocyte Harvesting

Oocytes were removed from Xenopus frogs, cut into clumps of 20-50 oocytes, and placed in a 50-ml polypropylene tube (Sarstedt) containing 580 units/ml type 1 collagenase (Worthington) in OR-2 (82.5 mM NaCl, 2.0 mM KCl, 1.0 mM MgCl(2), and 5 mM HEPES, pH 7.3). The tube was incubated for 1-2 h on a rotary shaker rotating at 50 rpm. Half-way through the incubation, the solution was exchanged with fresh collagenase solution. The oocytes were then washed with six to eight 50-ml volumes of OR-2, transferred to a 60 times 15-mm Petri dish containing ND-96 (96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl(2), 1.0 mM MgCl(2), 5 mM Hepes, pH 7.1-7.5)/Pen/Strep/Gent (100 units/ml penicillin G (Sigma), 100 µg/ml streptomycin (Sigma), and 100 µg/ml gentamycin (Life Technologies, Inc., Grand Island, NY)). The oocytes were visually examined and only healthy appearing oocytes were transferred to a second dish containing ND-96 and antibiotics. Oocytes were injected 1-2 days after harvesting and recordings were made 1-7 days after injection.

Voltage-clamp Recording

An injected oocyte was placed in a 30-µl recording chamber consisting of a cylindrical well (4 mm diameter times 2 mm deep) fabricated from Sylgard, and gravity-perfused with either ND96 or ND96 containing 1 µM atropine (ND96A) at a rate of 1 ml/min. All toxin solutions also contained 0.1 mg/ml bovine serum albumin to reduce nonspecific adsorption of toxin. The perfusion medium could be switched to one containing toxin or acetylcholine (ACh) by use of a distributor valve (SmartValve, Cavro Scientific Instruments, Sunnyvale, CA) and a series of three-way solenoid valves (model 161TO31, Neptune Research, Northboro, MA). ACh-gated currents were obtained with a two-electrode voltage-clamp amplifier (model OC-725B, Warner Instrument Corp., Hamden, CT) set for ``fast'' clamp and with clamp gain at maximum (times 2000). Glass microelectrodes, pulled from fiber-filled borosilicate capillaries (1 mm outer diameter times 0.75 mm inner diameter, WPI Inc., Sarasota, FL) and filled with 3 M KCl, served as voltage and current electrodes. Resistances were 0.5-5 megohm for voltage, and 0.5-2 megohm for current electrodes. The membrane potential was clamped at -70 mV, and the current signal, recorded through virtual ground, was low-pass filtered (5 Hz cut-off) and digitized at a sampling frequency of 20 Hz. The solenoid perfusion valves were controlled with solid state relays (model ODC5 in a PB16HC digital I/O backplane, Opto 22, Temecula, CA). A/D conversion and digital control of solenoid valves were performed with a Lab-LC or Lab-NB board (National Instruments, Austin, TX) in a Macintosh (Quadra 630 or IIcx) computer. The computer communicated with the distributor valve via its serial port. Data acquisition and activities of the distributor and solenoid valves were automatically controlled by a home-made virtual instrument constructed with the graphical programming language LabVIEW (National Instruments, Austin, TX). To apply a pulse of ACh to the oocyte, the perfusion fluid was switched to one containing ACh for 1 s. This was automatically done every 5 min. The concentration of ACh was 1 µM for oocytes expressing alpha1beta1, 1 mM for alpha7, and 300 µM for all others. The ACh was diluted in ND96A for all except alpha7, in which case the diluent was ND96. For control responses, the ACh pulse was preceded by perfusion with ND96 (for alpha7) or ND96A (all others). No atropine was used with oocytes expressing alpha7, since it has been demonstrated to be an antagonist of these receptors(30) . For responses in toxin (test responses), the oocyte was perfused with toxin solution until equilibrated (generally 5 min, but up to 25 min at lower toxin concentrations) before the ACh pulse was applied. All ACh pulses contained no toxin, for it was assumed that little, if any, bound toxin would have washed away in the brief time (<2 s) it takes for the responses to peak (see Fig. 3). The peak amplitudes of the ACh-gated current responses were measured by the virtual instrument. The average of three control responses just preceding a test response was used to normalize the test response to obtain ``% response.'' Each data point of a dose-response curve represents the average ± S.E. of at least three oocytes. Dose-response curves were fit, with Prism software (GraphPad Software Inc., San Diego, CA), to the equation: % response = 100/{1 + ([toxin]/IC)^n}, where n(H) is the Hill coefficient. All recordings were done at room temperature (22 °C).


