Gambierol acts as a functional antagonist of neurotoxin site 5 on voltage-gated sodium channels in cerebellar granule neurons.

The marine toxin gambierol, a polyether ladder toxin derived from the marine dinoflagellate Gambierdiscus toxicus, was evaluated for interaction with voltage-gated sodium channels (VGSCs) in cerebellar granule neuron (CGN) cultures. At concentrations ranging from 10 nM to 10 microM, gambierol alone had no effect on the intracellular Ca2+ concentration [Ca2+]i of exposed CGN cultures. Furthermore, there was no evidence of neurotoxicity in CGN cultures exposed for 2 h to gambierol (1 nM-10 microM). However, gambierol was a potent inhibitor (IC50 = 189 nM) of the elevation of [Ca2+]i that accompanies exposure of CGN cultures to the VGSC activator brevetoxin-2 (PbTx-2). To further explore the potential interaction of gambierol with VGSCs, the influence of gambierol on PbTx-2-induced neurotoxicity was assessed. Gambierol reduced the PbTx-2-induced efflux of lactate dehydrogenase in exposed CGN cultures in a concentration-dependent manner (IC50 = 471 nM). It is noteworthy that the potencies of gambierol as an inhibitor of both PbTx-2-induced Ca2+ influx and cytotoxicity were coincident. Finally, the inhibitory effects of gambierol on PbTx-2-induced elevation of [Ca2+]i were compared with those of brevenal, a natural inhibitor of the toxic effects of brevetoxin isolated from cultures of Karina brevis. Like gambierol, brevenal inhibited PbTx-2-induced elevation of [Ca2+]i in a concentration-dependent manner (IC50 = 108.6 nM). These results provide evidence for gambierol acting as a functional antagonist of neurotoxin site 5 on neuronal VGSCs.

The inhibition of CHO-K1-BH4 cell proliferation and induction of chromosomal aberrations by brevetoxins in vitro.

Brevetoxins (PbTxs) are highly potent trans-syn polyether neurotoxins produced during blooms of several species of marine dinoflagellates, most notably Karenia brevis. These neurotoxins act on voltage-sensitive sodium channels prolonging the active state. During red tides, the commercial fishing and tourism industries experience millions of dollars of lost revenue. Human consumption of shellfish contaminated with PbTxs results in neurotoxic shellfish poisoning (NSP). Additionally, blooms of K. brevis are potentially responsible for adverse human health effects such as respiratory irritation and airway constriction in coastal residents. There is little information regarding the full range of potential toxic effects caused by PbTxs. Recent evidence suggests that PbTxs are genotoxic substances. The purpose of this study was to determine if PbTxs could induce chromosomal aberrations and inhibit cellular proliferation in CHO-K1-BH4 cells, and if so, could the damage be negated or reduced by the PbTx antagonist brevenal. Results from the chromosomal aberrations assay demonstrated that PbTxs are potent inducers of CHO-K1-BH4 chromosome damage. Results from the inhibition of cellular proliferation assays demonstrated that PbTxs inhibit the ability of CHO-K1-BH4 cells to proliferate, an effect which can be reduced with brevenal.

Florida Red Tide: Inhalation Toxicity of Karenia brevis Extract in Rats.

Brevetoxins are neurotoxins produced by the marine dinoflagellate Karenia brevis. Histopathologic examination of marine mammals dying following repeated exposure of brevetoxins during red tide events suggests that the respiratory tract, nervous, hematopoietic, and immune systems are potential targets for toxicity in repeatedly exposed individuals. The purpose of this experiment was to evaluate the effects of repeated inhalation of K. brevis extract on these potential target systems in rats. Male Sprague-Dawley rats were exposed four hours/day, five days/week for up to four weeks to target concentrations of 200 and 1000 µg/L K. brevis extract (approximately 50 and 200 µg/L brevetoxin-like compounds; positive neurotoxicity in a fish bioassay). Control rats were sham exposed to air. Immunohistochemical staining of pulmonary macrophages indicated deposition of brevetoxin-like compound within the lung. However, exposure resulted in no clinical signs of toxicity or behavioral changes. There were no adverse effects on hematology or serum chemistry. No histopathological changes were observed in the nose, lung, liver, kidneys, lymph nodes, spleen, or brain of exposed rats. Immune suppression was suggested by reduced responses of spleen cells in the IgM-specific antibody-forming plaque cell response assay and reduced responses of lymphocytes to mitogen stimulation in vitro. Differences between responses observed in rats in this study and those observed in manatees may be a function of dose or species differences in sensitivity.

