From the
Conus peptides, including
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy µO-conotoxin MrVIA was
chemically synthesized and proved indistinguishable from the natural
product. Surprisingly, the µO-conotoxins show no sequence
similarity to the µ-conotoxins. However, ananalysis of cDNA clones
encoding the µO-conotoxin MrVIB demonstrated striking sequence
similarity to
Recent progress in molecular neuroscience has demonstrated that
all signaling components in the nervous system are represented by
multiple molecular forms (``subtypes''). The identification
of different receptor and ion channel subtypes by molecular cloning
proceeds at an ever-accelerating rate. In contrast, the structural and
functional definition of each subtype has lagged far behind. A major
approach to the functional biology of subtypes is to obtain highly
subtype-specific ligands that can be used to discriminate between
closely related molecular forms.
In this respect the conotoxins,
peptides found in the venoms of the predatory cone snails, constitute a
unique resource. For incidental biological reasons, it appears that
these peptides are under strong selection to be unusually
subtype-specific. Several Conus peptide families which our
laboratories have developed are already being used to discriminate
between closely related subtypes. For example, in the calcium channel
field, the
Conus peptides that target
voltage-sensitive sodium channels have also been described. The best
characterized of these are the µ-conotoxins, which selectively
target vertebrate skeletal muscle subtypes of voltage-sensitive sodium
channels(2, 3, 4) . A review of the present
state of knowledge regarding the functional role of the various sodium
channel subtypes (5) makes it clear that it is desirable to
develop other highly specific ligands for this group of channel
proteins.
In this paper, we describe the isolation,
characterization, and synthesis of novel peptides, µO-conotoxins
MrVIA and MrVIB, from the venom of the snail-hunting species Conus
marmoreus (Fig. 1). Although these peptides produce a
biological effect which is similar to that of the µ-conotoxins,
they are unrelated structurally. Thus, the µO-conotoxins define a
new family of peptides which block Na
Crude venom from dissected ducts of C. marmoreus was
collected in the Philippines, lyophilized, and stored at -70
°C until use. Lyophilized crude venom (500 mg) was
extracted(6) , placed in a Centriprep 30 microconcentrator, and
centrifuged at 1500
Peptides from the final purification were reduced and
pyridylethylated by previously described methods(7) . The
alkylated peptides were then purified by reversed phase high
performance liquid chromatography. Sequencing was performed with Edman
chemistry on an Applied Biosystems model 477A protein sequencer at the
Protein/DNA Core Facility at the University of Utah Cancer Center. Mass
spectra were measured with a Jeol JMS-HX110 double focusing
spectrometer fitted with a Cs
Synthesis was carried out by the strategy developed for
A cDNA clone encoding µO-conotoxin MrVIB was identified
from a size-fractionated cDNA library constructed from C. marmoreus venom duct mRNA as described previously(10) . The clones
were identified by colony hybridization screening the C. marmoreus cDNA library using an oligonucleotide probe specific for the
signal sequence of Conus peptides belonging to the
O-superfamily. The oligonucleotide probe with the sequence 5` ATG AAA
CTG ACG TGC ATG ATG 3` was radiolabeled with
[
20-100 µl
of toxin solution were applied to the experimental dish
(0.25-0.75 ml) using a Gilson tip pipette placed a few
millimeters away from the neuron under study. The indicated toxin
concentrations correspond to the final concentration in the
experimental dish.
To minimize potassium currents we used an external potassium
channel-blocking solution, in which KCl was substitute for by CsCl. In
addition, the solution contained 50 mM tetraethylammonium
chloride and 0.1 mM 3,4-diaminopyridine. The osmolarity of the
solution was adjusted by reducing the NaCl concentration to 410
mM.
To monitor sodium current, a calcium-free potassium
channel-blocking solution was used. In this solution, Ca
To monitor calcium currents the external
sodium ions were replaced by tetraethylammonium chloride. The patch
pipette contained 440 mM CsCl, 40 mM CsOH, 2 mM CaCl, 6.8 mM MgCl
To simultaneously monitor calcium and sodium
currents, the experiments were carried out in potassium
channel-blocking solution, and the patch pipette contained the same
internal solution used for calcium current monitoring supplemented by
20 mM NaCl.
