Conus Geographus Research Articles

Conantokin-G: A novel peptide antagonist to the [formula omitted] acid (NMDA) receptor

Conantokin-G is a 17 amino acid peptide isolated from the venom of the fish-eating snail Conus geographus which produces hyperactivity when injected into the brains of adult mice. We show that this peptide is a selective N- methyl- d-aspartate (NMDA) antagonist based on its ability to block NMDA-induced elevation of cGMP in rat cerebellar slices in vitro (IC 50 = 171 nM), but not kainic acid-induced elevations. This inhibition could not be overcome by increasing the NMDA concentration, indicating non-competitive inhibition. Conantokin-G displayed no affinity for binding sites for thienylcyclohexylpiperidine, various glutamate subclasses or those for several other neurotransmitters/neuromodulators. This peptide, however, enhanced [ 3H]glycine binding to rat forebrain membranes but not to spinal cord membranes. The activity profile of the peptide in various assays indicates that it is a novel type of non-competitive NMDA antagonist.

Conotoxin GIIIA: selective inhibition of 22Na influx via voltage-dependent Na channels in adrenal medullary cells

Conotoxin GIIIA and GIIIB from the marine snail Conus geographus have been reported to inhibit voltage-dependent Na channels in skeletal muscle and postganglionic sympathetic neuron, but have no effect on Na channels in brain, giant axon and heart. In eel electroplax, conotoxins were also shown to share the common binding sites with saxitoxin (see review Gray et al. 1988). In bovine adrenal medullary cells, conotoxin GIIIA inhibited veratridine-induced influx of 22Na, 45Ca and secretion of catecholamines with an IC50 of 6 mumols/l while saxitoxin suppressed veratridine-induced responses with an IC50 of 6.3 nmol/l. [3H]Saxitoxin binding to the cells was inhibited by unlabeled saxitoxin with an IC50 of 5.1 nmol/l, but was slightly reduced by 10 mumols/l conotoxin GIIIA. Conotoxin GIIIA, at 10 mumols/l, did not alter carbachol-induced influx of 22Na, 45CA and secretion of catecholamines as well as high K-induced 45Ca influx and catecholamine secretion. These results indicate that conotoxin GIIIA, at concentrations 950 fold higher than saxitoxin, inhibits Na influx via voltage-dependent Na channels, but has no effect on the nicotinic receptor-ion channel complex or the voltage-dependent Ca channels. Conotoxin GIIIA seems to bind at the sites which are distinct from saxitoxin, but are functionally linked to the voltage-dependent Na channels. Conotoxins may be useful for the classification of Na channels in excitable cell membranes.

Diversity of Conus Neuropeptides

Conus venoms contain a remarkable diversity of pharmacologically active small peptides. Their targets are ion channels and receptors in the neuromuscular system. The venom of Conus geographus contains high-affinity peptides that act on voltage-sensitive calcium channels, sodium channels, N-methyl-D-aspartate (NMDA) receptors, acetylcholine receptors, and vasopressin receptors; many more peptides with still uncharacterized receptor targets are present in this venom. It now seems that the Conus species (approximately 500 in number) will each use a distinctive assortment of peptides and that the pharmacological diversity in Conus venoms may be ultimately comparable to that of plant alkaloids or secondary metabolites of microorganisms. The cone snails may generate this diverse spectrum of venom peptides by a "fold-lock-cut" synthetic pathway. These peptides are specific enough to discriminate effectively between closely related receptor subtypes and can be used for structure-function correlations.

Disulfide pairings in geographutoxin I, a peptide neurotoxin from Conus geographus

The three intramolecular disulfide linkages of geographutoxin I, a peptidic neurotoxin isolated from the venom of the marine snail Conus geographus, were examined by a novel method for determination of the positions of disulfide linkages in peptides [(1989) Bull. Chem. Soc. Jp. 62, 1986–1994]. The disulfide bridges were found to be between Cys 3 and Cys 15, Cys 4 and Cys 20, and Cys 10 and Cys 21, indicating that geographutoxin I has a rigid conformation consisting of three loops stabilized by these three disulfide linkages.

