Brevetoxin versus Brevenal Modulation of Human Nav1 Channels.

Brevetoxins (PbTx) and brevenal are marine ladder-frame polyethers. PbTx binds to and activates voltage-gated sodium (Nav) channels in native tissues, whereas brevenal antagonizes these actions. However, the effects of PbTx and brevenal on recombinant Nav channel function have not been systematically analyzed. In this study, the PbTx-3 and brevenal modulation of tissue-representative Nav channel subtypes Nav1.2, Nav1.4, Nav1.5, and Nav1.7 were examined using automated patch-clamp. While PbTx-3 and brevenal elicit concentration-dependent and subtype-specific modulatory effects, PbTx-3 is >1000-fold more potent than brevenal. Consistent with effects observed in native tissues, Nav1.2 and Nav1.4 channels were PbTx-3- and brevenal-sensitive, whereas Nav1.5 and Nav1.7 appeared resistant. Interestingly, the incorporation of brevenal in the intracellular solution caused Nav channels to become less sensitive to PbTx-3 actions. Furthermore, we generated a computational model of PbTx-2 bound to the lipid-exposed side of the interface between domains I and IV of Nav1.2. Our results are consistent with competitive antagonism between brevetoxins and brevenal, setting a basis for future mutational analyses of Nav channels' interaction with brevetoxins and brevenal. Our findings provide valuable insights into the functional modulation of Nav channels by brevetoxins and brevenal, which may have implications for the development of new Nav channel modulators with potential therapeutic applications.

Cucumis sativus extract elicits chloride secretion by stimulation of the intestinal TMEM16A ion channel.

CONTEXT: Cucumber (Cucumis sativus Linn. [Cucurbitaceae]) is widely known for its purgative, antidiabetic, antioxidant, and anticancer therapeutic potential. However, its effect on gastrointestinal (GI) disease is unrecognised. OBJECTIVE: This study investigated the effect of C. sativus fruit extract (CCE) on intestinal chloride secretion, motility, and motor function, and the role of TMEM16A chloride channels. MATERIALS AND METHODS: CCE extracts were obtained from commercially available cucumber. Active fractions were then purified by HPLC and analysed by high resolution mass spectrometry. The effect of CCE on intestinal chloride secretion was investigated in human colonic T84 cells, ex vivo mouse intestinal tissue using an Ussing chamber, and the two-electrode voltage-clamp technique to record calcium sensitive TMEM16A chloride currents in Xenopus laevis oocytes. In vivo, intestinal motility was investigated using the loperamide-induced C57BL/6 constipation mouse model. Ex vivo contractility of mouse colonic smooth muscles was assessed by isometric force measurements. RESULTS: CCE increased the short-circuit current (ΔIsc 34.47 ± µA/cm2) and apical membrane chloride conductance (ΔICl 95 ± 8.1 µA/cm2) in intestinal epithelial cells. The effect was dose-dependent, with an EC50 value of 0.06 µg/mL. CCE stimulated the endogenous TMEM16A-induced Cl- current in Xenopus laevis oocytes. Moreover, CCE increased the contractility of smooth muscle in mouse colonic tissue and enhanced small bowel transit in CCE treated mice compared to loperamide controls. Mass spectrometry suggested a cucurbitacin-like analogue with a mass of 512.07 g/mol underlying the bioactivity of CCE. CONCLUSION: A cucurbitacin-like analog present in CCE activates TMEM16A channels, which may have therapeutic potential in cystic fibrosis and intestinal hypodynamic disorders.

Epicortical Brevetoxin Treatment Promotes Neural Repair and Functional Recovery after Ischemic Stroke.