Potassium-containing external and internal
solutions were used when outward currents were under study. Sodium
currents were eliminated by replacing the sodium ions with Trizma 7.4
(Sigma). In these experiments the patch pipette contained 480 mM KCl, 20 mM NaCl, 6.8 mM MgCl
Here, and throughout the manuscript the ``±''
stands for standard error of the mean.
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
Liquid secondary ion mass spectrometry indicated that Cys
residues are present as disulfides and that the COOH-terminal
Surprisingly,
the new conotoxins show no detectable amino acid sequence homology to
the well characterized sodium channel blocking peptides from Conus venoms, the µ-conotoxins from fish-hunting Conus snails. Instead, the cysteine pattern resembles that of the
To examine the effects
of µO-conotoxins MrVIA/B on the sodium current we carried out a
series of experiments (n = 6 for MrVIA and n = 3 for MrVIB) in which the potassium and calcium currents
were eliminated (see ``Experimental Procedures''). The
voltage clamp records of Fig. 4B and 5A show
that 150-250 nM MrVIA completely blocks the inward
sodium current within 30 s after toxin application. The blockade of
I
Complete
blockade of I
The results described above establish that two peptides from C. marmoreus venom, µO-conotoxins MrVIA and MrVIB potently
block the inward Na
The central nervous system of Aplysia is
extensively used for the study of various neurobiological questions,
yet no efficient sodium channel blocking agents are presently
available. The classical sodium channel blockers, tetrodotoxin and
saxitoxin, are inefficient in Aplysia neurons. For example,
tetrodotoxin blocks Aplysia sodium channels at concentrations
of 100-200 µM, 3-4 orders of magnitude higher
concentration than those required to block sodium channels in
vertebrate brain, in vertebrate muscles and even the less sensitive
channels in cardiac and denervated
muscles(5, 23, 24, 25, 26) . In
contrast, the new µO-conotoxins MrVIA and MrVIB block sodium
current in Aplysia channels at submicromolar concentrations.
They should thus provide useful tools for Aplysia neurobiological studies and probes for molecular biological
analysis of molluscan sodium channels in general.
Thus, the work described above suggests that
in this single genus there has been functional convergence of
peptides from two different superfamilies to produce toxins that
inhibit sodium channel function, but functional divergence within a single superfamily to produce functionally distinct toxin
families. Such an extreme interspecific diversification of venom
peptides may account in part for the unusual proliferation of species
in the genus Conus, which is presently believed to be the
largest living genus of marine invertebrates (approximately 500 living
species)(27) .
In vivo results already suggest that the
first peptides characterized from these two conotoxin families (µ
and µO) may have distinctly different sodium channel subtype
specificity in mammalian systems. Thus, relatively high doses of
µO-conotoxin MrVIA (>10 nmol/10 g) are inactive peripherally in
rodents, whereas µ-conotoxin GIIIB is a potent paralytic when
injected intraperitoneal(28) ; this is consistent with the well
established specificity of µ-conotoxins for voltage-sensitive
sodium channels from mammalian skeletal muscle. In contrast, low doses
of µO-conotoxin MrVIA cause ataxia and/or reversible coma in a few
minutes when injected intracranially, whereas no detectable symptoms
are observed after several hours of a similar central injection of
µ-conotoxin GIIIB. These results suggest that MrVIA is a potent
ligand in the mammalian central nervous system and may target central
sodium channels. In contrast, GIIIB targets the skeletal muscle subtype
with high affinity, but not neuronal sodium channels. We cannot exclude
the possibility, however, that the differing effects between these
toxins are due to differences in state-dependent channel block, rates
of absorption, or susceptibility to proteolysis. Preliminary
experiments with cloned sodium channels expressed in Xenopus oocytes are, however, consistent with the in vivo central
effects of µO-conotoxins.