Monoclonal antibodies against the presynaptic calcium channel antagonist ω-conotoxin GVI A from cone snail poison

Monoclonal antibodies have been prepared against co-conotoxin GVI A, a peptide isolated from marine snails of the genus Conus (Conus geographus and Conus magus). This toxin is a blocker of select presynaptic Ca 2+ channels in the central nervous system. Antigenic ω-conotoxin GVI A was synthesized as a covalent conjugate with bovine serum albumin and injected s.c. An ELISA assay combined with a competitive inhibition assay was used to select and characterize monoclonal antibodies able to recognize and bind the free toxin. Several of the antibodies were found to block ω-conotoxin GVI A inhibition of 45Ca transport into rat brain synaptosomes and to block ω-conotoxin GVI A binding to membranes from the same preparation. The antibodies recognize native, synthetic toxin, and are useful for analysis of toxin in biological fluids.

Selective neuromuscular blocking properties of α-conotoxins In vivo

Synthetic α-conotoxins GI and MI (based on the natural polypeptides isolated from Conus geographus and C. magus, respectively) were tested for effects on neuromuscular transmission and on the cardiovascular system in anaesthetised cats. Both produced neuromuscular blockade at doses of 20–80 μg/kg. Conotoxin GI was about 2.5 times more potent than MI. Both were rapidly reversed by the anticholinesterase neostigmine. At doses that abolished neuromuscular transmission, the α-conotoxins had no effects on arterial blood pressure, heart rate, or responses to vagal and preganglionic stimulation, indicating their specificity for nicotinic receptors at the neuromuscular junction.

Presynaptic α 2-adrenoceptor and κ-opiate receptor occupancy promotes closure of neuronal (N-type) calcium channels

Synaptosomes prepared from rat cerebral cortex by homogenization in isotonic sucrose and centrifugation on four-step discontinuous percoll density gradients were loaded with the fluorescent indicator fura-2 to allow measurement of intrasynaptosomal free calcium concentrations ([Ca 2+] i). Incubation of fura-2 loaded synaptosomes with either the κ-opiate agonist U-50,488H(0.1–100 μM) or the α 2-adrenoceptor agonist clonidine (0.1–100 μM), resulted in a dose-dependent reduction in [Ca 2+] i and these changes were completely antagonised by prior inclusion of naloxone (20 μM) or idazoxan (RX781094) (2 μM) respectively. When the 1,4-dihydropyridine Ca 2+-channel blocker nifedipine (1 μM) was incubated with synaptosomes for 1 min, there was a 17.0% decrease in [Ca 2+] i and when it was combined with either U-50,488H (1 μM) or clonidine (1 μM) there was a reduction in [Ca 2+] i of 35.0 and 48.1% respectively i.e. the effects were additive. The increase in the depression of [Ca 2+] i produced by these drug combinations were antagonised by the inclusion of naloxone (20 μM) or idazoxan (2 μM) which resulted in decrease in free [Ca 2+] i of 26.5 and 14.1% respectively. These data indicate that the effects of clonidine and U-50,488H are not mediated by L-type Ca 2+ channels. When synaptosomes were treated for 1 min with a low concentration ω-conotoxin fraction GVIA from Conus geographus (ω-CgTx), (93 nM) there was a 15.7% decrease in [Ca 2+] i and when they were co-incubated with ω-CgTx (93 nM) plus either U-50,488H (1 μM) or clonidine (1 μM); the reduction in [Ca 2+] i was 11.4 and 18.3% respectively i.e. there was no apparent additive effect on the reduction in [Ca 2+] i. The decrease in [Ca 2+] i following treatment of synaptosomes with ω-CgTx (93 nM) (which is reported to block both L- and N-type Ca 2+ channels) was surprising since the value was not greater than that with nifedipine (a blocker of only L-type Ca 2+ channels). However, addition of nifedipine (0.01–10.0 μM) to synaptosomes preincubated with the low concentration of ω-CgTx (93 nM) showed an additional reduction in [Ca 2+] i which suggest that at low concentrations ω-CgTx may not be a good blocker of L-type Ca 2+ channels. This was confirmed by studies in which synaptosomes were preincubated with a higher concentration of ω-CgTx (1.0 μM): under these circumstances no further reduction in [Ca 2+] i could be detected when nifedipine was added (0.10–10.0 μM). On the basis of the observations above we conclude that U-50,488H and clonidine elicit their effects on [Ca 2+] i and hence neurotransmitter release, by closing N-type Ca 2+ channels. In addition, our studies indicate that ω-CgTx at low concentration, is a more potent blocker at N-type rather than L-type Ca 2+ channels.