Figure 3: alpha-Conotoxin MII blocks ACh responses in oocytes expressing alpha3beta2 nicotinic acetylcholine receptors. Oocytes expressing alpha3beta2 nAChRs were voltage-clamped and the response to a 1-s pulse of ACh was measured (see ``Materials and Methods''). Panel A, 0.5 nM alpha-conotoxin-MII blocks 45% of the ACh-induced response. Panel B, in a different oocyte, 20 nM toxin blocks 98% of the ACh-induced response.



Bioassay

Intraperitoneal injections of toxin into Swiss Webster mice and intramuscular injections into goldfish were performed as described previously(17, 20) .


RESULTS

Purification and Characterization of alpha-Conotoxin MII

Serial dilutions of a 50 mg/ml ND96 buffer extract of crude C. magus venom were tested for their ability to block the ACh-induced current in Xenopus oocytes expressing alpha3beta2 nAChRs. Dose-dependent block was observed; 82% block was produced with 0.071 mg/ml crude venom extract solution (data not shown). C. magus venom was purified by RPLC as described under ``Materials and Methods.'' For the initial RPLC fractionation (Fig. 1), 5-ml fractions were collected. Aliquots of 0.2% of each fraction were pooled in groups of three, lyophilized, and 30% of each pool was tested on oocytes expressing alpha3beta2 subunits (see ``Materials and Methods''). Individual fractions of the active pool were then tested within the oocyte system and the active fraction purified to homogeneity via RPLC.

The purified peptide was reduced, alkylated, and sequenced as described under ``Materials and Methods.'' The sequence is: GCCSNPVCHLEHSNLC. Liquid secondary ion mass spectrometry indicated that Cys residues are present as disulfides and that the COOH-terminal alpha-carboxyl group is amidated (monoisotopic MH: calculated 1710.65, observed 1710.6). The sequence was further verified by total chemical synthesis (see below). The sequence resembles previously isolated alpha-conotoxins in its spacing of Cys residues, yet differs substantially in other amino acids. As will be shown below, the peptide potently targets the nAChR, and we have therefore named the peptide alpha-conotoxin MII in accordance with the nomenclature previously proposed for conotoxins (31) .

Chemical Synthesis

Solid phase chemical synthesis of alpha-conotoxin MII was undertaken to provide an abundant supply of peptide. It was assumed that the disulfide bridging of alpha-conotoxin MII would be analogous to all previously characterized alpha-conotoxins, i.e. 1st Cys-3rd Cys, 2nd Cys-4th Cys. Cys groups were protected in pairs to direct disulfide formation. The acid-labile trityl protecting groups were removed from Cys^2 and Cys^8 (i.e. the first and third cysteines) during the cleavage reaction which released linear peptide from the resin. Closure of the disulfide bridge was accomplished by air oxidation, and the monocyclic peptide was purified by RPLC. The acid-stable acetamidomethyl group was next removed from Cys^3 and Cys (i.e. the second and fourth cysteines), and the disulfide bridge closed by rapid iodine oxidation and the bicyclic peptide purified by RPLC. Peptide yield was 41.7 nmol/mg peptide resin.

The order of RPLC elution of the synthetic peptides is of note: linear first, followed by monocyclic and bicyclic last. With the formation of each disulfide bridge, alpha-conotoxin MII becomes increasingly hydrophobic. This is exactly the opposite behavior from alpha-conotoxin EI, where the formation of each disulfide bond results in decreased retention time on RPLC(17) , and may indicate that the disulfide bridges in alpha-conotoxin MII force hydrophobic residues to face outward. Synthetic peptide comigrated with native on RPLC (Fig. 2). Liquid secondary ionization mass spectrometry of synthetic alpha-conotoxin MII was consistent with the amidated sequence (monoisotopic MH: calculated 1710.65, observed 1710.8). Lyophilization or bath application of small (fmol - pmol) amounts of alpha-conotoxin MII resulted in apparent loss of peptide (data not shown). The hydrophobic nature of the peptide may account for this problem which was minimized by the use of carrier protein (bovine serum albumin) in all solutions and continuous perfusion rather than bath application of peptide.


Figure 2: Comparison of natural and synthetic alpha-conotoxin MII by RPLC. Native and synthetic peptide have similar elution times when chromatographed separately and comigrate when coinjected on RPLC. RPLC conditions were as described in the legend to Fig. 1, panel B, except that a 1-ml sample loading loop was used. Absorbance was monitored at 220 nM. Maximum OD readings were 0.0031, 0.16, and 0.012 absorbance units in the first, second, and third panels, respectively.