Brevetoxin Depuration in Shellfish via Production of Non-toxic Metabolites: Consequences for Seafood Safety and the Environmental Fate of Biotoxins.

During blooms of the dinoflagellate Karenia brevis, filter-feeders such as oysters and clams bioaccumulate brevetoxins, often to levels that are toxic to humans. In controlled aquarium experiments, we exposed live oysters to bloom levels of toxic K. brevis, followed by 10 weeks of exposure to non-toxic microalgae. Oysters were harvested weekly and analyzed for brevetoxins and brevetoxin metabolites to quantify toxin bioaccumulation and depuration. All of the PbTx-2 concentrated by oysters was immediately converted to a mixture of polar metabolites that were then slowly eliminated from the oysters. However, 90% of measured PbTx-3 was eliminated within two weeks of toxic exposure but without apparent biotransformation. Extracts of oysters containing high levels of PbTx-3 were toxic to mice by intraperitoneal (IP) injection. Extracts of oysters harvested after PbTx-3 had been eliminated were non-toxic despite high concentrations of PbTx-2 metabolites. Oysters collected in Florida during and after a bloom of K. brevis contained polar metabolites of PbTx-2 as well as PbTx-3, but no PbTx-2. Again, PbTx-3 concentration was a good predictor of mouse toxicity. One hundred percent conversion of PbTx-2 to polar metabolites was also accomplished in vitro by spiking oyster or clam homogenate with PbTx-2, followed by a brief incubation at room temperature. These PbTx-2 metabolites did not kill mice, either orally or by intraperitoneal injection, even at concentrations 30 times greater than toxic PbTx-3 levels.

Brevetoxin derivatives act as partial agonists at neurotoxin site 5 on the voltage-gated Na+ channel.

Brevetoxins (PbTx-1 to PbTx-10) are potent lipid-soluble polyether neurotoxins produced by the marine dinoflagellate Karina brevis, an organism associated with 'red tide' blooms in the Gulf of Mexico. Ingestion of shellfish contaminated with K. brevis produces neurotoxic shellfish poisoning (NSP) in humans. NSP symptoms emanate from brevetoxin activation of neurotoxin site 5 on voltage-gated sodium channels (VGSC) [Toxicon 20 (1982) 457]. In primary cultures of rat cerebellar granule neurons (CGN), brevetoxins produce acute neuronal injury and death. The ability of a series of naturally occurring and synthetic brevetoxins to trigger Ca(2+) influx in CGN was explored in the present study. Intracellular Ca(2+) concentration was monitored in fluo-3-loaded CGN using a fluorescent laser imaging plate reader. The naturally occurring derivatives PbTx-1, PbTx-2 and PbTx-3 all produced a rapid and concentration-dependent increase in cytosolic [Ca(2+)]. The maximum response to PbTx-1 was approximately two-fold greater than that of either PbTx-2 or PbTx-3. Two synthetic derivatives of PbTx-3, alpha-naphthoyl-PbTx-3 and beta-naphthoyl-PbTx-3, were also tested. Both alpha- and beta-naphthoyl-PbTx-3 stimulated a rapid and concentration-dependent Ca(2+) influx that was, however, less efficacious than that of PbTx-3. These data indicate that, analogous to neurotoxin site 2 ligands, activators of neurotoxin site 5 display a range of efficacies, with PbTx-1 being a full agonist and other derivatives acting as partial agonists.

Are Pfiesteria species toxicogenic? Evidence against production of ichthyotoxins by Pfiesteria shumwayae.