Calcium channels in solitary retinal ganglion cells from post‐natal rat.

1. Calcium currents from identified, post-natal retinal ganglion cell neurones from rat were studied with whole-cell and single-channel patch-clamp techniques. Na+ and K+ currents were suppressed with pharmacological agents, allowing isolation of current carried by either 10 mM-Ca2+ or Ba2- during whole-cell recordings. For cell-attached patch recordings, the recording pipette contained 96-110 mM-BaCl2 while the bath solution consisted of isotonic potassium aspartate in order to zero the neuronal membrane potential. 2. A transient component, present in approximately one-third of the whole-cell recordings resembles closely the T-type calcium current observed previously in other tissues. This component activates at low voltages (-40 to -50 mV from holding potentials negative to -80 mV), inactivates with a time constant of 10-30 ms at 35 degrees C, and is carried equally well by Ba2+ or Ca2+. In single-channel recordings small (8 pS) channels are observed whose aggregate microscopic kinetics correspond well to the macroscopic current obtained during whole-cell measurements. 3. During whole-cell recordings, a more prolonged component activates in all retinal ganglion cells at -40 to -20 mV from a holding potential of -90 mV. This component is substantially larger when equimolar Ba2+ replaces Ca2+ as the charge carrier, and is sensitive to the dihydropyridine agonist Bay K8644 (5 microM) and antagonists nifedipine (1-10 microM) and nimodipine (1-10 microM). Thus, the dihydropyridine pharmacology of this prolonged component resembles that of the L-type calcium current found in dorsal root ganglion neurones and in heart cells. Also reminiscent of the L-current, the prolonged component in this preparation is less inactivated at depolarized holding potentials (-60 to -40 mV) than the transient component. In cell-attached recordings, large (20 pS) channels are observed with activation properties similar to those of the prolonged portion of the whole-cell current. 4. omega-Conotoxin fraction GVIA (omega-CgTX VIA), a peptide from the venom of the snail Conus geographus, produces a readily reversible blockade of all components of the calcium current in these central mammalian neurones. This finding is in contrast to that of other preparations in which this toxin is responsible for an ephemeral block of T-current but a long-lasting block of other components of calcium current. 5. In summary, at least two components of calcium current with discrete underlying unitary events are present in post-natal retinal ganglion cells from rat. One component closely resembles the T or transient current observed in other cell types.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of a locality in snake venomα-neurotoxins with a singificant compositional similarity to marine snailα-conotoxins: Implications for evolution and structure/activity