Emerging literature suggests that after a stroke, the peri-infarct region exhibits dynamic changes in excitability. In rodent stroke models, treatments that enhance excitability in the peri-infarct cerebral cortex promote motor recovery. This increase in cortical excitability and plasticity is opposed by increases in tonic GABAergic inhibition in the peri-infarct zone beginning three days after a stroke in a mouse model. Maintenance of a favorable excitatory-inhibitory balance promoting cerebrocortical excitability could potentially improve recovery. Brevetoxin-2 (PbTx-2) is a voltage-gated sodium channel (VGSC) gating modifier that increases intracellular sodium ([Na+]i), upregulates N-methyl-D-aspartate receptor (NMDAR) channel activity and engages downstream calcium (Ca2+) signaling pathways. In immature cerebrocortical neurons, PbTx-2 promoted neuronal structural plasticity by increasing neurite outgrowth, dendritogenesis and synaptogenesis. We hypothesized that PbTx-2 may promote excitability and structural remodeling in the peri-infarct region, leading to improved functional outcomes following a stroke. We tested this hypothesis using epicortical application of PbTx-2 after a photothrombotic stroke in mice. We show that PbTx-2 enhanced the dendritic arborization and synapse density of cortical layer V pyramidal neurons in the peri-infarct cortex. PbTx-2 also produced a robust improvement of motor recovery. These results suggest a novel pharmacologic approach to mimic activity-dependent recovery from stroke.

Bidirectional influence of sodium channel activation on NMDA receptor-dependent cerebrocortical neuron structural plasticity.

Neuronal activity regulates brain development and synaptic plasticity through N-methyl-D-aspartate receptors (NMDARs) and calcium-dependent signaling pathways. Intracellular sodium ([Na(+)](i)) also exerts a regulatory influence on NMDAR channel activity, and [Na(+)](i) may, therefore, function as a signaling molecule. In an attempt to mimic the influence of neuronal activity on synaptic plasticity, we used brevetoxin-2 (PbTx-2), a voltage-gated sodium channel (VGSC) gating modifier, to manipulate [Na(+)](i) in cerebrocortical neurons. The acute application of PbTx-2 produced concentration-dependent increments in both intracellular [Na(+)] and [Ca(2+)]. Pharmacological evaluation showed that PbTx-2-induced Ca(2+) influx primarily involved VGSC activation and NMDAR-mediated entry. Additionally, PbTx-2 robustly potentiated NMDA-induced Ca(2+) influx. PbTx-2-exposed neurons showed enhanced neurite outgrowth, increased dendritic arbor complexity, and increased dendritic filopodia density. The appearance of spontaneous calcium oscillations, reflecting synchronous neuronal activity, was accelerated by PbTx-2 treatment. Parallel to this response, PbTx-2 increased cerebrocortical neuron synaptic density. PbTx-2 stimulation of neurite outgrowth, dendritic arborization, and synaptogenesis all exhibited bidirectional concentration-response profiles. This profile paralleled that of NMDA, which also produced bidirectional concentration-response profiles for neurite outgrowth and synaptogenesis. These data are consistent with the hypothesis that PbTx-2-enhanced neuronal plasticity involves NMDAR-dependent signaling. Our results demonstrate that PbTx-2 mimics activity-dependent neuronal structural plasticity in cerebrocortical neurons through an increase in [Na(+)](i), up-regulation of NMDAR function, and engagement of downstream Ca(2+)-dependent signaling pathways. These data suggest that VGSC gating modifiers may represent a pharmacologic strategy to regulate neuronal plasticity through NMDAR-dependent mechanisms.

Development of a fluorescence assay for the characterization of brevenal binding to rat brain synaptosomes.

