| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
AG Molekulare und Zelluläre Neuropharmakologie, Max-Planck-Institut für Experimentelle Medizin, Göttingen, Germany; and Department of Biology, University of Utah, Salt Lake City, Utah
ABSTRACT I. OVERVIEW OF CONE SNAILS AND CONOTOXINS A. Introduction B. Cone Snails: Historical Perspectives and Biology 1. Conus in human history 2. Natural history of Conus: overview of Conus evolution 3. Biology of cone snails C. Overview of Conus Venom Components 1. Biochemistry 2. Molecular genetics 3. Overview of pharmacology and physiology II. CONOPEPTIDES TARGETED TO VOLTAGE-GATED ION CHANNELS A. Introduction and Overview B. Na Channel-Targeted Toxins 1. µ-Conotoxins 2. µO- and {delta}-Conotoxins C. K Channel-Targeted Toxins: {kappa}-, {kappa}A-, and {kappa}M-Conotoxins 1. {kappa}-Conotoxins 2. {kappa}A- and {kappa}M-conotoxins D. Ca Channel-Targeted {omega}-Conotoxins E. State Dependence of Block: Transitions Between States III. CONUS PEPTIDES TARGETED TO LIGAND-GATED ION CHANNELS A. Overview B. {alpha}-Conotoxins and nAChRs: Matching a Family of Conopeptides to a Family of Ion Channel Targets C. {alpha}-Conotoxins and Subtype Selectivity D. Defining Molecular Isoforms of Nicotinic Receptors: Combining {alpha}-Conotoxins With Knockout Mutants E. Other Conopeptide Families Targeted to Ligand-Gated Ion Channels IV. OVERVIEW OF CONUS VENOM COMPONENTS: PERSPECTIVES A. Other Venom Components B. Perspectives
 |
ABSTRACT |
|---|
 Top NextReferences |
|---|
100 different conotoxins, with little overlap between species. An overwhelming majority of these peptides are probably targeted selectively to a specific ion channel. Because conotoxins discriminate between closely related subtypes of ion channels, they are widely used as pharmacological agents in ion channel research, and several have direct diagnostic and therapeutic potential. Large conotoxin families can comprise hundreds or thousands of different peptides; most families have a corresponding ion channel family target (i.e., 
-conotoxins and Ca channels, 
-conotoxins and nicotinic receptors). Different conotoxin families may have different ligand binding sites on the same ion channel target (i.e., µ-conotoxins and 
-conotoxins to sites 1 and 6 of Na channels, respectively). The individual peptides in a conotoxin family are typically each selectively targeted to a diverse set of different molecular isoforms within the same ion channel family. This review focuses on the targeting specificity of conotoxins and their differential binding to different states of an ion channel. |
I. OVERVIEW OF CONE SNAILS AND CONOTOXINS |
|---|
|
TopPreviousNextReferences |
|---|
The first Conus venom peptides were isolated and characterized two decades ago (32, 143), and the systematic investigation of cone snail toxins has continued at an accelerating pace. These studies have revealed that each of the 500 different species of predatory cone snails (genus Conus) (156) has its own distinctive, complex, and peptide-rich venom. Venom is the primary weapon used by these carnivorous molluscs to capture prey and is also believed to be used defensively and competitively, and possibly for other biological purposes as well (137). Because different cone snail species have divergent biotic interactions, a corresponding divergence in Conus venoms is the expected consequence. Cone snails thrive in tropical marine habitats; the complete web of biological interactions in such marine communities provides a general rationale for why each Conus species has evolved its own large molecular repertoire of venom components, different from that of every other Conus species (139).
The detailed interactions between an individual Conus species and other animals in its environment are mostly unknown; thus the pharmacological spectrum to be found in any venom cannot be predicted a priori. There are probably >100 different venom components per species, leading to an estimate of >50,000 different pharmacologically active components present in venoms of all living cone snails. Conus venom components have become known as conotoxins or conopeptides, terms used interchangeably in this review. In the literature, disulfide-rich peptides are referred to as conotoxins; conopeptide is used as a more general term for all peptides found in Conus venoms.
This review focuses on the neurophysiology of Conus venom components. Since only a miniscule fraction of the total conopeptide diversity has been characterized in detail, a few broad themes are emphasized that should be relevant even for venom peptides yet to be characterized. The molecular targets of individual Conus venom components are functionally diverse, including G protein-coupled receptors and neurotransmitter transporters; some Conus venom components even have enzymatic activity. However, the majority of biologically active Conus venom constituents that are characterized at present are small, structured peptides that target ion channels, either of the ligand-gated or voltage-gated class. It is highly likely that most Conus peptides have a specific ion channel as the physiologically relevant target. Thus the work that will primarily be reviewed is the effects of Conus peptides on ion channels.
In the last 50 years, the dominant intellectual influence of the Hodgkin-Huxley formulation (70, 71) which led to "the Na channel" and "the K channel" as conceptualized components for action potential generation has given way to a rather messier molecular reality. The K channel is really >80 different genes that can assort in a combinatorial fashion to generate an amazing diversity of tetrameric K channel isoforms (67, 79). A similar situation exists for other ion channel types.
The remarkable molecular complexity of ion channels has been a rich evolutionary substrate for generating effective offensive, defensive, and competitive weaponry for Conus. Because these predatory snails are not notable for either speed or mechanical weaponry, to compensate they have evolved rapidly acting, potent venoms. Targeting ion channels is a sensible venom strategy in this biological context: the rich molecular diversity of potential ion channel targets is one factor that leads to molecular complexity of peptides in cone snail venoms.
Two features of individual conopeptides make them of particular interest to the community of neurophysiologists. The first is their ability to discriminate between closely related molecular isoforms of members of a particular ion channel family. Their unprecedented selectivity makes conopeptides an increasingly important tool for defining ion channel function. Most ion channel families have a bewildering array of molecular forms, with the true extent of molecular complexity being undefined in many cases. Pharmacological agents that discriminate between members of a large ion channel family will become increasingly essential for differentiating the physiological roles of closely related ion channel isoforms. A second emerging aspect of conotoxins is their potential utility for investigating different states of the targeted ion channel and transitions between these states. Although such studies are in their infancy, the experimental evidence demonstrating that conotoxin interactions are state dependent and may affect transitions between states is compelling, and will be reviewed below.
Two major sections of this review focus on ligand-gated and voltage-gated ion channels. A theme in the ligand-gated ion channel section is the subtype selectivity of Conus peptides for their targets. The section on the interaction of Conus peptides with voltage-gated ion channels reviews data that illustrate the state dependence of conotoxin interactions.