The functional differentiation of sodium channel subtypes is much
more poorly understood; the paucity of sodium channel subtype-specific
ligands is a major reason for this situation. This report describes a
new family of Conus peptides, the µO-conotoxins, which
potently block voltage-sensitive sodium currents. The µO-conotoxins
are potentially as important to sodium channel subtype differentiation
as the
Toxin was
injected intracranially (i.c.) or intraperitoneally (i.p.) into young
(5-8 g) mice (29). NE, no effect; NT, not tested.
Mass spectrometry was performed by A. G. Craig at the
Clayton Foundation Laboratories of the Salk Institute. Construction of
the peptide resin and sequence analysis were carried out by R.
Schackmann at the Utah Regional Cancer Center. Dr. K.-J. Shon
synthesized one of the synthetic MrVIA samples used in these studies.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-conotoxins and
-conotoxins (targeting calcium channels and nicotinic
acetylcholine receptors, respectively) have been useful ligands in
neuroscience. In this report, we describe a new family of sodium
channel ligands, the µO-conotoxins. The two peptides characterized,
µO-conotoxins MrVIA and MrVIB from Conus marmoreus potently block the sodium conductance in Aplysia neurons.
This is in marked contrast to standard sodium channel blockers that are
relatively ineffective in this system. The sequences of the peptides
are as follows.
- and
-conotoxin precursors. Together, the
-,
-, and µO-conotoxins define the O-superfamily of Conus peptides. The probable biological role and evolutionary
affinities of these peptides are discussed.
-conotoxins have provided the means for discriminating
between various channel subtypes that play a role in neurotransmitter
release (see Ref. 1 for review).
currents of
voltage-sensitive sodium channels. The discovery of this novel family
provides a new opportunity to obtain a large armamentarium of
subtype-specific sodium channel-targeted Conus peptides.
Figure 1:
The marble cone C. marmoreus and other snail-hunting Conus species. Top
left, an etching of C. marmoreus by Rembrandt. Note that
Rembrandt neglected to reverse the picture, so that when printed, the
helical shell comes out sinistral, instead of dextral. He did, however,
remember to render his signature as a mirror image so the correct
signature impression is made. Top right, a photograph of Conus marmoreus. The photographer has signed as Rembrandt did.
In a mirror, the photograph has the handedness of the etching and vice versa. Lower panels show members of two major
clades of snail-hunting Conus. In the C. marmoreus clade, left, the following species and forms are
included: top row, left to right, Conus vidua and C. marmoreus (both Philippines). Middle row,
a dwarf, melanistic C. marmoreus specimen (for nigrescens)
(Samoa) and Conus bandanus (Hawaii). Bottom row, Conus nicobaricus (Sulu Sea), Conus nocturnus (Palawan Island), and Conus araneosus (India). In the lower right panel is the C. textile clade of
snail-hunting species. Top row, left to right, Conus gloriamaris (Philippines), Conus retifer (Okinawa), and Conus natalis (South Africa). Middle
row, C. textile and Conus legatus (both Philippines). Bottom row, Conus eutrios (Mozambique), Conus dalli (Mexico), and Conus bengalensis (Bay of Bengal).
Photographs by Kerry Matz.
Peptide Purification
g for 8 h at 4 °C. Filtrate
was purified on a C18 Vydac column (22
250 mm) using a gradient
system. Buffer A = 0.1% trifluoroacetic acid and buffer B
= 0.1% trifluoroacetic acid, 60% acetonitrile. 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 10 min. Flow rate was 10 ml/min. For
subsequent purification steps Vydac C8 columns (10
250 mm, or
4.6
250 mm, 5 µm particle size) were used with buffer A as
above and buffer B = 0.1% trifluoroacetic acid, 90%
acetonitrile. The gradient was 5-55% buffer B/15 min, followed by
55-70% buffer B/45 min. Flow rate was 5 ml/min for the
semipreparative column and 1 ml/min for the analytical column.
Absorbance was monitored at 220 and 280 nm.
Sequence Analysis
gun.
