The muscle subtype of nicotinic acetylcholine receptor (nAChR) (
)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
, and 5-HT
receptors(1) . The mammalian nAChR has the subunit
composition (
1)
1
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
subunits, designated
2-
7 and
9 (in chick
there is also an
8 subunit), and three
subunits,
2-
4. The
2,
3, and
4 subunits can each combine
with
2 or
4 subunits to form functional channels when
expressed in Xenopus oocytes, e.g.
2
2,
3
2,
2
4, etc. In addition,
7 and
9
subunits can be expressed as functional homooligomers in this system.
Studies employing either nucleotide probes or antibodies indicate that
each of these
and
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-
-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
-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
2
2,
3
2, and
4
2 neuronal
nAChRs(8) . Neosurugatoxin, a glycoside from the gastropod Babylonia japonica(9) , potently but nonselectively
blocks
x
2 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,
-bungarotoxin, in addition to blocking the muscle receptor,
potently blocks
7 subunit-containing neuronal nAChRs(10) .
Methyllycaconitine, an alkaloid toxin from the seeds of Delphinium
brownii has markedly greater affinity for the
I-
-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,
-bungarotoxin (also known as neuronal
bungarotoxin, toxin F, or Bgt 3.1) preferentially blocks
3
2
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
-conotoxins. In contrast to snake
-toxins (
60-80
amino acids),
-conotoxins are much smaller (
12-25 amino
acids), a feature which has allowed them to be readily chemically
synthesized (13) .
-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
H-1 cell line
-conotoxins MI, GI, and SIA (respectively from Conus magus,
Conus geographus, and Conus striatus) selectively bind to
the ACh binding site at the
/
interface with more than
10
-fold greater affinity than the site at the
/
interface(14, 15) . With Torpedo nAChR, the
situation is reversed.
-Conotoxins MI and GI bind at the
/
interface with approximately 2 orders of magnitude greater
affinity than the
/
interface(15, 16) . Like
-conotoxins MI, GI, and SIA,
-conotoxin EI, from Conus
ermineus, prefers the
/
to the
/
interface of
receptors in BC
H-1 cells, but with only 30-fold difference.
In contrast to these other
-conotoxins, with Torpedo receptors,
-conotoxin EI preferentially binds the
/
versus the
/
interface by a 400-fold difference in
affinity and is the only ligand known to possess this
selectivity(17) . Thus, these
-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
-conotoxin ImI which, unlike other
-conotoxins,
selectively targets
7, and to a lesser degree
9,
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
3
2 nAChRs were used in the assay to isolate
-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
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
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
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
-conotoxin MII.
RPLC Purification
All RPLC columns were from
Rainin Instruments. Crude venom extract was fractionated on a
semipreparative Vydac C18 column (10 mm
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
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
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
-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
and Cys
were protected as the
stable Cys(S-acetamidomethyl), while Cys
and Cys
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
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
and Cys
(i.e. the first
and third cysteines), the major peptide fraction from the preparative
RPLC (see above) was diluted to 1 liter with H
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
-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
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:
1,
1,
,
(22) ;
2(23) ;
3(24) ;
4(25) ;
7(
);
9(26) ;
2(27) ; and
4(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
, 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
15-mm Petri dish containing ND-96 (96.0
mM NaCl, 2.0 mM KCl, 1.8 mM CaCl
, 1.0 mM MgCl
, 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
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 (
2000). Glass microelectrodes, pulled from fiber-filled
borosilicate capillaries (1 mm outer diameter
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
1
1
, 1 mM for
7, and 300 µM for all others. The ACh was diluted in ND96A for all except
7, in which case the diluent was ND96. For control responses, the
ACh pulse was preceded by perfusion with ND96 (for
7) or ND96A
(all others). No atropine was used with oocytes expressing
7,
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
)
},
where n
is the Hill coefficient. All recordings
were done at room temperature (
22 °C).
Figure 3:
-Conotoxin MII blocks ACh responses
in oocytes expressing
3
2 nicotinic acetylcholine receptors.
Oocytes expressing
3
2 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
-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
-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
3
2
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
3
2 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
-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
-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
-conotoxin MII in accordance with
the nomenclature previously proposed for conotoxins (31) .
Chemical Synthesis
Solid phase chemical synthesis
of
-conotoxin MII was undertaken to provide an abundant supply of
peptide. It was assumed that the disulfide bridging of
-conotoxin
MII would be analogous to all previously characterized
-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
and Cys
(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
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,
-conotoxin MII
becomes increasingly hydrophobic. This is exactly the opposite behavior
from
-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
-conotoxin MII force
hydrophobic residues to face outward. Synthetic peptide comigrated with
native on RPLC (Fig. 2). Liquid secondary ionization mass
spectrometry of synthetic
-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
-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
-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
-Conotoxin MII purification
by RPLC was guided by an assay which used Xenopus oocytes
expressing
3
2 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
3
2 nAChRs with equal potency (data not shown). Due to very
limited availability of native toxin, synthetic toxin was used for all
subsequent experiments. Synthetic
-conotoxin MII showed
dose-dependent block of
3
2 receptors at low nanomolar
concentrations (Fig. 3). This block slowly reversed with washing (Fig. 4).