alpha-neurotoxins from elapid snake venoms and alpha-conotoxins from marine snails bind specifically and with high affinity to nicotinic cholinoceptors. Although both types of toxin are polypeptides, there is more than a fourfold difference in size between the two and no clear sequence homology is evident. A systematic computer search of the three-dimensional structure of erabutoxin b (an alpha-neurotoxin from the false sea snake Laticauda semifasciata) was performed to identify the locality that most closely matched the amino acid compositions of the smaller alpha-conotoxins (from the marine snails Conus magus and Conus geographus). The area of greatest similarity centered on residue position 25 of erabutoxin b, a locale that is conserved throughout the snake alpha-neurotoxins and their homologues. Six proteins unrelated to erabutoxin b were compared to the alpha-conotoxins to show that the extent of the erabutoxin b/alpha-conotoxin match was too high to be coincidental. Homologues of erabutoxin b, namely alpha-cobratoxin from Naja naja siamensis and cytotoxin VII4 from Naja mossambica mossambica, were also analyzed. The extent of the matching with the alpha-conotoxins decreased in the series erabutoxin b greater than alpha-cobratoxin greater than cytotoxin VII4, and this also relates the order of similarity to the pharmacological properties of the alpha-conotoxins. The alpha-conotoxin-like area of the snake alpha-neurotoxins is peripheral to the site previously considered important for binding to the cholinoceptor, even though it seems to represent the focus of evolutionary convergence between the two types of neurotoxin. The area of resemblance does, however, have strong associations with the conformational behavior of the snake toxins. Hence, the outcome of this study has important consequences for the current ideas on snake alpha-neurotoxin structure/activity relationships and the evolutionary origins of neurotoxicity.

Geographutoxin-sensitive and insensitive sodium currents in mouse skeletal muscle developing in situ.

1. The whole-cell voltage-clamp technique was used to examine developmental changes of Na+ current properties in single fibres of mouse flexor digitorum brevis muscles developing in situ from birth to 20 days post-natal. 2. Geographutoxin II (GTX II), a novel polypeptide toxin from the marine snail Conus geographus, distinguished two different types of voltage-sensitive Na+ currents: GTX II-sensitive and GTX II-insensitive currents, which corresponded respectively to currents with high or low TTX sensitivity. 3. Voltage-dependent activation and inactivation of the GTX II-insensitive currents occurred at membrane potentials 10-20 mV more negative than those for the GTX II-sensitive currents. 4. The GTX II-insensitive current in fibres from mice older than 8 days inactivated more slowly than the GTX II-sensitive current. However, in fibres from younger mice, the two currents decayed with similar speed. 5. The mean specific Na+ conductance (gNa) for the total (GTX II-sensitive plus GTX II-insensitive) Na+ channels was 0.22 mS/muF at a Na+ concentration of 5 mM at birth. The total gNa increased 6-fold to 1.32 mS/muF during the first 20 days after birth. 6. The mean specific gNa for the GTX II-insensitive channels was 0.15 mS/muF at birth, remained at approximately the same level for the first 8 days, and then decreased progressively to become undetectable by day 16. 7. In muscle fibres denervated 12 days after birth, the GTX II-insensitive gNa increased over the next 8 days, whereas the total gNa increased less than normal. 8. By contrast, in fibres denervated on day 4, the total gNa increased more than normal in the following 8 days, and the GTX II-insensitive specific gNa increased above the level seen at birth. 9. Half-maximal activation and inactivation potentials of the total and the GTX II-insensitive currents shifted in the negative direction by 9-17 mV in the first 8 days after birth. 10. We conclude that the regulatory effects of innervation on the total gNa are either suppressive or enhancing depending on the stage of development. On the other hand, denervation elicits an increase in GTX II-insensitive Na+ currents at all ages studied.

Omega-conotoxin GVIA specifically blocks neuronal mechanisms in rat ileum

Omega Conotoxin is a peptide venom of the marine snail Conus geographus which has been postulated as a neural specific calcium channel blocker in several systems. In the present study in rat ileum strips omega-conotoxin GVIA specifically blocked neuronal responses evoked by electrical field stimulation in a dose-dependent manner, but did not block responses induced by carbachol or direct depolarization with KCl. On the other hand the dyhydropyridine calcium channel blocker nitrendipine (10 −7 M) showed no specificity and blocked electrical field stimulation as well as carbachol- and KCl-induced contractions. This indicates that contractions of intestinal smooth muscle which can be blocked by dihydropyridine calcium channel blockers or Ca 2+-free medium are not reduced by the neurotoxin omega-conotoxin. Omega-conotoxin caused even a little increase of the responses elicited by depolarisation with KCL. Omega-conotoxin blocked however neural mediated contractions which might suggest that it interacts with a distinct neuronal calcium channel and, in addition to tetrodotoxin, could provide a useful pharmacological tool to discriminate between muscular and neural sites of action of excitatory or inhibitory agents.