Chemical Synthesis
-conotoxin MVIID(8) . Briefly, standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry was used, with
single couplings using dicyclohexylcarbodiimide and
hydroxybenzotriazole. All amino acid derivatives were purchased from
Bachem (Torrance, CA) and were loaded into cartridges for use in an ABI
model 430A peptide synthesizer. Side chain protection used was t-butoxycarbonyl (Lys), t-butyl (Glu, Tyr, Ser), and
pentamethylchromansulfonyl (Arg). Cysteine was protected as S-trityl (residues 2, 19, 20, 30) and S-acetamidomethyl (residues 9 and 25). Cleavage from the resin
and subsequent workup followed essentially the same procedures as for
conotoxin GmVIA(9) .
Molecular Cloning
-
P]ATP using T4 polynucleotide kinase.
Hybridization was carried out using duplicate nylon filters (Hybond-N,
Amersham Corp.) to which colonies of the C. marmoreus cDNA
library were bound for 16 h at 42 °C using standard hybridization
conditions (5
Denhardt's solution, 6
NET (100
mM NaCl, 1 mM EDTA, 10 mM Tris, (pH 8.0),
0.1% SDS, 100 µg/ml salmon sperm DNA). The filters were washed at
51 °C in 0.1
SSC, 0.1% SDS, and positive colonies were
identified by autoradiography. A second round of screening was carried
out on 50 putative clones that were radiolabeled in the first round of
colony hybridization. Clones which were positive in both screening
rounds were sequenced. Plasmid DNA (5 µg) was prepared for
sequencing by adding 0.1 volume of 2 M NaOH, 2 mM EDTA at 37 °C for 30 min (thereby denaturing the DNA), and the
mixture was then neutralized with 0.1 volume of 3 M sodium
acetate (pH 4.5). DNA was precipitated with 2 volumes of ethanol,
pelleted, washed with 70% ethanol, dried, and redissolved in distilled
H
O (7 µl). Sequenase reaction buffer (2 µl) and the
oligonucleotide indicated above (1 µl) were added, the mixture
heated to 65 °C for 5 min and cooled to 30 °C to allow
annealing of the oligonucleotide. One DNA strand from each clone was
then sequenced using the Sequenase version 2.0 DNA sequencing kit and
S-dATP (Sequenase version 2.0 seventh edition protocol).
The MrVIB sequence was found in four independent clones.
Procedures for Electrophysiology
Neuronal Culture
RB neurons from the abdominal
ganglion of the sea hare Aplysia oculifera collected in the
Gulf of Eilat or Aplysia californica shipped from the
University of Miami (Aplysia Resource Facility) were isolated
and grown in culture(11, 12, 13) . The neurons
were cultured at very low densities of three to five cells per culture
dish, to prevent synaptic interactions between them. Experiments were
performed on neurons 1-4 days in culture.
Electrophysiology
Analysis of the toxin action was
carried out using both intracellular current clamp recording and
stimulation as well as whole-cell patch clamp techniques(14) .
For the intracellular current clamp experiments a micro-electrode was
inserted into the cell body. This electrode was used for both current
injection and voltage recordings. An EPC-9 computerized amplifier (Heka
Electronics, Lembracht, Germany) was used to record from neurons in the
whole cell patch configuration. Patch pipettes had a typical resistance
of 1-1.5 megaohms. The series resistance never exceeded 2.5
megaohms and was stable throughout the experiments. The series
resistance was compensated by 70-90%. Experiments in which
voltage-current relationship was monitored were carried out only when
the series resistance was less than 2.5 megaohms. Thus, the maximal
voltage error in these experiments was smaller than 3 mV. Leak and
capacitance currents were subtracted. To achieve adequate space clamp,
the large axon of the neuron was trimmed off about 30 min prior to the
experiment (11, 15). The experiments were carried out at room
temperature ranging between 20-24 °C.
Solutions
Control experiments were carried out in
artificial sea water composed of: 460 mM NaCl, 10 mM KCl, 11 mM CaCl, 55 mM MgCl
, and 10 mM HEPES. The pH was adjusted to
7.6.
was substituted for by Mg
, and the solution was
supplemented by 8 mM Co
to prevent any
Ca
influx. For these experiments the patch pipette
contained 440 mM CsCl, 40 mM CsOH, 20 mM NaCl, 6.8 mM MgCl
, 10 mM EGTA, 100
mM HEPES, and 3 mM adenosine 5`-triphosphate (ATP).