B. Cone Snails: Historical Perspectives and Biology
The present-day research on cone snails and cone snail venoms is a culmination of the long documented history of human interest of this group of molluscs. The most familiar molluscs are those that are eaten (e.g., oysters, escargots); although cone snails are harvested for food in some Pacific islands, they are not abundant enough to be a notable culinary resource. However, the strikingly beautiful patterns on their shells have attracted human interest from the earliest times, and in a wide variety of cultures. A particularly striking example that may be more than 5,000 years old (shown in Fig. 1) is a Conus necklace excavated from a Mesopotamian tomb in Uruk, the city presently believed to be the first urban settlement. Cone snails were known to Roman scholars and natural history collectors, but it was not until the age of European colonization that they attracted wide attention. European collectors were so intrigued by cone snails that several of the rare and striking species shown in Figure 1 became among the most valuable of all natural history objects. The matchless cone, Conus cedonulli, even outsold a painting by Vermeer at auction in the late 18th century (148).
|
The other aspect of cone snails that has attracted human interest is that they can be deadly to humans (35). One species, Conus geographus, has caused multiple human fatalities. Small Pacific island communities were clearly aware of this potential, and two reports from the 19th century (26, 112) show that in some island cultures, an appropriate medical response to a cone snail sting was practiced. The first scientific record of a fatality from a cone snail sting is in the monumental work of Rumphius (157). A few dozen stinging cases by C. geographus have been detailed in the medical literature, and the high frequency of fatality from untreated stings (70%) of this species has been noted (194). The recognition that this snail could kill people led to the initial scientific investigation of cone snail venoms (47, 98). Earlier scientific work on cone snails that was not purely taxonomic focused on the pharmacological and physiological properties of whole venom. Since these studies were recently reviewed (139), the present article reviews work involving individual venom components.
The increasing interest in cone snail venom components arises in part from their pharmacological potential. Not only are conopeptides used as basic science tools for neurobiologists, but several cone snail toxins are being directly developed for therapeutic applications (86, 127, 138). At present, at least three conopeptides have reached human clinical trials as drug candidates, and several others are being actively explored. The scientific basis for the therapeutic potential of conopeptides is described in the sections that follow.
2. Natural history of Conus: overview of Conus evolution
All cone snails are conventionally included in a single large genus (Conus); however, all venomous snails presently known, including Conus, are usually included in a larger taxon, the superfamily Conacea (also known as the suborder Toxoglossa), collectively referred to as the toxoglossate gastropods (an overview of the group is provided by Taylor et al., Ref. 178). In shallow-water tropical marine environments, cone snails are the most numerous and diverse toxoglossates. However, viewed from a different perspective, Conus are only a minor component of venomous molluscan diversity, probably 10 times as many toxoglossate gastropod species exist outside the genus Conus as within it.
The fossil record of the toxoglossate gastropods suggests origins in the Cretaceous period, but true Conus are known only after the Cretaceous extinction (96). The first fossils that unquestionably belong to the genus Conus are found in the early Eocene. Thus it is generally considered that like many other carnivorous snails, this is a group that had an ecological opportunity with the massive marine extinction at the K-T (Cretaceous/Tertiary) boundary. Predatory Mesozoic marine molluscs, particularly the ammonites, disappeared at the same time as the dinosaurs on land. This major extinction probably made the first radiation of cone snails possible, and a second radiation, at the beginning of the Miocene, basically continues to the present day. It is likely that there are as many living species of cone snails as there ever have been, since the genus appears to have continually expanded. These radiations of Conus in particular, and toxoglossate molluscs in general, presumably require the evolution of novel venoms and efficient delivery systems for the venoms.
The genus Conus is a large and successful group of 500 species (156) of carnivorous predators found in all tropical marine habitats. All members of this species-rich group use venom as the major weapon for prey capture and have a delivery system consisting of a venom duct, where the venom is synthesized and stored; a venom bulb, believed to transfer venom from the duct; and most remarkably, hollow, harpoonlike teeth which serve as a hypodermic needle for injecting the venom. Most cone snails have a long distensible proboscis, and when they forage for prey, a single harpoon tooth is transferred into the lumen of the proboscis. Once the extended proboscis touches prey, the tooth is propelled out, grasped by circular muscles at the anterior tip of the proboscis, and the venom injected into the prey through the hollow tooth. This efficient venom delivery system is characteristic of all cone snails (98), as well as certain other toxoglossate molluscs.
Cone snails are generally divided into three groups, depending on the prey envenomated. The largest class are the vermivorous (worm-hunting) species; most of these feed on polychaetes (a class of segmented marine worms in the phylum Annelida), but a number of species will also attack hemichordate and echiuroid worms. A second major group are the molluscivorous (snail-hunting) species that hunt other gastropods. The final, most remarkable group are the piscivorous (fish-hunting) cone snails; these have venoms that very rapidly immobilize fish (95, 137, 143, 180).
Most cone snails are nocturnal. Although they have two eye stalks, their vision is poor, and their considerable chemosensory prowess is used to track prey. Even though a few species have been adapted to cooler waters, and some Conus species, including some of the largest forms are collected only at depths >100 m, Conus diversity is greatest in the shallow marine habitats of the tropics. In a single coral reef in the central Indo-Pacific, over 30 different species of Conus can be found (97).
The description of cone snails above does not begin to give the reader a sense of their remarkable biological diversity. For example, while all cone snails harpoon their prey, fish-hunters use a single harpoon to capture a fish, while many molluscivorous species repeatedly inject venom into prey after the first attack and have been observed to use over half a dozen harpoons to capture a single prey snail. Even among fish-hunters, the detailed strategy for prey capture is species specific; some of these more subtle aspects of the biology of Conus, described in a recent review (139), can be used to rationalize the biochemical and pharmacological diversity of conopeptides (see sect. IC3).