The estuarine genus Pfiesteria has received considerable attention since it was first identified and proposed to be the causative agent of fish kills along the mid-Atlantic coast in 1992. The presumption has been that the mechanism of fish death is by release of one or more toxins by the dinoflagellate. In this report, we challenge the notion that Pfiesteria species produce ichthyotoxins. Specifically, we show that (i) simple centrifugation, with and without ultrasonication, is sufficient to "detoxify" water of actively fish-killing cultures of Pfiesteria shumwayae, (ii) organic extracts of lyophilized cultures are not toxic to fish, (iii) degenerate primers that amplify PKS genes from several polyketide-producing dinoflagellates failed to yield a product with P. shumwayae DNA or cDNA, and (iv) degenerate primers for NRPS genes failed to amplify any NRPS genes but (unexpectedly) yielded a band (among several) that corresponded to known or putative PKSs and fatty acid synthases. We conclude that P. shumwayae is able to kill fish by means other than releasing a toxin into bulk water. Alternative explanations of the effects attributed to Pfiesteria are suggested.

Brevetoxin derivatives that inhibit toxin activity.

BACKGROUND: The brevetoxins are marine neurotoxins that interfere with the normal functions of the voltage-gated Na(+) channel. We have identified two brevetoxin derivatives that do not exhibit pharmacological properties typical of the brevetoxins and that function as brevetoxin antagonists. RESULTS: PbTx-3 and benzoyl-PbTx-3 elicited Na(+) channel openings during steady-state depolarizations; however, two PbTx-3 derivatives retained their ability to bind to the receptor, but did not elicit Na(+) channel openings. alpha-Naphthoyl-PbTx-3 acted as a PbTx-3 antagonist but did not affect Na(+) channels that were not exposed to PbTx-3. beta-Naphthoyl-PbTx-3 reduced openings of Na(+) channels that were not exposed to PbTx-3. CONCLUSIONS: Some modifications to the brevetoxin molecule do not alter either the binding properties or the activity of these toxins. Larger modifications to the K-ring sidechain do not interfere with binding but have profound effects on their pharmacological properties. This implies a critical function for the K-ring sidechain of the native toxin.

Uptake, tissue distribution, and excretion of brevetoxin 3 administered to rats by intratracheal instillation.

Brevetoxins are cyclic polyether neurotoxins produced by the marine dinoflagellate Ptychodiscus brevis. Blooms of P. brevis (red tides) are toxic to fish, marine mammals, and humans. Humans exposed to seaspray aerosols containing brevetoxins may experience respiratory tract irritation. Because a major route of human exposure to brevetoxins is via the respiratory tract, the objective of this study was to examine the toxicokinetics of brevetoxin 3 (PbTx-3) administered to the lung by intratracheal instillation. Twenty-one male F344/Crl BR rats, 12 wk of age, were administered 3H-PbTx-3 (1 microCi, 6.6 microg PbTx-3/kg) by intratracheal instillation. Groups of 3 rats were sacrificed at 0.5, 3, 6, 24, 48, and 96 h after exposure, and tissues were collected. Three additional rats were placed in glass metabolism cages for collection of urine and feces over a 7-d period. PbTx-3-associated activity was cleared rapidly from the lung and distributed throughout the body, chiefly to the carcass, intestines, and liver. Blood, brain, and fat contained the lowest percentages of the administered dose. Although a majority of the PbTx-3 was cleared rapidly from lung, liver, and kidneys, approximately 20% of the initial concentration present in each organ was retained for 7 d. Concentrations of PbTx-3 in brain and fat were low, but remained relatively constant over time. Approximately twice as much PbTx-3-associated activity was excreted in feces than in urine, with the majority of excretion occurring within 48 h after instillation. The results of this study indicate that over 80% of the PbTx-3 is rapidly absorbed from the lung to the blood and distributed to all tissues. The tissues containing the greatest amount of PbTx-3-associated activity reflect the compound's site of deposition, storage compartment, and major route of metabolism and excretion. These results illustrate that brevetoxin exposure by the respiratory route results in systemic distribution of brevetoxin and suggest that the initial respiratory irritation and bronchoconstriction may only be a part of the overall toxicological consequences associated with brevetoxin inhalation.