The marine dinoflagellate Karenia brevis produces a family of neurotoxins known as brevetoxins. Brevetoxins elicit their effects by binding to and activating voltage-sensitive sodium channels (VSSCs) in cell membranes. K. brevis also produces brevenal, a brevetoxin antagonist, which is able to inhibit and/or negate many of the detrimental effects of brevetoxins. Brevenal binding to VSSCs has yet to be fully characterized, in part due to the difficulty and expense of current techniques. In this study, we have developed a novel fluorescence binding assay for the brevenal binding site. Several fluorescent compounds were conjugated to brevenal to assess their effects on brevenal binding. The assay was validated against the radioligand assay for the brevenal binding site and yielded comparable equilibrium inhibition constants. The fluorescence-based assay was shown to be quicker and far less expensive and did not generate radioactive waste or need facilities for handling radioactive materials. In-depth studies using the brevenal conjugates showed that, while brevenal conjugates do bind to a binding site in the VSSC protein complex, they are not displaced by known VSSC site specific ligands. As such, brevenal elicits its action through a novel mechanism and/or currently unknown receptor site on VSSCs.

Development and utilization of a fluorescence-based receptor-binding assay for the site 5 voltage-sensitive sodium channel ligands brevetoxin and ciguatoxin.

Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Consumption of fish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of toxins has historically been measured using a radioligand competition assay that is fraught with difficulty. In this study, we developed a novel fluorescence-based binding assay for the brevetoxin receptor. Several fluorophores were conjugated to polyether brevetoxin-2 and used as the labeled ligand. Brevetoxin analogs were able to compete for binding with the fluorescent ligands. This assay was qualified against the standard radioligand receptor assay for the brevetoxin receptor. Furthermore, the fluorescence-based assay was used to determine relative concentrations of toxins in raw extracts of K. brevis culture, and to determine ciguatoxin affinity to site 5 of VSSCs. The fluorescence-based assay was quicker, safer, and far less expensive. As such, this assay can be used to replace the current radioligand assay and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels.

Brevenal, a brevetoxin antagonist from Karenia brevis, binds to a previously unreported site on mammalian sodium channels.

Brevetoxins are a family of ladder-frame polyether toxins produced by the marine dinoflagellate Karenia brevis. During blooms of K. brevis, inhalation of brevetoxins aerosolized by wind and wave action can lead to asthma-like symptoms in persons at the beach. Consumption of either shellfish or finfish contaminated by K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to binding at a defined site on, and subsequent activation of, voltage-sensitive sodium channels (VSSCs) in cell membranes (site 5). In addition to brevetoxins, K. brevis produces several other ladder-frame compounds. One of these compounds, brevenal, has been shown to antagonize the effects of brevetoxin. In an effort to further characterize to effects of brevenal, a radioactive analog ([3H]-brevenol) was produced by reducing the side-chain terminal aldehyde moiety of brevenal to an alcohol using tritiated sodium borohydride. A KD of 67 nM and Bmax of 7.1 pmol/mg protein were obtained for [3H]-brevenol in rat brain synaptosomes, suggesting a 1:1 matching with VSSCs. Brevenal and brevenol competed for [3H]-brevenol binding with Ki values of 75 nM and 56 nM, respectively. However, although both brevenal and brevenol can inhibit brevetoxin binding, brevetoxin was completely ineffective at competition for [3H]-brevenol binding. After examining other site-specific compounds, it was determined that [3H]-brevenol binds to a site that is distinct from the other known sites including the brevetoxin site (site 5) although some interaction with site 5 is apparent.

Development of a competitive fluorescence-based synaptosome binding assay for brevetoxins.

Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Inhalation of brevetoxins aerosolized by wind and wave action can lead to asthma-like symptoms in beach goers. Consumption of either shellfish or finfish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of brevetoxin analogs and competitors to site 5 on these channels has historically been measured using a radioligand competition assay that is fraught with difficulty, including slow analysis time, production of radioactive waste, and cumbersome and expensive methods associated with the generation of radioactive labeled ligands. In this study, we describe the development of a novel fluorescent synaptosome binding assay for the brevetoxin receptor. BODIPY(®)-conjugated to PbTx-2 was used as the labeled ligand. The BODIPY(®)-PbTx-2 conjugate was found to displace [(3)H]-PbTx-3 from its binding site on VSSCs on rat brain synaptosomes with an equilibrium inhibition constant of 0.11 nM. We have shown that brevetoxin A and B analogs are all able to compete for binding with the fluorescent ligand. Most importantly, this assay was validated against the current site 5 receptor binding assay standard, the radioligand receptor assay for the brevetoxin receptor using [(3)H]-PbTx-3 as the labeled ligand. The fluorescence based assay yielded equilibrium inhibition constants comparable to the radioligand assay for all brevetoxin analogs. The fluorescence based assay was quicker, far less expensive, and did not generate radioactive waste or need radioactive facilities. As such, this fluorescence-based assay can be used to replace the current radioligand assay for site 5 on voltage-sensitive sodium channels and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels.