C. Overview of Conus Venom Components
An overview diagram of peptidic Conus venom components is shown in Figure 2. Two broad divisions of venom components are shown: disulfide-rich conotoxins and peptides that lack multiple disulfide cross-links. In the disulfide-rich conotoxins, Cys residues may be found at an unprecedentedly high frequency; most cysteine residues are separated by 0–6 amino acids from the next Cys residue in the primary sequence. In many conotoxins, multiple pairs of adjacent cysteine residues are found. The arrangement of cysteines in the primary sequence is restricted to only a few patterns; in general, each pattern corresponds to a specific disulfide connectivity. Furthermore, the Cys pattern is diagnostic of the gene superfamily that encodes the peptide (see sect. IC2), and in many cases can be indicative of the pharmacological target of the conopeptide.
|
Several biochemical features of Conus peptides are distinctive. Conopeptides are unusually small; most disulfide-rich conotoxins are 12–30 amino acids. In contrast, the size range of polypeptidic toxins from other venoms is typically 40–80 amino acids. Despite their small size, there is a remarkable interspecific sequence divergence, even between homologous conopeptides from closely related Conus species (149, 193). Another striking feature of conopeptides is the presence of an unusually diverse complement of posttranslationally modified amino acids, found at a high frequency in some conopeptide families (27). These include some that are well known and widely distributed [hydroxyproline, O-glycosylated Ser or Thr (30)] as well as others that are unusual [6-Br-Trp (82), 
-carboxy-Glu (123), D-amino acids (84), sulfated-Tyr (109)]. However, all conopeptides are translated through the conventional ribosomal mechanism, and mRNAs encoding each peptide can be identified in the venom duct.
Characterization of venom duct cDNA clones established that Conus peptides are initially translated as prepropeptide precursors. The canonical organization of conopeptide precursors consists of a typical signal sequence at the NH2-terminal end of the open reading frame (the "pre" region), followed by an intervening "pro" region, and at the COOH-terminal end of the open reading frame, the mature toxin region, always in single copy. Thus proteolytic cleavage of the precursor to generate the final functional toxin is an obligatory step in the maturation of all Conus peptides.
A comparison of homologous cDNA sequences from different Conus species revealed an unusual and striking pattern; peptides with similar arrangements of Cys residues in the primary sequence of the mature toxin share a highly conserved signal sequence. These features define members of a conopeptide gene superfamily. A relatively small number of gene superfamilies have undergone extensive proliferation and diversification in the genus Conus to generate the majority of the >50,000 different conopeptides found in the venoms of living Conus today (137).
Some of the more well-characterized gene superfamilies with the corresponding arrangement of cysteine residues found in superfamily peptides are shown in Figure 2. The mechanisms that lead to the conservation of the signal sequences (a conservation that extends even to the third position of codons, making even silent mutations underrepresented in this region) juxtaposed with the hyperdivergence observed in the mature toxin region (149, 193) remain a subject for speculation; a variety of mechanisms leading to the observed differences in the differential rate of divergence observed for the signal sequence region, the pro region, and the mature toxin region have been proposed (25, 40, 49, 137, 149).
Insights into the mechanism of posttranslational modification of conopeptides have also been obtained, mostly from studies of the best-characterized Conus posttranslational modification, the -carboxylation of glutamate to the unusual amino acid -carboxyglutamate (8, 123). Cone snails express the posttranslational modification enzyme in their venom ducts (9, 174); the enzyme has a recognition signal in the pro region of the precursor. The presence of such a signal in a precursor peptide recruits the posttranslational enzyme and instructs the enzyme to modify specific amino acid residues in the mature toxin region. Thus the pro region of conopeptide precursors provides potential anchor binding sites for posttranslational modification enzymes (72).
3. Overview of pharmacology and physiology
As a group, the cone snails are specialists in neuropharmacology. There are many parallels between conopeptide evolution and modern pharmacological practices. The hypermutation that occurs in conopeptide-encoding regions that accompanies Conus speciation is the evolutionary equivalent of a combinatorial library strategy for drug development. Posttranslational modification of conopeptides achieves the same ends as the medicinal chemistry carried out after an initial lead candidate for drug development is identified. A final pharmacological insight regarding the mode of action of conopeptides is that cone snails are sophisticated practitioners of combination drug therapy. To efficiently impose the desired physiological effect on the injected prey, predator, or competitor, multiple conopeptides act together in a synergistic fashion to affect the targeted animal in a manner that benefits the cone snail. The term toxin cabal has been applied to an assemblage of conopeptides acting coordinately to a specific physiological end point.
The systematic analysis of the venom components of the fish-hunting species Conus purpurascens, the purple cone, defined the presence of two different toxin cabals in the venom (180), whose effects are separable in both time and space. The first, the "lightning-strike cabal," causes immediate immobilization of the injected prey; components of the lightning-strike cabal include peptides that inhibit voltage-gated Na channel inactivation as well as peptides that block K channels. Together, this combination would result in the massive depolarization of any axons in the immediate vicinity of the venom injection site, causing an effect similar to electrocuting the fish. Thus the characteristic tetanic state elicited by the peptides of the lightning-strike cabal are manifest immediately after venom injection.
The second physiological end point is achieved more slowly: a total inhibition of neuromuscular transmission. This is effected by the venom through the "motor cabal" of conopeptides. These peptides act at sites that are remote from the venom injection site, e.g., at neuromuscular junctions. Since transport of the peptides to the remote target sites is required before the desired physiological effects are achieved, the motor cabal's effects are observed after those of the lightning-strike cabal. The motor cabal, found in all fish-hunting Conus venoms examined so far, includes peptides that inhibit the presynaptic Ca channels that control neurotransmitter release, the postsynaptic nicotinic receptors, and the Na channels that underlie the muscle action potential.
There is evidence that different fish-hunting Conus species may have a divergent spectrum of toxin cabals as part of their repertoire for prey capture. In part, this divergence can be correlated to different behavioral strategies for capturing fish. The combination of lightning-strike and motor cabals is found in the venoms of species that extend their proboscis to initially approach and then harpoon the fish prey. Several species use an alternative strategy for capturing fish; such Conus can greatly distend their "false mouths," effectively using them as a net. These Conus engulf the fish with the false mouth before they inject venom. Thus the venom of one such species, C. geographus, appears to have novel conopeptides that jam the sensory circuitry of the targeted prey; these have been referred to as the "nirvana cabal" (140). It has been proposed that such venom components may be released by the snail to make a school of fish more quiescent, making capture by a net strategy more facile (139).
The combination drug strategy employed by cone snails is presumably one underlying explanation for the complexity of Conus venoms, as well as for the surprising pharmacological diversity of Conus venom components. Furthermore, different toxin cabals may include peptides that act on the same general class of targets, but through different, and sometimes seemingly incompatible mechanisms. Thus, to interfere with neuromuscular transmission, the motor cabal typically contains a Na channel-targeted peptide that blocks conductance. In contrast, the lightning-strike cabal that causes an immediate immobilization of prey by axonal hyperexcitation requires one or more conopeptides that inhibit Na channel inactivation, thereby increasing the total Na flux with each channel opening event. The former would, on the surface, appear to cancel out the effects of the latter. The evolutionary solution to this problem is of great significance: each peptide is selectively targeted to a specific and different voltage-gated Na channel subtype. As a result, the two peptides are effective components of different cabals, acting within different time windows, without functional cross-interference.