Electrophysiology

alpha-Conotoxin MII purification by RPLC was guided by an assay which used Xenopus oocytes expressing alpha3beta2 receptors. RPLC fractions were tested for their ability to block the ACh-induced response in this assay. We also examined the effect of the toxin on other nAChR subunit combinations expressed in oocytes. Both native and synthetic toxin blocked the alpha3beta2 nAChRs with equal potency (data not shown). Due to very limited availability of native toxin, synthetic toxin was used for all subsequent experiments. Synthetic alpha-conotoxin MII showed dose-dependent block of alpha3beta2 receptors at low nanomolar concentrations (Fig. 3). This block slowly reversed with washing (Fig. 4). alpha-Conotoxin MII blocks alpha3beta2 nAChRs with an IC of 0.5 nM, with an apparent Hill coefficient, n(H), of 0.8 (Fig. 5). alpha-Conotoxin MII was also tested on other nAChR subunit combinations. Results are shown in Fig. 5and indicate that alpha-conotoxin MII is 2-4 orders of magnitude more potent at alpha3beta2 nAChRs than at other nAChR subtypes.


Figure 4: nAChR block by alpha-conotoxin MII is slowly reversible. 20 nM toxin was applied to alpha3beta2-expressing oocytes for 5 min. The oocytes were then continuously perfused with buffer without toxin. Responses to ACh from a single experiment are shown. The experiment was repeated twice with similar results.




Figure 5: alpha-Conotoxin MII selectively blocks alpha3beta2 nAChRs. Oocytes expressing various nAChR subunit combinations were voltage clamped and the response to ACh measured. The IC for alpha-conotoxin MII block of the alpha3beta2 receptor is 0.5 nM (n(H) = 0.8). Oocytes expressing other nAChR subunit combinations were perfused with 200 nM alpha-conotoxin MII (400 times the IC on alpha3beta2 receptors). The mean ± S.E. response to ACh was as follows: , alpha4beta4, 96 ± 1; , alpha2beta4, 96 ± 6; bullet, alpha1beta1, 89 ± 3; box, alpha3beta4, 85 ± 5; Delta, alpha2beta2, 80 ± 3; down triangle, alpha4beta2, 70 ± 1; , alpha7, 44 ± 8. Data represent the mean for 3-5 oocytes.



We have previously shown that alpha-conotoxin MI (also from C. magus venom), alpha-conotoxin GI, and alpha-conotoxin ImI have no effect on alpha3beta2 receptors at up to 5 µM concentration (21) . Thus, alpha-conotoxin MII is the only conotoxin known to potently block this neuronal receptor subtype.

In Vivo Activity

Intraperitoneal injections of 10 nmol of alpha-conotoxin MII into 8-10-g mice did not result in any signs of paralysis (n = 3). This is in contrast to alpha-conotoxin MI, 0.67 nmol of which kills a 20-g mouse in 20 min(32) . Intramuscular injection of 5 nmol of alpha-conotoxin MII into fish did not result in any signs of paralysis (n = 3). This is in contrast to alpha-conotoxin MI where 0.5 nmol is paralytic.


Discussion

nAChR Selectivity

Xenopus oocytes expressing mammalian neuronal alpha3beta2 nAChRs were used in an assay which successfully guided the isolation of the novel 16-residue peptide, alpha-conotoxin MII. This is significant in that it is the first alpha-conotoxin known to target alpha3beta2 receptors. Most previously reported alpha-conotoxins target the muscle nAChR. Exceptions are alpha-conotoxin ImI, which selectively blocks homomeric alpha7 and alpha9 receptors(20, 21) , and alpha-conotoxins PnIA/B, which block molluscan neuronal nAChRs(33) . We have shown elsewhere that alpha-conotoxins MI and GI potently target muscle nAChRs expressed in Xenopus oocytes, but are inactive at all neuronal nAChRs tested, including alpha3beta2 receptors(21) . As demonstrated in this report, alpha3beta2 receptors are blocked by alpha-conotoxin MII with an IC of 0.5 nM. The effectiveness of the toxin on the other nAChR subunit combinations tested is 2-4 orders of magnitude less. Thus, alpha-conotoxin MII has an entirely unique activity profile among the alpha-conotoxins (Table 1), and represents a potent and selective new probe for studying nAChRs. Notably, the small size of alpha-conotoxin MII has allowed it to be chemically synthesized and thus readily available.