The pH was adjusted to 7.3.
, 100 mM HEPES, 10
mM Cs
BAPTA
(
)(Molecular
Probes, Eugene, OR), and 5.6 mM glucose. In order to prevent
run-down of calcium channels(16, 17) , the internal
solution was supplemented with 0.5 mM guanosine
5`-triphosphate (GTP) and ``ATP-regenerating system,'' 10
mM ATP, 20 mM creatine phosphate (Sigma) and 50
units/ml of phosphocreatine kinase (Sigma). In those conditions,
run-down of calcium current was less than 10% per hour. The pH was
adjusted to 7.3.
, 10
mM EGTA, and 100 mM HEPES. The pH was adjusted to
7.3.
Purification and Characterization of µO-conotoxin
MrVIA
We screened venom fractions from C. marmoreus for
potential high affinity blockers of the tetrodotoxin-insensitive inward
sodium current which underlies the generation of propagating action
potentials in cultured Aplysia neurons. Two strongly
hydrophobic fractions of this venom potently abolished the sodium
component of the action potential. The active fractions were purified
to homogeneity as is shown in Fig. 2.
Figure 2:
Isolation and characterization of
µO-conotoxins MrVIA and MrVIA from C. marmoreus venom. A, RPLC of crude venom filtrate. The very hydrophobic MrVIA
and MrVIB elute as the last two major peaks at 109.4 and 110.3 min. B, RPLC of the fraction eluting at 109.4 min separates MrVIA
from MrVIB. An analogous RPLC was performed to isolate MrVIB (not
shown). C, rechromatography of the MrVIA (arrow) and
MrVIB (not shown) containing fractions shows homogeneous appearing
peaks. D, the complete sequences are shown using single-letter
amino acid code.
The purified components
were analyzed by amino acid sequencing as described under
``Experimental Procedures,'' revealing two novel peptides,
µO-conotoxins MrVIA and MrVIB. Their sequences are as follows.-carboxyl group is the free acid for both peptides (monoisotopic
MH
: MrVIA calculated 3487.66, observed 3487.8; MrVIB
calculated 3404.58, observed 3404.8). The sequence was further verified
by chemical synthesis and cDNA cloning (see below).
-conotoxins, the large family of Conus peptides which
are specifically targeted to calcium channels and of the
-conotoxins that slow down the inactivation rate of sodium
channels (1, 4, 9, 18-21).
Chemical Synthesis
Solid-phase chemical synthesis
of µO-conotoxins MrVIA and MrVIB was undertaken by the two-stage
strategy of Monje et al.(8) , as modified for the
hydrophobic -conotoxins TxVIA and GmVIA(9) . On the
assumption that the disulfide bridging would be conserved, Cys
and Cys
were protected as the stable
Cys(S-acetamidomethyl), whereas the other four Cys residues
were protected as the acid-labile Cys(S-trityl). After
cleavage from the resin, the linear peptide was purified by RPLC, and
then oxidized to a mixture of three bicyclic peptides. Trial
experiments indicated which of these corresponded to the correct
isomer, and the main batch of peptide was subjected to further
oxidation by iodine in 10% trifluoroacetic acid. After destroying
excess iodine with ascorbic acid, the tricyclic peptide was purified by
RPLC (0.1% trifluoroacetic acid with an acetonitrile gradient of
32-68% in 40 min). Synthetic peptide was shown to co-elute with
the natural material and was equipotent in biological assays.
Precursor Sequence
In order to determine the
precursor sequence of these peptides, a cDNA library was prepared from
venom ducts of Conus marmoreus and clones related to the new
peptides were identified. Two cDNA clones for µO-conotoxin MrVIB
were characterized. The nucleotide sequence and predicted amino acid
sequence of the encoded precursor is shown in Fig. 3. It is seen
that the last 31 residues exactly match the experimentally determined
sequence of µO-conotoxin MrVIB. The putative 82-amino acid
precursor sequence is presumably cleaved after Arg-51 to yield the
mature 31-amino acid peptide. The cDNA sequence also verifies the
biochemical assignment for a free carboxyl terminus for MrVIB.