Thus at least two factors favor subtype selectivity of conopeptides. First, cone snails are unusually slow predators lacking effective offensive mechanical weaponry; as a consequence, there is strong selective pressure for the venoms to act very quickly. Binding to an irrelevant target, including those closely related to the physiologically relevant target, would functionally slow down an individual venom component. A second factor is the toxin cabal/combination drug therapy characteristic of the Conus strategy; the more complex the venom, the greater the selective pressure for subtype specificity on a newly evolving venom component. The less specific a conopeptide is for its physiologically relevant target, the greater the potential that it would interfere with the activity of another cabal in the venom. The overall complexity of the venom may therefore generally select for individual conopeptides with high targeting specificity.
Thus the biology and evolutionary history of cone snails has led to the unique biochemistry, genetics, pharmacology, and physiology of conopeptides: unusually small, mostly disulfide-rich peptides with a high frequency of nonstandard amino acids, encoded by a few gene superfamilies that have rapidly generated an extraordinarily diverse targeting specificity for ion channels.
|
II. CONOPEPTIDES TARGETED TO VOLTAGE-GATED ION CHANNELS |
|---|
|
TopPreviousNextReferences |
|---|
The voltage-gated ion channel superfamily comprises a large set of structurally similar membrane-bound proteins activated by a change in the transmembrane voltage. These proteins exhibit different selectivity for monovalent cations and are conventionally divided into Na, K, and Ca channels (67). The most important physiological role of voltage-gated ion channels is the generation, shaping, and transduction of the electrical signals of cells. The main pore-forming -subunit of voltage-gated ion channels consists of either a single subunit containing four homologous domains (Na and Ca channels) or four distinct subunits (K channels); the latter may be either homomeric or heteromeric. The -subunits interact with auxiliary subunits that are not integral to forming a pore, but which can alter the properties of the -subunit. Upon activation, voltage-gated ion channels undergo a conformational change, which under physiological conditions results in the selective permeation of cations through the pore of the channel protein. From this open state, voltagegated ion channels can be either inactivated by an additional conformational change, thereby entering a nonconducting state, or they may be deactivated, returning to a closed state.
Voltage-gated ion channels are targets of toxins from a great variety of different organisms. In this section, we describe the properties of conotoxins interacting with the pore-forming -subunit of Na, K, and Ca channels; an increasing number of such conotoxin families have been identified (see Fig. 2). Three different Conus peptide families are known to target voltage-gated sodium channels: the µ-conotoxins that are channel blockers, the µO-conotoxins that inhibit Na channel conductance, and the -conotoxins that delay or inhibit fast inactivation.
One of the -conotoxins that block voltage-gated calcium channels, -conotoxin GVIA, is probably the most widely used Conus peptide in neuroscience; over 2,000 papers in the literature have used -GVIA as a pharmacological tool, primarily to inhibit synaptic transmission. Conus peptides that target K channels have just begun to be characterized; two peptides, 
-conotoxin PVIIA and M-conotoxin RIIIK, have been investigated in considerable detail. However, a number of Conus peptide families have been shown to also target K channels, but the molecular specificity of most of these has not yet been defined. A striking contrast between peptides that target K channels and those that target Na channels is that the Na channel-targeted Conus peptide families appear to be widely conserved over a broad range of Conus species. In marked contrast, structurally and genetically divergent Conus peptide families have been shown to target K channels in different groups of Conus species.
The endogenous functions of peptides that target voltage-gated ion channels are likely to be extremely diverse. In fish-hunting Conus venoms, µ-conotoxins targeted to the muscle subtype of sodium channels have a major role in prey paralysis, as do the Conus peptides that block the presynaptic calcium channels at the neuromuscular junction of the prey. These are therefore major components of the motor cabal of toxins, which cause an irreversible block of neuromuscular transmission. The -conotoxins that inhibit Na channel inactivation are a major component of the lightning-strike cabal of conopeptides, as are at least some of the K channel blockers (see sect. IC3). Thus prey immobilization and paralysis are likely functions of many of these peptides. However, the enormous complexity of Na channel- and K channel-targeted Conus peptides suggests that these are likely to have many other endogenous biological functions; for example, some -conotoxins could conceivably play a role in predator deterrence, since if targeted to the appropriate Na channel subtype, they would be expected to cause intense pain in an injected animal.
Voltage-sensitive Na channels are key molecules for generating action potentials in electrically excitable cells by forming the action potential upstroke. To date, 10 different isoforms of -subunits with different developmental and tissue distribution have been identified, and a standardized nomenclature (Nav1.x) has recently been proposed (18, 57, 58, 136). Voltage-gated Na channels have conventionally been divided in two pharmacological classes, tetrodotoxin (TTX)-sensitive and TTX-insensitive channels, based on their sensitivity to the classical blocker of Na currents TTX. The interaction site of TTX with the channel protein has been called site I and is postulated to be at the extracellular end of the ion channel pore. In addition to this site, up to five other interaction sites for different substances with different modes of activity on the channel protein can be distinguished (2, 17). Most ligands that target these other sites cause a net increase in Na currents either by shifting the current-voltage relationship or by blocking the fast inactivation of the channels. Na channel-targeted toxins have been important compounds in the historical development of neuroscience and remain indispensable pharmacological tools for neuroscience research.
µ-Conotoxins were originally isolated from the venom of the fish-hunting snail C. geographus. These peptides are 22–25 amino acids with 6 cysteine residues arranged in a class III framework (see Fig. 2) and belong to the M-superfamily of conopeptides (see Fig. 2). µ-Conotoxins block Na currents by acting at site I of Na channels, the interaction site of TTX. So far, µ-conotoxins are the only polypeptide toxins known that affect Na currents through this site.
The µ-conotoxins GIIIA and GIIIB from the venom of C. geographus are the best characterized. µ-GIIIA is known to specifically block skeletal muscle Na channels (Nav1.4) and exhibits significantly decreased affinity for any other Na channel subtype (10, 34, 60). It is known that µ-GIIIA interacts with the pore region of the ion channel and therefore competes with TTX for binding to site I. Structure-function studies have revealed a rather complex interaction of µ-GIIIA and µ-GIIIB with the ion channel pore, with several amino acids at different positions of the peptide contributing to the high affinity of binding. These data indicate that binding sites for TTX and µ-conotoxins overlap, but are not identical (42, 129); some mutations that have strong effects on the TTX sensitivity of Nav1.4 lead to only a minor increase in the IC50 of µ-GIIIA to the channel (19, 21, 175).