Structural Relationships among alpha-Conotoxins

Reported alpha-conotoxins can be classified into two main groups based on the spacing of the cysteine residues. One group has a ``3,5 spacing'' where the numerals indicate the number of amino acids between the second and third Cys and the third and fourth Cys, respectively (see Table 2). The other group, which includes alpha-conotoxin MII, has a ``4,7 spacing.'' Individual toxins from both groups can potently block the muscle nAChR, suggesting that it is not the Cys spacing which is responsible for alpha-conotoxin MII's selectivity. Aside from the Cys residues, the only completely conserved residue in all reported alpha-conotoxins is a proline between the second and third Cys. The other non-Cys residues in alpha-conotoxin MII are strongly divergent from all other alpha-conotoxins. Of the non-Cys residues in the 4,7 group, alpha-conotoxin MII from C. magus shares only 4 of 12 residues with alpha-conotoxin PnIB from C. pennaceus and only 1 of 12 residues with alpha-conotoxin EI from C. ermineus. Furthermore, except for the proline, alpha-conotoxin MII shares little if any homology with alpha-conotoxin MI (which has a 3,5 spacing) although both are from the same Conus species. However, despite the difference in Cys spacing and strong divergence in other amino acids, the disulfide bridges are exactly analogous between the two toxins, 1st Cys to 3rd Cys, 2nd Cys to 4th Cys, and this arrangement is also conserved in all other alpha-conotoxins where the disulfide linkages have been studied (Table 2). Recently, a new family of Conus peptides targeted to nAChRs was reported. These alphaA-conotoxins have a distinctly different structure including three disulfide bridges instead of two(34) .



Multiple nAChR-targeted toxins have previously been isolated from two other Conus species, C. geographus and C. striatus (see Table 2). In both of these cases, however, the alpha-conotoxins have considerable structural homology and all target the muscle nAChR in contrast to the MI and MII peptides isolated from C. magus that differ substantially in both structure and function. It will be of interest to determine which residues in MII confer alpha3beta2 nAChR selectivity and which in MI confer alpha1beta1 selectivity.

Biological Role

Injection of venom by Conus snails results in prey immobilization and capture. Nevertheless, the majority of the 100-200 peptides present in the venom of fish-hunting Conus do not induce paralysis when injected into fish. (^3)The functional targets and roles of these non-paralytic peptides are under investigation. However, all previously reported alpha-conotoxins from fish-hunting Conus do cause rapid paralysis when injected into fish. alpha-Conotoxin MII is unique in not causing paralysis in this assay. It has recently been shown that only as few as three amino acid substitutions in the or subunit of mouse nAChR can result in a 10^4-fold change in the affinity of this receptor for alpha-conotoxin MI(18) . It has also been shown that a single amino acid substitution in alpha-conotoxin SI increases its affinity for mouse muscle nAChRs by 2 orders of magnitude (35) . It is possible, therefore, that alpha-conotoxin MII does potently target the muscle nAChR of its natural tropical fish prey, and that a few amino acid substitutions in the goldfish nAChR used in our assay may be responsible for the observed substantial differences in toxin potency. Poor dispersal of the more hydrophobic alpha-conotoxin MII might also lead to an apparent lack of activity in our assay. Since C. magus already has a toxin which potently blocks muscle nAChRs in the form of alpha-conotoxin MI, another possibility is that C. magus uses alpha-conotoxin MII to selectively target ganglionic or adrenal nAChRs in fish to lessen the sympathetically mediated fight or flight response. In frog, alpha-conotoxin MII blocks ganglionic neurotransmission. (^4)A related example may be neosurugatoxin. This glycoside, isolated from mid-gut gland of the Japanese ivory shell, appears to be responsible for human poisonings following ingestion of this carnivorous gastropod(9) . Poisoning symptoms are consistent with blockade of autonomic ganglia. Like alpha-conotoxin MII, neosurugatoxin preferentially targets neuronal versus muscle nAChRs. In contrast, however, neosurugatoxin is non-selective among alphaxbeta2 nAChRs(4, 8) .

The discovery of alpha-conotoxin MII, which differs substantially in both structure and function from other alpha-conotoxins, provides further evidence of the enormous diversity of nAChR-targeted toxins present in Conus. This report demonstrates the feasibility of using specific nAChR subunit combinations expressed in oocytes as a functional screen to initially detect and ultimately guide the purification of these peptides.


FOOTNOTES

*
This work was supported by Scientist Development Award for Clinicians Grant MH00929 (to J. M. M.) and National Institutes of Health Grant PO1 48677 (to B. M. O. and D. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: University of Utah, 201 S. Biology, Salt Lake City, Utah 84112.

(^1)
The abbreviations used are: nAChR, nicotinic acetylcholine receptors; RPLC, reverse phase liquid chromatography; Fmoc, N-(9-fluorenyl)methoxycarbonyl.

(^2)
J. Boulter, unpublished data.

(^3)
B. M. Olivera and J. M. McIntosh, unpublished observations.

(^4)
S. Tavazoie, M. Tavazoie, J. M. McIntosh, B. M. Olivera, and D. Yoshikami, manuscript in preparation.


ACKNOWLEDGEMENTS

Mass spectrometry was performed by A. Gray Craig of the Salk Institute. We thank Stephen F. Heinemann and David S. Johnson for providing nAChR subunit clones and John Syphers for technical assistance.


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