.mu.-Conotoxin GIIIA, a peptide ligand for muscle sodium channels: chemical synthesis, radiolabeling and receptor characterization

The peptide conotoxin GIIIA from Conus geographus L. venom, which specifically blocks sodium channels in muscle, has been synthesized by a solid-phase method. The three disulfide bridges were formed by air oxidation. After HPLC purification, the synthetic product was shown to be identical with the native conotoxin GIIIA from Conus geographus. A high specific activity, 125I derivative of mu-conotoxin was prepared and used for binding assays to the Na channel from Electrophorus electric organ. Specific binding could be abolished by competition with tetrodotoxin. The radiolabeled toxin was specifically cross-linked to the Na channel. These studies demonstrate that mu-conotoxin GIIIA can be used to define the guanidinium toxin binding site and will be a useful ligand for understanding functionally important differences between Na channel subtypes.

Intrasynaptosomal free calcium concentration is increased by phorbol esters via a 1,4-dihydropyridine-sensitive (L-type) Ca 2+ channel

Incubation of non-depolarised fura-2-loaded rat cortical synaptosomes with 12-tetradecanoylphorbol-13-monoacetate (TPA) results in a dose-dependent increase in calcium concentration (to a maximum of 140%). It is dependent on extrasynaptosomal Ca 2+, is partially blocked by 1 μM verapamil and effectively blocked by 100 μM verapamil (⩾90%). Nifedipine (1 μM), nicardipine (1 μM) and ω-conotoxin fraction GVIA from Conus geographus (50 nM) (ω-CgTx) also cause blockade (⩾ 90%) of the increase. The sensitivity of the TPA-induced increase in calcium concentration to ω-CgTx, nicardipine and nifedipine, but not to low concentrations of verapamil (1 μM), suggests that the TPA-induced rise in calcium concentration is mediated by increased Ca 2+ influx through 1,4-dihydropyridine-sensitive Ca 2+ channels. Incubation of synaptosomes with the inactive phorbol ester phorbol-13-monoacetate (TMA) does not result in any significant dose-dependent increase in calcium concentration. The data which are presented are consistent with (i) the proposal that phorbol ester-induced increases in calcium concentration are the result of Ca 2+ influx through an L-type Ca 2+ channel and (ii) the existence of functioning L-type Ca 2+ channels on rat brain synaptosomes.

Imaging and Correlative Physicochemical Techniques

A novel peptide toxin, which causes a sleep-like state upon intracerebral injection in mice, has been purified to homogeneity from the venom of the piscivorous marine snail Conus geographus L. It elicits no obvious effects when injected i.p. into either mice or fish. The purified toxin is a highly acidic heptadecapeptide with no cystine residues (Lys1, Arg1, Asx2, Ser1, Glx7–8, Gly1, Ile1, Leu2). This composition is in marked contrast to those of other conotoxins, which are basic and disulphide-bridged. The N-terminal residue is Gly and the COOH-terminal sequence is Ser-Asn-NH2.

A novel sodium channel inhibitor from Conus geographus: purification, structure, and pharmacological properties.