From a set of different studies including modeling work, a detailed picture of how µ-conotoxins plug the ion channel pore has been hypothesized (74). The amino acid that likely occludes the pore is Arg-13 of µ-conotoxin GIIIA (10, 56); this residue seems to act as a steric and electrostatic barrier (42, 56, 74). Some mutations at this locus result in analogs of µ-GIIIA/µ-GIIIB that only partially block the channel, presumably because occlusion of the ion channel pore is incomplete. Such analogs are useful as a starting point for evaluating the contributions of other residues within the peptide. Furthermore, µ-GIIIA and µ-GIIIB have been heavily used as tools to investigate structure-function relationships of Na channels. For example, pairwise interactions between the residues of µ-GIIIA and those of Nav1.4 have been investigated by the double mutant cycle method (65, 163). Together with the three-dimensional structure of the peptide, the data clearly indicate that the four repeats within the -subunit of Na channels have a clockwise orientation (41). Furthermore, a mutant µ-GIIIA (R13Q) has been used to study how the conformation of the pore region near the selectivity filter affects channel dynamics. The data indicate a conformation change in the P-loop of domain IV of Na channels during activation (66).
µ-Conotoxin PIIIA was isolated from the venom of C. purpurascens. This peptide also contains an Arg residue (Arg-14), shown to be a key residue for binding, at a position homologous to Arg-13 of µ-GIIIA (168). Therefore, µ-PIIIA shares the critical guanidinium group postulated to be important for interaction with site I of Na channels. Although quite similar to µ-GIIIA, µ-PIIIA has features that make it particularly useful for studying Na channels in situ. In amphibian systems, µ-PIIIA, unlike µ-GIIIA, irreversibly blocks muscle Na channels, providing a useful tool for studying synaptic electrophysiology. When the effects of the peptide on cloned mammalian Na channels are evaluated, the specificity of µ-conotoxin PIIIA is not as highly focused for Nav1.4 as is observed for µ-GIIIA; µ-PIIIA blocks neuronal Nav1.2 Na channels with affinities in the submicromolar range, although Nav1.4 skeletal muscle Na channels are clearly the highest affinity target (168). This feature can be used to subdivide TTX-sensitive Na channels into three categories: 1) sensitive to µ-GIIIA and to µ-PIIIA (i.e., Nav1.4); 2) insensitive to µ-GIIIA, but sensitive to µ-PIIIA (Nav1.2 and perhaps others); and 3) insensitive to µ-GIIIA and µ-PIIIA.
The differential properties of µ-PIIIA have been used for further discrimination of different Na channel subtypes in a number of systems. A recent example is the demonstration that Nav1.2 and Nav1.7 channels appear sequentially during neuronal differentiation of PC12 cell lines (158). Interestingly, in rat brain neurons µ-PIIIA seems to preferentially inhibit persistent over transient voltage-activated Na currents (135).
Recently, the solution structure of µ-PIIIA has been solved by NMR techniques. It was shown that µ-PIIIA in solution adopts two conformations due to a cis/trans-isomerization at hydroxyproline-8 (135). The three-dimensional structure of the trans-conformation is significantly different from the previously solved structures of µ-GIIIA and µ-GIIIB. These underlying structural differences may be one factor in the divergent pharmacological properties of these peptides.
A new µ-conotoxin that has neuronal subtype specificity for voltage-gated sodium channels was recently reported; this peptide, µ-conotoxin SmIIIA from Conus stercusmuscarum, has several distinctive sequence features (see Table 1) (186). Uniquely among Na channel ligands, this peptide inhibited most TTX-resistant Na current irreversibly in voltage-clamped dissociated neurons from frog sympathetic and dorsal root ganglia. The TTX-sensitive Na currents in these neurons were largely unaffected by the peptide. Although the effect of this novel µ-conotoxin on other voltage-gated Na channel subtypes, particularly those neurons from mammalian systems, has not yet been reported, this peptide is clearly different from other µ-conotoxins reported so far; neither µ-conotoxin GIIIA nor PIIIA had an effect on the amphibian TTX-resistant channels. The results suggest that the µ-conotoxin family is a potential source of subtype-specific voltage-gated Na channel antagonists. Presumably, all µ-conotoxins act on site I; the arginine residue believed to occlude the pore in µ-conotoxins GIIIA and PIIIA is conserved in µ-conotoxin SmIIIA (see Table 1).
|
Both the µO- and -conotoxins are unusually hydrophobic peptides that belong to the O-superfamily (see Fig. 2), and the pattern of disulfide bonds gives these peptides an inhibitory cysteine knot (ICK) motif.
The µO-conotoxins are a Conus peptide family that inhibits Na channel conductance like µ-conotoxins, but most likely through a different mechanism. In contrast to µ-conotoxins, no competition for binding with saxitoxin (STX) has been observed for µO-conotoxins, indicating that these peptides do not interact with site I (181). So far, the mechanism of interaction with Na channels is not understood. Two closely related 31-amino acid peptides, µO-MrVIA and µO-MrVIB (see Table 1) isolated from the venom of the snail hunting species C. marmoreus, have a disulfide bonding pattern that more closely resembles the -conotoxins (see below) than the µ-conotoxins.
µO-MrVIA blocks Nav1.2 channels expressed in Xenopus oocytes in the nanomolar range. Na currents recorded from hippocampal pyramidal neurons in culture are also inhibited by the peptide (181). The blocking effect of µO-MrVIA was readily reversible for the Na currents recorded from hippocampal cells in culture; in contrast, it was only very slowly reversible for Nav1.2 currents recorded in the Xenopus expression system. Unlike TTX/STX, µO-MRVIA did not show any phasic or use-dependent inhibition of Na currents, but the steady-state inactivation curve of the Na currents is shifted to more hyperpolarized potentials in the presence of the peptide. µO-MrVIA appears to block both TTX-sensitive and TTX-insensitive Na channels, but the complete subtype specificity of the peptide remains to be established.
