©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A New Family of Conotoxins That Blocks Voltage-gated Sodium Channels (*)

J. Michael McIntosh (3) (1)(§), Arik Hasson (4), Micha E. Spira (4), William R. Gray (1), Wenqin Li (1), Maren Marsh (2), David R. Hillyard (1) (2), Baldomero M. Olivera (1)

From the (1)Departments of Biology, (2)Pathology, and (3)Psychiatry, University of Utah, Salt Lake City, Utah, 84112, the (4)Department of Neurobiology, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904 Israel, and The Interuniversity Institute for Marine Sciences, Eilat 88103, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Conus peptides, including -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.

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 - 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.


INTRODUCTION

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 -conotoxins have provided the means for discriminating between various channel subtypes that play a role in neurotransmitter release (see Ref. 1 for review).

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 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.




EXPERIMENTAL PROCEDURES

Peptide Purification

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 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

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 gun.

Chemical Synthesis

Synthesis was carried out by the strategy developed for -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

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 [-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 HO (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.

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.

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.

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 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.

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, 100 mM HEPES, 10 mM CsBAPTA()(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.

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, 10 mM EGTA, and 100 mM HEPES. The pH was adjusted to 7.3.

Here, and throughout the manuscript the ``±'' stands for standard error of the mean.


RESULTS

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.

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 -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).

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 -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.

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

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.


DISCUSSION

The results described above establish that two peptides from C. marmoreus venom, µO-conotoxins MrVIA and MrVIB potently block the inward Na 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.

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.

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.

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) .

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.

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.()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) .

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 -conotoxins are to calcium channels.

  
Table: Bioassay µO-conotoxin MrVIA

Toxin was injected intracranially (i.c.) or intraperitoneally (i.p.) into young (5-8 g) mice (29). NE, no effect; NT, not tested.



FOOTNOTES

*
This work was supported by National Institutes of Health Grant PO1 GM28677 and by a Scientist Development Award for Clinicians K20 MH 00929 (to J. M. M.), as well as grants from the Israel Ministry of Sciences and Arts (4977/93 to M. E. S.). 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 So. Biology, Salt Lake City, UT 84112. Tel.: 801-585-3622; Fax: 801-585-5010.

The abbreviations used are: BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N`,N`-tetraacetic acid; RPLC, reversed phase liquid chromatography.

M. Marsh and D. Hillyard, unpublished results.

H. Terlau, B. M. Olivera, and W. Stühmer, unpublished results.


ACKNOWLEDGEMENTS

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.


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