A novel toxin, tentatively named conotoxin GS (CGS), has been isolated from a marine snail, Conus geographus. CGS was found to exist as a single polypeptide chain, consisting of 34 amino acid residues, cross-linked by three disulfide bonds. Its amino acid sequence was shown to be Ala-Cys-Ser-Gly-Arg-Gly-Ser-Arg-Cys-Hyp-Hyp-Gln-Cys-Cys-Met-Gly-Leu-Arg- Cys-Gly - Arg-Gly-Asn-Pro-Gln-Lys-Cys-Ile-Gly-Ala-His-Gla-Asp-Val. In competition experiments, CGS inhibited the bindings of [3H]Lys-tetrodotoxin ([3H]Lys-TTX) and [3H]propionylconotoxin GIIIA to Electrophorus electricus electroplax membranes, with Ki values of 34 nM and 24 nM, respectively. The toxin inhibited the binding of [3H]Lys-TTX (1 nM) to rat skeletal muscle homogenates with an IC50 value of 880 nM but showed very little effect on this binding to the rat brain P2 fraction at 10 microM. These binding studies indicate that CGS belongs to the same group of Na channel inhibitors as TTX, STX (saxitoxin), and mu-conotoxins. Although CGS, like the mu-conotoxins, is a pharmacological probe for distinguishing between neuronal and muscle Na channel subtypes, the homology in the sequences of CGS and mu-conotoxins is very limited.

Total synthesis and further characterization of the gamma-carboxyglutamate-containing "sleeper" peptide from Conus geographus venom.

The total synthesis of the Gla-containing "sleeper" peptide (Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2 ) from Conus geographus is described. A new strategy for the synthesis of acid-sensitive peptide amides was developed, which allowed complete deprotection and cleavage of the L-gamma-carboxyglutamate-containing peptide from the 2,4-dimethoxybenzhydrylamine resin. Synthetic sleeper peptide, after preparative high-performance liquid chromatography (HPLC) purification, was shown to be identical with the native peptide by all criteria (coelution experiments of HPLC, sequence analysis, and biological activity). In addition, a developmental switch in the behavioral symptoms induced by the peptide after intracerebral administration in mice was documented. At low doses of the peptide (4-30 pmol/g), a sleeplike state was induced in mice under 2 weeks old; in contrast, older mice became markedly hyperactive. It is proposed that, in the presence of Ca2+, the sleeper peptide assumes an alpha-helical configuration in which all the gamma-carboxyglutamate residues are located on the same side of the alpha-helix.

Invertebrate vasopressin/oxytocin homologs. Characterization of peptides from Conus geographus and Conus straitus venoms.

The vasopressin-oxytocin family of peptides is of very ancient lineage, found in organisms as diverse as hydra and man. Although these peptides have been intensively studied in vertebrates, the presumably more extensive invertebrate series was defined primarily by immunological methods. In this report, we describe the purification and structures of two peptides of the vasopressin-oxytocin family from molluscs ("Conopressins"), which were found in the venom of fish-hunting marine snails of the genus Conus. The biological activity observed when the two snail peptides are injected intracerebrally into mice is very similar to that elicited by the vertebrate neurohypophyseal hormones and presumably reflects their actions upon a common receptor in the brain. The sequences of the purified peptides reveal unique features not found in the vertebrate peptide series, most notably an additional positive charge. These are the first members of the invertebrate series of the vasopressin-oxytocin family to be characterized biochemically. The sequences of these peptides are: from Conus geographus venom, Lys-conopressin-G, Cys-Phe-Ile-Arg-Asn-Cys-Pro-Lys-Gly-NH2; and from Conus striatus venom, Arg-conopressin-S, Cys-Ile-Ile-Arg-Asn-Cys-Pro-Arg-Gly-NH2.