Figure 3:
The precursor sequence of µO-conotoxin
MrVIB. Nucleic acid sequence and inferred amino acid sequence of
MrVIB-encoding cDNA clone. A C. marmoreus cDNA library was
prepared as described by Colledge et al. (10). Fifteen clones
were identified in two rounds of colony hybridization of this library
using a probe specific for -conotoxin signal sequence. The 15
clones were then sequenced with this same oligonucleotide. Of these,
four encoded µo-conotoxin MrVIB.
Electrophysiology
Action potentials generated by
intracellular stimulation of cultured Aplysia neurons were
rapidly (30 s) blocked by bath application of 350 nM conotoxin
MrVIA (Fig. 4, A and B). The action potential
blockade was not associated with changes in the transmembrane potential
or input resistance. An increase in the intracellular stimulus
intensity failed to evoke a full-blown action potential, but induced a
small local response (not shown). The local response was most likely
generated by unblocked voltage-gated calcium channels, as it could be
abolished by the addition of 8 mM cobalt ions to the bath
solution (see also Fig. 5A).
Figure 4:
Blockade of action potential and inward
sodium current by µO-conotoxin MrVIA as revealed by current and
voltage clamp experiments. A (control), the action
potential was generated by an intracellular rectangular depolarizing
pulse. A
, 10 s after toxin application to reach a
final bath concentration of 350 nM, the action potential was
blocked. An increase in the stimulus intensity after the blockade
failed to elicit a regenerative response. The inward
I
Figure 5:
Effect of MrVIA on the sodium and calcium
current. For the experiment the potassium currents were blocked. The
neuron was depolarized from a holding potential of -50 to 20 mV.
In the control (A), the depolarization induced an early
rapidly inactivating sodium current and a plateau of calcium current.
Application of 150 nM MrVIA rapidly blocked the early sodium
current (second trace in A and see inset). The rate
at which I
To identify the
mechanisms underlying the action potential blockage by
µO-conotoxins MrVIA/B, we used experimental protocols that
permitted us to examine the isolated macroscopic currents of either
K, Ca
, or Na
. The
toxins had no effects on the voltage-gated potassium currents at
concentrations of up to 10 µM.
µO-conotoxin MrVIA Is Active in Vertebrate
Systems
The results above demonstrate that µO-conotoxins
purified from the venom of a mollusc-hunting Conus potently
block voltage-sensitive sodium channels in molluscan systems. The
activity of µO-conotoxin MrVIA was also examined using injections
into mice; results are shown in . The data reveal that the
peptide is extremely potent when injected into the rodent central
nervous system. Dramatic symptomatology is observed (ataxia, coma),
even upon injection of doses of MrVIA as low as 0.1 nmol. In contrast,
no effects are observed at doses up to 100 greater when the
toxin is injected intraperitoneally.
current of cultured Aplysia neurons. As will be discussed below, the µO-conotoxins show
great potential as general pharmacological tools for discriminating
among sodium channel subtypes, and together with the
- and
-conotoxins, define a ``superfamily'' of Conus peptides. We also comment on the probable biological role of these
peptides.
Superfamilies of Conus Peptides
Surprisingly, although
the new conotoxins clearly block Na currents elicited
through voltage-sensitive sodium channels, they show no detectable
amino acid sequence identity to the µ-conotoxins from fish-hunting Conus, the other group of Conus peptides that block
sodium currents. Instead, the MrVIA/B peptides have a cysteine pattern
similar to that of the
-conotoxins from fish-hunting snails and
the
-conotoxins from mollusc-hunting snails. An examination of the
precursor sequence of the MrVIB peptide reveals considerable homology
not only to the
-conotoxin family, but to the
-conotoxins
which slow down the inactivation rate of sodium channels (9,
19-21). In fact there is greater similarity between the new
conotoxin and the
-conotoxins (35 out of 51 residues identical in
the prepro region) than there is to the
-conotoxins (24 out of 51
residues identical). A comparison of the predicted precursor sequences
of these peptides, deduced from cDNA clones, is shown in Fig. 6.