Brevetoxin modulates neuronal sodium channels in two cell lines derived from rat brain.

Single Na+ channel currents were recorded from cell-attached membrane patches from two neuronal cell lines derived from rat brain, B50 and B104, and compared before and after exposure of the cells to purified brevetoxin, PbTx-3. B50 and B104 Na+ channels usually exhibited fast activation and inactivation as is typical of TTX-sensitive Na+ channels. PbTx-3 modified channel gating in both cell lines. PbTx-3 caused (1) significant increases in the frequency of channel reopening, indicating a slowing of channel inactivation, (2) a change in the voltage dependence of the channels, promoting channel opening during steady-state voltage clamp of the membrane at voltages throughout the activation range of Na+ currents, but notably near the resting potential of these cells (-60 - -50 mV), and (3) a significant, 6.7 mV hyperpolarized shift in the threshold potential for channel opening. Na+ channel slope conductance did not change in PbTx-3-exposed B50 and B104 neurons. These effects of Pbx-3 may cause hyperexcitability as well as inhibitory effects in intact brain.

Brevetoxicosis in manatees (Trichechus manatus latirostris) from the 1996 epizootic: gross, histologic, and immunohistochemical features.

In 1996, at least 149 manatees (Trichechus manatus latirostris) died in an unprecedented epizootic along the southwest coast of Florida. At about the same time, a bloom of the brevetoxin-producing dinoflagellates, Gymnodinium breve, was present in the same area. Grossly, severe nasopharyngeal, pulmonary, hepatic, renal, and cerebral congestion was present in all cases. Nasopharyngeal and pulmonary edema and hemorrhage were also seen. Consistent microscopic lesions consisted of catarrhal rhinitis, pulmonary hemorrhage and edema, multiorgan hemosiderosis, and nonsuppurative leptomeningitis. Immunohistochemical staining using a polyclonal primary antibody to brevetoxin (GAB) showed intense positive staining of lymphocytes and macrophages in the lung, liver, and secondary lymphoid tissues. Additionally, lymphocytes and macrophages associated with the inflammatory lesions of the nasal mucosa and meninges were also positive for brevetoxin. These findings implicate brevetoxicosis as a component of and the likely primary etiology for the epizootic. The data suggest that mortality resulting from brevetoxicosis may not necessarily be acute but may occur after chronic inhalation and/or ingestion. Immunohistochemical staining with interleukin-1-beta-converting enzyme showed positive staining with a cellular tropism similar to GAB. This suggests that brevetoxicosis may initiate apoptosis and/or the release of inflammatory mediators that culminate in fatal toxic shock.

Brevetoxin-3 (PbTx-3) and its derivatives modulate single tetrodotoxin-sensitive sodium channels in rat sensory neurons.

Brevetoxin-3 (PbTx-3), produced by marine dinoflagellates (Ptychodiscus brevis), is a lipophilic 11-ring polyether molecule that binds with high affinity to site 5 of the voltage-sensitive sodium (Na+) channel. The effects of PbTx-3 and its derivatives were studied in cell-attached membrane patches on neurons dissociated from neonatal rat nodose ganglia by the patch-clamp technique. PbTx-3 (30-500 nM) produced a shift in activation to more negative membrane potentials whereby single-channel activity was observed under steady-state conditions (maintained depolarization at -50 mV). The unitary current-voltage relationship is linear, which exhibits a reversal potential of approximately +60 mV. Two unitary current amplitudes could be observed in the presence of PbTx-3, with slope conductances of 10.7 pS and 21.2 pS. PbTx-3 inhibits the inactivation of Na+ channels and prolongs the mean open time of these channels. Unitary Na+ currents could be blocked by 1 microM tetrodotoxin (TTX) added to the pipette solution, which indicates that the single-channel currents are caused by the opening of TTX-sensitive Na+ channels. The PbTx-3 molecule is proposed to have multiple active centers (A-ring lactone, C-42 of R side chain) interacting with the Na+ channel binding site. Modification of the molecular structure of PbTx-3 at these centers produced derivatives (PbTx-6, 2,3,41,43-tetrahydro-PbTx-3, 2,3,27,28,41, 43-hexahydro-PbTx-3 and 2,3-dihydro-PbTx-3 A-ring diol), which were less potent than PbTx-3 in producing similar effects on Na+ channel kinetics. PbTx-3 and its derivatives may provide insight into the mechanics of voltage-sensitive Na+ channel gating.