The Conus toxin geographutoxin IL distinguishes two functional sodium channel subtypes in rat muscle cells developing in vitro

Sodium currents in cultured rat muscle cells converted to myoballs by treatment with colchicine were recorded using a giga-ohm seal voltage-clamp procedure in the whole-cell configuration. Geographutoxin II (GTX II), a novel polypeptide toxin from the piscivorous marine snail Conus geographus, reduces sodium currents in rat myoballs without marked alteration of the time course or voltage dependence of activation of the remaining current. Titration of the inhibition of sodium currents by GTX II showed that, in individual myoballs, a fraction of the sodium current averaging 49 +/- 9% (SEM) was inhibited by saturating (25 microM) concentrations of GTX II. The concentration-effect curve fit a noncooperative, 1:1 binding isotherm with a single KD for GTX II of 19 nM characteristic of inhibition of the TTX-sensitive sodium channels of adult rat muscle. Titration of the sodium current remaining in the presence of 2.5 microM GTX II with TTX gave complete inhibition. The dose-response curve fit a noncooperative, 1:1 binding isotherm with a single KD for TTX of 1.3 microM characteristic of TTX-insensitive sodium channels of embryonic muscle. The action of GTX II was not frequency dependent. The all-or-none inhibition of these 2 sodium channel subtypes by GTX II suggests substantial structural differences in the region of neurotoxin receptor site 1 on TTX-sensitive and -insensitive sodium channels and provides definitive evidence that these 2 sodium channel subtypes function in parallel in muscle cells developing in the absence of innervation.

Omega-conotoxin: direct and persistent blockade of specific types of calcium channels in neurons but not muscle.

Blockade of Ca2+ channels by omega-conotoxin GVIA, a 27 amino acid peptide from the venom of the marine snail Conus geographus, was investigated with patch-clamp recordings of whole-cell and unitary currents in a variety of cell types. In dorsal root ganglion neurons, the toxin produces persistent block of L- and N-type Ca2+ channels but only transiently inhibits T-type channels. Its actions appear to be neuron-specific, since it blocks high-threshold Ca2+ channels in sensory, sympathetic, and hippocampal neurons of vertebrates but not in cardiac, skeletal, or smooth muscle cells. Block occurs through direct interaction of the toxin with an external site closely associated with the Ca2+ channel, without apparent involvement of a second messenger or dependence on channel gating. The tissue and channel-type specificity and the directness and slow reversibility of the block are features that favor use of omega-conotoxin as a tool for purifying particular neuronal Ca2+ channels and defining their physiological function.

Mu-conotoxins share a common binding site with tetrodotoxin/saxitoxin on eel electroplax Na channels

The binding characteristics of conotoxin GIIIA purified from the venom of a marine snail, Conus geographus, with regard to electroplax membranes from Electrophorus electricus were studied using a radiolabeled monopropionyl derivative of the toxin (3H-Pr-CGIIIA). 3H-Pr-CGIIIA bound specifically to a single class of saturable binding sites in electroplax membranes with a dissociation constant of 1.1 +/- 0.2 nM and a maximal binding capacity of 11 +/- 2 pmol/mg of protein. The latter value was similar to the number of specific binding sites (10 +/- 2 pmol/mg of protein) for 3H-lysine-tetrodotoxin (3H-Lys-TTX). Monopropionyl CGIIIA and CGIIIA had similar inhibitory effects on the binding of 3H-Lys-TTX (1 nM) to electroplax membranes with IC50 values of 3.5 and 0.9 nM, respectively. The association and dissociation of 3H-Pr-CGIIIA and electroplax membranes were much slower than those of 3H-Lys-TTX and the membranes. Mu-Conotoxins (CGIIIA and CGIIIB) and guanidinium toxins (TTX and saxitoxin) inhibited 3H-Pr-CGIIIA (1 nM) binding to electroplax membranes with IC50 values of 0.6, 1.1, 7.1, and 2.2 nM, respectively. However, several other kinds of neurotoxins and local anesthetics known to interact with Na channels did not affect 3H-Pr-CGIIIA binding. These findings indicate that mu-conotoxins must be classified in the same group of Na channel inhibitors as guanidinium toxins, since they competed with guanidinium toxins for binding sites on the Na channel. The peptide mu-conotoxins should be useful in studies on the functional and structural domains of Na channel proteins.