The boxes indicate sequence identities.
Figure 6:
Comparison of O-superfamily precursor
sequences. The inferred sequence of the µO-conotoxin MrVIB
precursor sequence (see Fig. 3) is aligned with the prepropeptide
sequences of -conotoxin TxVIA (formerly called the King-Kong
peptide) and
-conotoxin GVIA. The signal sequence and mature
toxin regions are indicated, with an intervening proregion. The
conserved amino acids are boxed. Note the almost total
conservation of signal sequences versus the striking
divergence of the mature toxins.
Thus, three
distinctive pharmacological groups of Conus peptides appear to
belong to the same structural group, which we will define as the
``O-superfamily'' of Conus peptides: the
-conotoxins which inhibit calcium channels, the
-conotoxins
which slow down the inactivation rate of voltage-sensitive sodium
channels, and the µO-conotoxins which block voltage-sensitive
sodium currents. By contrast, the µ-conotoxins from fish-hunting Conus, which show the same general physiological mechanism as
the µO-conotoxins (i.e. blockage of voltage-sensitive
sodium currents), have no detectable homology to any of the peptides in Fig. 6. They clearly belong to a different Conus peptide
superfamily. By definition, the O-superfamily comprises those peptides
with strong similarity to the omega-conotoxins in their precursor
sequences; a curious feature is that the signal sequences region is the
most highly conserved.
Biological Role
Our results suggest that
snail-hunting Conus have evolved at least two solutions to the
same biological problem of feeding on snails. When harpooned, the prey
would naturally retract into their shells to escape from a predatory Conus snail. Our observations of feeding in aquaria suggest
that two snail-hunting Conus groups have generated very
different ways to prevent prey from becoming paralyzed inside the
shell. Conus textile, after envenomating a snail (such as the Kelletia species), induces convulsive contractures, with the
victim moving into and out of the shell. In contrast, envenomation by C. marmoreus elicits a slow relaxation. Purified
-conotoxin TxVIA induces the convulsive contractures in snails
observed with whole C. textile venom, whereas µO-conotoxin
MrVIA causes the flaccid relaxation characteristic of crude C.
marmoreus venom. In either case, access to the prey is ensured by
the cone snail after immobilization of the victim, albeit by very
different means.
The Potential of Conus Peptides for Generating a
Subtype-specific Sodium Channel Pharmacology
µO-conotoxins
MrVIA and MrVIB are harbingers of a potentially much wider range of
peptides active against sodium channels. The availability of the cloned
precursor sequence has allowed us to identify potential homologs from
venoms of other Conus species.(
)The
sequences available suggest that there is considerable sequence
hypervariability in this family of Conus peptides, making it
likely that different members may have differing sodium channel subtype
specificity profiles. Similarly, although the structurally unrelated
µ-conotoxins have only been characterized from a single
fish-hunting Conus species, Conus geographus, a
molecular genetic analysis has revealed that a number of other
fish-hunting Conus species also have µ-conotoxins; the
predicted peptides are presently being synthesized and will be further
characterized.
(
)Many more members
of the µ- and µ0-conotoxin families can be identified by
molecular genetic techniques and the predicted peptides chemically
synthesized. By screening for those peptides that induce different in vivo symptoms, an expanding set of promising ligands for
establishing a subtype-specific pharmacology for sodium channels should
be established.
General Perspectives
Different ion channel
subtypes might be expected to play distinctive functional roles in
nervous systems. For voltage-sensitive calcium channels, this
functional differentiation of subtypes has become increasingly well
documented. This has been made possible in large part by the
availability of a family of Conus peptides, the
-conotoxins: thus,
- and
-containing calcium channel complexes are
differentially targeted by different
-conotoxins(1) .
-conotoxins are to calcium channels.
Table: Bioassay µO-conotoxin MrVIA
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.