The main effect of conopeptides belonging to the -conotoxin family is the inhibition of the fast inactivation of Na currents, one of the key mechanisms underlying the proper shape and duration of action potentials. This results in a hyperexcited state of the affected cells, which can eventually lead to a massive electrical hyperexcitation of the complete organism. The molecular mechanism of this action is not yet clear, but extracellular binding of the peptides seems to affect events in the intracellular part of the Na channel important for fast inactivation. -Conotoxins have the same cysteine framework as the µO- and -conotoxins and belong to the same O-conotoxin superfamily (Fig. 2)
The high degree of hydrophobicity that is a biochemical hallmark of both the µO- and -conopeptides might be important for the mechanism of action. The most recent structure information on gating of voltage-activated K channels (80, 81) is likely to be highly relevant in providing a rationale for the observed hydrophobicity of these peptides. However, similarly detailed structural information of comparable resolution for voltage-gated sodium channels is unavailable, and the peptide binding sites have not been directly localized; thus any detailed hypothesis would be conjectural. Several -conotoxins from fish- and snail-hunting species have been identified (see Table 1).
The observed effects of the -conotoxins can depend enormously on the system being investigated. -TxVIA from Conus textile (originally called the "King Kong" peptide because it causes a characteristic behavior upon injection into lobster) was shown to prolong Na currents in molluscan neuronal membranes. In vertebrate systems, this peptide binds to Na channels but without any toxic effects. -PVIA was isolated from the venom of C. purpurascens, a fish-hunting Conus species; the peptide elicits excitatory symptoms in mice and fish but is inactive in molluscs even at doses 100-fold higher. When administered to fish, it causes specific muscle contraction resulting in a characteristic extension of the mouth, which was termed "the lockjaw syndrome" (166). -PVIA slows fast inactivation of Nav1.2-mediated currents expressed in the Xenopus expression system, as well as of Na currents recorded from hippocampal neurons in culture (180). More recently, it was demonstrated that the peptide also affects Nav1.4- and Nav1.6-mediated currents (158). In rat brain synaptosomes, -PVIA competes with -TxVIA for the same binding site, despite the latter being nontoxic in vertebrate systems (166).
-PVIA is one of the major constituents of the lightning strike cabal in C. purpurascens venom. The excitatory effects of -PVIA act synergistically with the K channel-blocking peptide -PVIIA, resulting in the almost immediate immobilization of the prey (see sect. IIC1). -GmVIA isolated from Conus gloriamaris causes action potential broadening in Aplysia neurons (64, 167). Furthermore, this peptide has micromolar affinity for Nav1.2 and Nav1.4 but does not affect Nav1.6-mediated Na currents.
C. K Channel-Targeted Toxins: -, A-, and M-Conotoxins
K channels are not only important for the repolarization phase of action potentials, but are also involved in setting the resting membrane potential, bursting activity, and have a variety of specialized purposes in a wide range of cell types (67). In accordance with this broad spectrum of different physiological functions, more than 80 genes encoding different K channels have been identified, and several families of voltage-gated K channels (KV1.x, KV2.x, etc.) are known. The -subunits of voltage-gated K channels, the first class to be elucidated, are proteins with six transmembrane domains. The functional pore-forming protein complex may either be homomeric (with 4 identical subunits) or heteromeric (with 2 or more different -subunits). The first K channel-targeting conotoxin was identified relatively recently (180), and only a few K channel-targeted conopeptides have been extensively characterized. This may have to do with the potential high specificity anticipated for these peptides: if the target of a conopeptide were a heteromeric K channel, it will be a challenge to define the high-affinity K channel target affected by the conopeptide. Recent results indicate that the cone snails have evolved many different families of K channel-targeting peptides, although at present, the pharmacological range and specificity of any family, particularly in mammalian systems, is largely undefined
The first conotoxin shown to target voltage-gated K channels was -conotoxin PVIIA, isolated from the fish-hunting cone C. purpurascens (169, 180). With the use of the Xenopus expression system, it was demonstrated that this peptide blocks Shaker K current with relatively high affinity (IC50 50 nM). Because the Shaker channel was cloned from Drosophila, this is not the natural target of -PVIIA; the latter has not been identified. Nevertheless, -PVIIA seems to be quite subtype selective; it was shown to differentiate between splice variants of the Shaker channel homolog from lobster (91). Injection of -PVIIA leads to excitatory symptoms in mice, but to date no cloned mammalian K channel (>20 tested so far) has been shown to be affected by this toxin.
The K channel block of -PVIIA is physiologically significant for prey capture as it is a key venom component in the rapid immobilization of the fish prey. The activity of -PVIIA acts in a synergistic manner with -conotoxin PVIA, leading to massive hyperexcitation of the injected animal, resulting in an almost instant tetanic paralysis. Therefore, -PVIIA plays a very critical role in the lightning-strike cabal of conopeptides.
The interaction of the peptide with the ion channel pore is a bimolecular reaction. The analysis of Shaker K channel block by -PVIIA revealed that the conopeptide binds to the extracellular mouth of the ion channel pore (see Fig. 3). With the use of alanine-scanning mutagenesis, the interaction surface of -PVIIA with the Shaker channel pore has been identified. Mutations within the P-loop of the channel protein have major effects on the binding of -PVIIA (169); a double mutant cycling analysis revealed that -PVIIA, like other K channel blocking peptides, contains a critical dyad motif of amino acids important for K channel blockade (78). This dyad consists of lysine-7 and a phenylalanine-9, which are 6 Å apart. K7 is believed to occlude the ion permeation pathway, and the hydrophobic amino acid F9 may be important for hydrophobic interactions of the peptide with the ion channel.
|
These data support the critical dyad model developed by Menez and co-workers (37, 162) for polypeptide antagonists of K channels. Thus, although -PVIIA has no obvious sequence homology to polypeptide toxins from other venomous animals that interact with voltage-gated K channels, there appear to be convergent functional features in diverse K channel polypeptide antagonists. Several peptides containing this structural feature have been identified from different venomous organisms, demonstrating that peptides with quite different cysteine backbones that interact with K channels can all have the dyad motif.
The A- and M-conotoxins are structurally unrelated to each other; A-conotoxins are O-glycosylated peptides that belong to the A-superfamily, while M-conotoxins have a disulfide framework similar to the µ-conotoxins and belong to the M-superfamily (see Fig. 2).
The first peptide of the A-conotoxin family was identified from the venom of the fish-hunting cone snail Conus striatus. This peptide, A-conotoxin SIVA, causes spastic paralytic symptoms when injected into mice. The disulfide arrangement of A-SIVA is similar to the pharmacologically distinct A-conotoxins. Electrophysiological tests on diverse preparations provide evidence that A-SIVA blocks K channels. Recordings from frog cutaneus pectoris muscle and principal neurons from frog sympathetic ganglion reveal that this peptide induces repetitive activity in these cells. Furthermore, Shaker channels expressed in Xenopus oocytes are blocked by micromolar concentrations of A-SIVA. However, the molecular identity of the vertebrate high-affinity K channel target of this peptide has not yet been identified. Interestingly, it was shown that A-SIVA in its active form contains one O-glycosylated serine at position 7, which was the first evidence for O-glycosylation as a posttranslational modification in a biologically active conotoxin. A peptide called CcTx from Conus consors, which has the same cysteine scaffold as A-SIVA, was suggested to activate neuronal voltage-gated Na channels at resting membrane potential (101). If confirmed, this might indicate that similar peptides may act on different pharmacological targets, but a more thorough analysis of the A-conotoxins and related peptides needs to be carried out.