Brevetoxin-6 (PbTx-6), a nonaromatic marine neurotoxin, is a ligand of the aryl hydrocarbon receptor.

Brevetoxins (PbTx) are a family of marine polyether toxins that exert their toxic action by activating voltage-sensitive sodium channels. Two forms of brevetoxin, PbTx-2 and -3, induce hepatic cytochrome P4501A1, measured as ethoxyresorufin O-deethylase (EROD) activity, in redfish and striped bass. P4501A1 induction is transcriptionally regulated through the binding of a ligand, typically a planar aromatic compound, to the aryl hydrocarbon receptor (AhR) and subsequent complex formation with the dioxin response element (DRE), an upstream regulatory region of the CYP1A1 gene. To determine if PbTx, a nonaromatic compound, induced EROD by this mechanism, two sets of experiments were performed. Initially, saturation binding assays with PbTx-2, -3, and -6 were carried out to determine if PbTx-2, -3, or -6 was an AhR ligand. Results showed that PbTx-6 inhibited specific binding of dioxin to the AhR, whereas PbTx-2 and -3 had no effect. Subsequently, gel retardation assays showed that PbTx-6 caused a concentration-dependent increase in AhR-DRE complex formation. The most abundant and neurotoxic forms of brevetoxin, PbTx-2 and -3, did not appear to be involved in this process. However, PbTx-6, the epoxide which is a likely biotransformation product, is at least one of the forms of PbTx involved in EROD induction.

Complex behavior of marine animal tissue extracts in the competitive binding assay of brevetoxins with rat brain synaptosomes.

Brevetoxins are produced by the marine dinoflagellate Ptychodiscus brevis, an organism linked to red tide outbreaks, and the accompanying toxicity to marine animals and to neurotoxic shellfish poisoning in humans. Brevetoxins bind with high affinity to voltage-sensitive sodium channels and cause increased sodium ion conductance and nerve cell depolarization. The brevetoxin competitive binding assay with tritium-labeled brevetoxin 3 (3H-PbTx-3) and rat brain synaptosomes is a sensitive and specific assay for pure brevetoxins. Here we report that extracts of manatee, turtle, fish, and clam tissues contain components that interfere with the assay by cooperative, noncompetitive inhibition of 3H-PbTx-3 specific binding and increased nonspecific binding to synaptosomes. By determining the "apparent" toxin concentration ("[Toxin]") in the extract at several assay concentrations, a reasonable correction for the complex inhibition could be made using a semilog plot to extrapolate [Toxin] to zero extract concentration to obtain [Toxin]0. Spiking 4 extracts with 60 nM PbTx-3 caused [Toxin]0 to increase by 41 +/- 8 nM, indicating that the noncompetitive components did not prevent the assay of toxin but did reduce the accuracy of the result. Fourfold repetition of the assay of 4 samples gave standard deviations of 25 to 60% of the value of [Toxin]0, so the error can be fairly large, especially for samples with little toxin. Purification of an extract with a 1 g sample prep column of C-18 decreased the complex inhibition by about 3-fold but did not eliminate interference in the assay.

Complex association and dissociation kinetics of brevetoxin binding to voltage-sensitive rat brain sodium channels.