-Conotoxin MII blocks
3
2 nAChRs with an
IC
of 0.5 nM, with an apparent Hill coefficient, n
, of 0.8 (Fig. 5).
-Conotoxin MII was
also tested on other nAChR subunit combinations. Results are shown in Fig. 5and indicate that
-conotoxin MII is 2-4 orders
of magnitude more potent at
3
2 nAChRs than at other nAChR
subtypes.
Figure 4:
nAChR block by
-conotoxin MII is
slowly reversible. 20 nM toxin was applied to
3
2-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:
-Conotoxin MII selectively blocks
3
2 nAChRs. Oocytes expressing various nAChR subunit
combinations were voltage clamped and the response to ACh measured. The
IC
for
-conotoxin MII block of the
3
2
receptor is 0.5 nM (n
= 0.8).
Oocytes expressing other nAChR subunit combinations were perfused with
200 nM
-conotoxin MII (400
the IC
on
3
2 receptors). The mean ± S.E. response to ACh was as
follows:
,
4
4, 96 ± 1;
,
2
4,
96 ± 6;
,
1
1
, 89 ± 3;
,
3
4, 85 ± 5;
,
2
2, 80 ± 3;
,
4
2, 70 ± 1;
,
7, 44 ± 8.
Data represent the mean for 3-5
oocytes.
We have previously shown that
-conotoxin MI (also
from C. magus venom),
-conotoxin GI, and
-conotoxin
ImI have no effect on
3
2 receptors at up to 5 µM concentration (21) . Thus,
-conotoxin MII is the only
conotoxin known to potently block this neuronal receptor subtype.
In Vivo Activity
Intraperitoneal injections of 10
nmol of
-conotoxin MII into 8-10-g mice did not result in
any signs of paralysis (n = 3). This is in contrast to
-conotoxin MI, 0.67 nmol of which kills a 20-g mouse in 20
min(32) . Intramuscular injection of 5 nmol of
-conotoxin
MII into fish did not result in any signs of paralysis (n = 3). This is in contrast to
-conotoxin MI where 0.5
nmol is paralytic.
Discussion
nAChR Selectivity
Xenopus oocytes
expressing mammalian neuronal
3
2 nAChRs were used in an assay
which successfully guided the isolation of the novel 16-residue
peptide,
-conotoxin MII. This is significant in that it is the
first
-conotoxin known to target
3
2 receptors. Most
previously reported
-conotoxins target the muscle nAChR.
Exceptions are
-conotoxin ImI, which selectively blocks homomeric
7 and
9 receptors(20, 21) , and
-conotoxins PnIA/B, which block molluscan neuronal
nAChRs(33) . We have shown elsewhere that
-conotoxins MI
and GI potently target muscle nAChRs expressed in Xenopus oocytes, but are inactive at all neuronal nAChRs tested, including
3
2 receptors(21) . As demonstrated in this report,
3
2 receptors are blocked by
-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,
-conotoxin MII has an entirely unique
activity profile among the
-conotoxins (Table 1), and
represents a potent and selective new probe for studying nAChRs.
Notably, the small size of
-conotoxin MII has allowed it to be
chemically synthesized and thus readily available.
Structural Relationships among
-Conotoxins
Reported
-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
-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
-conotoxin MII's selectivity. Aside from the Cys residues,
the only completely conserved residue in all reported
-conotoxins
is a proline between the second and third Cys. The other non-Cys
residues in
-conotoxin MII are strongly divergent from all other
-conotoxins. Of the non-Cys residues in the 4,7 group,
-conotoxin MII from C. magus shares only 4 of 12 residues
with
-conotoxin PnIB from C. pennaceus and only 1 of 12
residues with
-conotoxin EI from C. ermineus. Furthermore, except for the proline,
-conotoxin MII shares
little if any homology with
-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
-conotoxins where the
disulfide linkages have been studied (Table 2). Recently, a new
family of Conus peptides targeted to nAChRs was reported.
These
A-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
-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
3
2 nAChR selectivity and which in MI confer
1
1
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. (
)The functional targets and roles of these
non-paralytic peptides are under investigation. However, all previously
reported
-conotoxins from fish-hunting Conus do cause
rapid paralysis when injected into fish.
-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
-fold change in
the affinity of this receptor for
-conotoxin MI(18) . It
has also been shown that a single amino acid substitution in
-conotoxin SI increases its affinity for mouse muscle nAChRs by 2
orders of magnitude (35) . It is possible, therefore, that
-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
-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
-conotoxin MI, another possibility is that C. magus uses
-conotoxin MII to selectively target ganglionic or adrenal nAChRs
in fish to lessen the sympathetically mediated fight or flight
response. In frog,
-conotoxin MII blocks ganglionic
neurotransmission. (
)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
-conotoxin MII, neosurugatoxin preferentially targets
neuronal versus muscle nAChRs. In contrast, however,
neosurugatoxin is non-selective among
x
2
nAChRs(4, 8) .The discovery of
-conotoxin
MII, which differs substantially in both structure and function from
other
-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.