A new family of conotoxins, the M-conotoxins, has the same class III scaffold as the µ- and 
-conotoxins, but an entirely different target specificity: voltage-gated K channels (55). The first peptide belonging to this family, M-RIIIK, was cloned from the venom duct of Conus radiatus. Unlike the structurally related µ-conotoxins, the peptide had no effect when tested on Na channels expressed in Xenopus oocytes. However, M-RIIIK blocks Shaker K channels with an IC50 of 1 µM and has an even higher affinity for TSha1, a Shaker homolog K channel from trout (IC50 20 nM).
The interaction of the peptide with the ion channel is a bimolecular reaction. Mutations of residues within the pore of the Shaker channel drastically affect the affinity of M-RIIIK, indicating that like -conotoxin PVIIA, the peptide interacts with the ion channel pore. These results show that conotoxins with similar cysteine frameworks can have entirely different pharmacological properties. M-RIIIK does not contain any phenylalanine or tyrosine present in other K channel-targeted peptides as part of the functional dyad, and it will be interesting to see whether a dyad motif is important for block of K channels by this conopeptide.
At least three additional families of conotoxins have been identified that are likely to be targeted to voltage-activated K channels (unpublished results). Interestingly, these peptides have entirely different cysteine connectivity from the three families above, which opens the possibility for novel polypeptidic ligands for investigating K channel structure and function. One group is peptides belonging to the I-superfamily (see Fig. 2); evidence that the family is extremely diverse has been obtained (85). Recently, an I-superfamily peptide (called -BtX) was shown to upmodulate the Ca- and voltage-sensitive BK currents measured from rat adrenal chromaffin cells and did not affect other voltage-gated channels (54), and another member of the I-superfamily designated VcTx was shown to inhibit Kv1.1 and Kv1.3 subtypes, but not Kv1.2 (88).
D. Ca Channel-Targeted -Conotoxins
Ca signaling is involved in a great variety of different physiological processes including neurotransmitter release. Voltage-gated Ca channels, which mediate the Ca influx in response to depolarization, are heteromeric protein complexes with four or five different subunits. The observed physiological and pharmacological diversity of Ca channels is mainly due to the properties of the pore-forming 1-subunits. Based on their different physiological and pharmacological properties, voltage-activated Ca channels have been categorized into L-, N-, P-, Q-, R-, and T-type channels. More recently, a standardized, more genetically derived nomenclature has been proposed for Ca channels, which mainly adopts the system originally developed for K channels. According to this nomenclature, the Cav1 family includes the channels that mediate L-type Ca currents; the Cav2 family includes the channels which mediate the P/Q-type, N-type, and R-type Ca currents; and the Cav3 family includes the channels that mediate T-type Ca currents.
Conotoxins that target Ca channels were among the first conotoxins characterized and are the peptides from Conus venoms most intensively used in neuroscience research. The selective inhibition of different Ca channels specifically located at presynaptic endings was made possible with the discovery of -conotoxins. To date, -conotoxins have been largely identified from fish-hunting cone snails, although it seems likely that worm- or mollusc-hunting Conus species have developed an equally rich repertoire of peptides to target voltage-gated Ca channels. A peptide that inhibits molluscan Ca channels that corresponds to the L-type mammalian subtype has been described (51). The venoms of fish-hunting Conus that have been systematically studied will typically have multiple -conotoxin isoforms with major differences in their amino acid sequences. These are likely to be functionally selected for targeting different Ca channels. This was clearly demonstrated in Conus magus venom (68, 141); one peptide, -conotoxin MVIIA, is highly specific for N-type calcium channels (Cav2.2), while another peptide from the same venom, -conotoxin MVIIC, preferentially targets P/Q channels (Cav2.1). The structure of several -conotoxins has been solved. A characteristic feature of -conotoxins is their high content of basic amino acid residues that are known to play an important role for the inhibition of Ca channels (132). Besides these positive charges, it is known that a tyrosine residue (Tyr-13 in -MVIIA and -MVIIC) is important for the binding to Cav2.2 and Cav2.1 channels (90, 102, 134).
Several -conotoxins have been identified and functionally characterized, and there is an extensive literature on these peptides (for overviews, see Refs. 43, 122, 147). Since this field has previously been reviewed comprehensively, the reader is referred to these reviews for the earlier literature on -conotoxins. The sequences of the most widely used -conotoxins are shown in Table 2.
|
The therapeutic potential of -conotoxins that specifically target N-type Ca channels (Cav2.2) is an important application of conopeptide research. One of these peptides, -conotoxin MVIIA from C. magus (141), has been through extensive human clinical trials and was given "approvable" status for the treatment of intractable pain by the United States Food and Drug Administration (127, 138). This peptide has been provided with a generic name, ziconotide, and a commercial name, Prialt, by its developer, Elan Pharmaceuticals. Another peptide, -conotoxin CVID from Conus catus (103), is being explored as an antinociceptive agent (173); it has been given the pharmaceutical designation AM336 and was slated for clinical development by Amrad, an Australian pharmaceutical company.
The antinociceptive potential of Cav2.2-targeted -conotoxins has two important scientific facets. The first is that in the mammalian spinal cord, the synapses with incoming C-fibers that carry nociceptive signals are particularly sensitive to block by these -conotoxins; autoradiographic studies revealed an enrichment in Cav2.2 channels at these sites in the dorsal horn. Furthermore, because these peptides are antagonists of voltage-gated ion channels, in contrast to opioids which are agonists of G protein-coupled receptors, they do not have the problems encountered upon continued exposure of the latter receptors to agonists that cause downregulation. This downregulation is the basis for development of tolerance to opioid drugs that occurs in patients; this does not occur in patients treated with the peptide. Thus Cav2.2-targeted conopeptides are envisioned to be useful in clinical situations where opioid drugs are no longer (or have never been) effective.