Brevetoxins bind with high affinity to the voltage-sensitive sodium channel and cause nerve cell depolarization and increased sodium ion conductance. Using 0.6 nM tritium-labeled brevetoxin-3 and freshly prepared synaptosomes from fresh or frozen rat brain, binding results at 6 degrees C fit well to a curve for 2-phase association with 65% of the binding in the rapid phase and t1/2 values of 11 and 74 min for the rapid and slow phases, respectively. Both phases were accelerated at higher toxin concentrations, binding of 9 nM brevetoxin-3 (PbTx-3) was close to equilibrium within 1 hr. The slow phase was not apparent when binding was done at 20 degrees C or when binding was done at 6 degrees C after the membrane sample had been preincubated at 4 degrees C for 1 day or at 22 degrees C for 1 hr. The 2-phase nature of association was not affected by substitution of KCl for choline chloride in the assay medium to produce sodium channel in the inactive state. Dissociation kinetics at 6 degrees C were also complex; the results fit well to a 2-phase curve with 55% of the dissociation in the rapid phase and t1/2 values of 13 and 64 min for the rapid and slow phases, respectively. The 2-phase nature did not change significantly after preincubation at 4 degrees C for 1 day. However, dissociation at 20 degrees C was rapid and fit a curve for 1-phase dissociation with a t1/2 of 2-6 min. At higher concentrations of PbTx-3, the binding is further complicated by the presence of 2-4 low-affinity binding sites with Kd values near 700 nM. In conclusion the association and dissociation of PbTx-3 with sodium channel from rat brain are complex processes that may involve changes in sodium channel conformation or interactions with other membrane (or membrane-associated) components.

The relationship of brevetoxin 'length' and A-ring functionality to binding and activity in neuronal sodium channels.

BACKGROUND: Brevetoxins are polyether ladder toxins that are ichthyotoxic at nanomolar concentrations. They bind to voltage-gated sodium channels, causing four distinct electrophysiological effects: (i) a shift of activation potential; (ii) occurrence of subconductance states; (iii) induction of longer mean open times of the channel; and (iv) inhibition of channel inactivation. We set out to determine whether these functions all require the same structural elements within the brevetoxin molecules. RESULTS: Several synthetically prepared structural analogs of brevetoxin B were examined in synaptosome receptor binding assays and by functional electrophysiological measurements. A truncated analog is not ichthyotoxic at micromolar concentrations, shows decreased receptor-binding affinity, and causes only a shift of activation potential without affecting mean open times or channel inactivation. An analog with the A-ring carbonyl removed binds to the receptor with nanomolar affinity, produces a shift of activation potential and inhibits inactivation, but does not induce longer mean open times. An analog in which the A-ring diol is reduced shows low binding affinity, yet populates five subconductance states. CONCLUSIONS: Our data are consistent with the hypothesis that binding to sodium channels requires an elongated cigar-shaped molecule, approximately 30 A long. The four electrophysiological effects of the brevetoxins are not produced by a single structural feature, however, since they can be decoupled by using modified ligands, which are shown here to be partial sodium channel agonists. We propose a detailed model for the binding of brevetoxins to the channel which explains the differences in the effects of the brevetoxin analogs. These studies also offer the potential for developing brevetoxin antagonists.

Modified immunoassays for polyether toxins: implications of biological matrixes, metabolic states, and epitope recognition.

Polyether marine toxins are responsible for the seafood intoxication phenomena known as neurotoxic shellfish poisoning (due to brevetoxins), ciguatera (due to ciguatoxin), and diarrheic shellfish poisoning (due to okadaic acid). Using traditional techniques of hapten (pure toxin) conjugation to protein to create complete antigen, animal immunization and antibody isolation, and specific antibody subpopulation purification, discriminating antibodies have been isolated that detect brevetoxins and ciguatoxin, but not okadaic acid, in a dose-dependent fashion. Using microorganic chemistry and purified toxins, a unique set of tools has been created for the study of polyether ladder toxin accumulation; depuration; and specific site localization in tissues, food sources, and clinical samples. Developed test protocols can detect toxin in dinoflagellate cells, in extracts from food sources, in seawater and culture media, and in human serum samples. Enzyme-linked immunosorbent assay protocols developed for eventual collaborative testing have been successful in limited applications within the laboratory (correlation coefficient of 0.92 excluding 2 outliers), and alternative formats are being developed to optimize the basic test for use in research laboratories, regulatory laboratories, and field inspections.

Radioimmunoassay for PbTx-2-type brevetoxins: epitope specificity of two anti-PbTx sera.

Antiserum against PbTx-2-type brevetoxins was produced by immunizing rabbits with a PbTx-2-bovine serum albumin (BSA) conjugate. This serum had a higher affinity, but lower titer, than our current goat serum. Using 4 natural brevetoxins and 6 synthetic derivatives as competitors in our brevetoxin radioimmunoassay, we determined the epitope specificity of both sera. Modification of the backbone structure at C-42 on the K-ring had little or no effect on the antigen-binding capability of either serum. Reduction of the double bond between C-2 and C-3 on the A-ring by reduction of the lactone decreased binding 500 to 750-fold. Epoxidation of the double bond between C-27 and C-28 on the H-ring did not affect binding, which suggested that the goat serum is specific for the A-ring region of the brevetoxin backbone. In contrast, modifying the A-ring had no effect on rabbit serum binding. However, epoxidation of the H-ring decreased binding 5 to 20-fold, which suggested that the rabbit antiserum is specific for the H-ring region of the molecule. These results suggest that assays utilizing only one antibody may not adequately detect toxin metabolites if molecules are altered in the critical region of antibody recognition.

Detection of marine toxins using reconstituted sodium channels.

Specific binding of the marine toxins saxitoxin, tetrodotoxin, and brevetoxin to the rat brain sodium channel is demonstrated using purified sodium channels reconstituted into phospholipid vesicles. Restoration of sodium channel function and binding activity by incorporation into phospholipid vesicles provides the only rigorous proof that the purified protein contains the neurotoxin receptor sites. In addition, reconstitution provides a valuable experimental preparation for biochemical analysis of neurotoxin binding sites and may facilitate the development of a specific toxin detection system.

Identification of peptide components of the brevetoxin receptor site of rat brain sodium channels.

To identify the binding domain for brevetoxins, a family of lipid-soluble neurotoxins acting at Na+ channel receptor site 5, purified and reconstituted rat brain Na+ channels were photolabeled with p-azidobenzoyl tritium-labeled brevetoxin, and the labeled peptides were identified. A radiolabeled band with an apparent molecular mass of 250 kDa corresponding to the Na+ channel alpha-subunit was revealed using both gel slicing and fluorography techniques. Regions of the alpha-subunit specifically photolabeled by this ligand were then identified by antibody mapping of proteolytic fragments. Even after extensive proteolysis, anti-peptide antibodies recognizing amino acid sequences within or adjacent to Na+ channel transmembrane segments IS6 and IVS5 were each able to immunoprecipitate up to 40% of the labeled peptides. A more extensive tryptic digest was obtained with a preparation in which the brevetoxin photolabel was incorporated into the alpha-subunit of purified Na+ channel in detergent solution. The identification of a specifically immunoprecipitated 6-kDa peptide containing transmembrane segment S6 from domain I restricted the labeled peptide fragment to residues Thr-400 to Lys-443 if tryptic digestion was complete or Ala-396 to Lys-455 if tryptic cleavage was incomplete. Similarly, the identification of a specifically immunoprecipitated 6-kDa peptide from domain IV restricted the labeled peptide to residues Glu-1738 to Lys-1785 or Glu-1738 to Lys-1793 on the extracellular side of transmembrane segment S5. These results provide direct evidence for close association of transmembrane segments IS6 and IVS5 in the native conformation of the Na+ channel alpha-subunit and implicate their region of interaction as an important component of the brevetoxin receptor site.

Brevetoxin PbTx-2 immunology: differential epitope recognition by antibodies from two goats.

The epitopic regions of the brevetoxin PbTx-3 molecule, produced by the marine dinoflagellate Ptychodiscus brevis, have been identified by structural modification at three distinct regions of the toxin. These are: the A-ring lactone region of the molecule, the K-ring side-chain and the H-ring. The modified PbTx-3 derivatives were tested for their ability to bind brevetoxin goat antisera directed against the PbTx-3 molecule, by radioimmunoassay. The results showed that at least two major epitopes and one minor epitope are recognized: the A-ring lactone region of the molecule and the K-ring side-chain, and the H-ring. The results illustrate the variety of antibodies which may be produced, even within a species, and suggests that epitope characterization is important in the development of assays which are to be employed in seafood safety issues.