E. State Dependence of Block: Transitions Between States
Because voltage-gated ion channels must necessarily undergo conformational changes to carry out their functions, the binding of pharmacologically active substances may differ depending on the state of the ion channel. Such state or use dependence was previously demonstrated; for example, the block of Na channels by TTX or STX has a tonic component and a phasic component, which depends on the activation of the Na channels (e.g., Refs. 7, 14, 23, 24, 44, 108, 159, 160). However, few investigations on state dependence for polypeptide antagonists of voltage-gated ion channels have been reported.
Recently, the binding of -PVIIA to Shaker K channels was investigated, primarily using electrophysiology. The observed changes in current kinetics in the presence of the toxin were explained by differences in both the steady-state affinity as well as binding kinetics to the open and closed state of the channel (179). Furthermore, it was shown that -PVIIA binding to closed but not to open Shaker channels depended on the extracellular K concentration: an increase leads to a reduction of the steady-state affinity to the closed state (see Fig. 4). The observed acceleration of -PVIIA dissociation from open channels in the presence of physiological levels of extracellular K concentration was attributed to the voltage-dependent occupancy by K ions of a site at the outer end of the conducting pore. Occupancy of this site by external cations most likely antagonizes on-binding to closed channels, whereas the apparent competition disappears in open channels if the competing cation can move along the pore. Some of the experimental results that led to this picture are shown in Figure 4.
|
The investigation of the effects of the ionic milieu on -PVIIA binding further revealed that binding to the closed channel is independent of the intracellular cation. Additionally, it was found that equally permeant cations may have quite different occupancy configurations within the pore permeation pathway (11). Studies on mutant Shaker channels in which C-type inactivation is accelerated demonstrated that -PVIIA binding and C-type inactivation are mutually exclusive (94). This is functionally significant, since under certain conditions, the binding of -PVIIA leads to an increase in the evoked currents (instead of the block of K currents that would generally be expected for this peptide).
These results show that binding of -PVIIA to Shaker K channels depends on and may affect the conformational state of the channel in a way that is functionally, and thereby physiologically, relevant. This analysis suggests that a detailed study of the interaction of substances blocking ion channels with permeant ions may provide important information regarding properties of the ion-conduction pathway and might add complementary data to the recent advances made on the knowledge about the structure of the permeation pathway of K channels.
State-dependent binding has recently been shown for binding of M-RIIIK to its target K channels (55); peptide neurotoxins from other organisms have been demonstrated to also interact in a state-dependent fashion (5). This opens up the possibility that the state dependence might be a common feature of the interaction of biologically active substances with voltage-gated ion channels.
|
III. CONUS PEPTIDES TARGETED TO LIGAND-GATED ION CHANNELS |
|---|
|
TopPreviousNextReferences |
|---|
Ligand-gated ion channels are proteins that mediate fast synaptic transmission (for an overview, see Kandel et al., Ref. 87). Because many of these membrane-bound proteins have been cloned, they are grouped according to their structural and functional similarities. One major group of ligand-gated ion channels, all belonging to the same gene superfamily, are those activated by acetylcholine, serotonin, GABA, or glycine. The functional channel protein complexes are composed of five subunits, with each subunit containing four transmembrane helices. Besides their ligand specificity, these proteins differ in their selectivity for permeant ions. The other gene superfamily of ligand-gated ion channels is the glutamate receptors, usually subdivided into N-methyl-D-aspartate (NMDA) and non-NMDA (kainate/AMPA) receptors. A third family of ligand-gated ion channels involved in synaptic transmission at certain synapses is the ATP receptors.
Conus peptides targeted to three different families of ligand-gated ion channels have been identified. Conus peptides that target the major ligand-gated ion channel superfamily are ubiquitously distributed in Conus venoms. One peptide has been shown to target the 5-hydroxy-tryptamine (5-HT3) receptor (48), with a large number known to target nicotinic acetylcholine receptors (124). Competitive nicotinic agonists are particularly well represented among Conus peptides, but noncompetitive nicotinic antagonists have also been characterized. An unusual family of Conus peptides, the conantokins, are antagonists of NMDA receptors, a subclass of the glutamate receptor superfamily. These peptides are biochemically distinctive in their high content of the modified amino acid -carboxyglutamate and lack of Cys residues.
The most widely distributed of the nicotinic antagonists are the -conotoxins, multiple representatives of which are expressed in the venom ducts of most, if not all, Conus species. Thus we expect that the total number of -conotoxins in Conus venoms will be >>1,000 different peptides. As discussed below, -conotoxins are highly subtype-selective nicotinic antagonists and are proving to be valuable pharmacological reagents for discriminating between closely related neuronal nicotinic acetylcholine receptor isoforms.
The endogenous functions of Conus peptides targeted to various ligand-gated ion channels can be speculated on. The major nicotinic acetylcholine receptor antagonists found in a venom are usually those used to disrupt neuromuscular transmission to paralyze prey; these are presumably targeted to the main nicotinic acetylcholine receptor subtype(s) present at the neuromuscular junction of the prey. This is clearly the case for fish-hunting (piscivorous) cone snails, where the quantitatively most abundant peptides of the -conotoxin family in the venom are paralytic to fish (and other vertebrates). The endogenous functions of nicotinic antagonists that are not targeted to the neuromuscular nicotinic acetylcholine receptors (nAChR) are far less clear. It has been hypothesized that a subset of these may be involved in suppressing the fight-or-flight response of the prey (177). Several neuronal nAChR subtypes are present in the autonomic circuitry of vertebrates.
An entirely different set of functions has been hypothesized for the glutamate and 5-HT3 receptor antagonists (conantokins and 
-conotoxin). It has been suggested that the snails from which these have been isolated use the peptides as part of the nirvana cabal whose overall function is to deaden the sensory circuitry of the prey (140). This elicits a sedated, opium den-like state, making the prey easier to capture, and to handle once captured. The use of a nirvana cabal is clearest in net-hunting piscivorous species (that probably try to capture schools of small fish in the wild).
B. -Conotoxins and nAChRs: Matching a Family of Conopeptides to a Family of Ion Channel Targets
Of all conopeptide families, the diverse targeting specificity of the -conotoxins is the best characterized at present. The -conotoxins were among the first conopeptides discovered; however, the first family members characterized were all targeted to the nicotinic receptor subtype at the neuromuscular junction (see Ref. 124 for a review). Beginning with the discovery of -conotoxin ImI, McIntosh et al. (125) have characterized individual -conotoxins that are selectively targeted to diverse nicotinic receptor subtypes. Some -conotoxins of potential importance for basic neuroscience research or with potential for clinical development are shown in Table 3.
6–46 channels. Two-electrode voltage-clamp recordings from an oocyte expressing Shaker-