Brevetoxicosis: red tides and marine mammal mortalities.

Potent marine neurotoxins known as brevetoxins are produced by the 'red tide' dinoflagellate Karenia brevis. They kill large numbers of fish and cause illness in humans who ingest toxic filter-feeding shellfish or inhale toxic aerosols. The toxins are also suspected of having been involved in events in which many manatees and dolphins died, but this has usually not been verified owing to limited confirmation of toxin exposure, unexplained intoxication mechanisms and complicating pathologies. Here we show that fish and seagrass can accumulate high concentrations of brevetoxins and that these have acted as toxin vectors during recent deaths of dolphins and manatees, respectively. Our results challenge claims that the deleterious effects of a brevetoxin on fish (ichthyotoxicity) preclude its accumulation in live fish, and they reveal a new vector mechanism for brevetoxin spread through food webs that poses a threat to upper trophic levels.

Brevetoxin inhalation alters the pulmonary response to influenza A in the male F344 rat.

Epidemiological studies demonstrated that the number of emergency-room visits for respiratory indications increases during periods of Florida Red Tides. The purpose of this study was to examine whether or not repeated brevetoxin inhalation, as may occur during a Florida Red Tide, affects pulmonary responses to influenza A. Male F344 rats were divided into four groups: (1) sham aerosol/no influenza; (2) sham aerosol/influenza; (3) brevetoxin/no influenza; and (4) brevetoxin/influenza. Animals were exposed by nose-only inhalation to vehicle or 50 µg brevetoxin-3/m3, 2 h/d for 12 d. On d 6 of aerosol exposure, groups 2 and 4 were administered 10,000 plaque-forming units of influenza A, strain HKX-31 (H3N2), by intratracheal instillation. Subgroups were euthanized at 2, 4, and 7 d post influenza treatment. Lungs were evaluated for viral load, cytokine content, and histopathologic changes. Influenza virus was cleared from the lungs over the 7-d period; however, there was significantly more virus remaining in the group 4 lungs compared to group 2. Influenza virus significantly increased interleukins-1α and -6 and monocyte chemotactic protein-1 in lung; brevetoxin exposure significantly enhanced the influenza-induced response. At 7 d, the severity of perivascular and peribronchiolar inflammatory cell infiltrates was greatest in group 4. Bronchiolitis persisted, with low incidence and severity, only in group 4 at d 7. These results suggest that repeated inhalation exposure to brevetoxin may delay virus particle clearance and recovery from influenza A infection in the rat lung.

Repeated exposure to aerosolized brevetoxin-3 induces prolonged airway hyperresponsiveness and lung inflammation in sheep.

CONTEXT: During a Florida red tide, brevetoxins (PbTxs) produced by Karenia brevis become aerosolized and can cause both immediate and prolonged airway symptoms in humans, especially in those with preexisting airway disease (e.g., asthma). Although environmental monitoring indicates that toxins remain airborne for up to 4 consecutive days, there is little information on airway responses after multiple-day exposures. OBJECTIVES: To delineate putative mechanisms leading to pulmonary dysfunction after PbTx exposure, we studied airway responses before and after multiple exposures to aerosol PbTx-3, the most potent PbTx produced, in nonallergic (healthy) and in allergic sheep, which serve as a surrogate for patients with compromised airways. METHODS: Both groups were exposed to 20 breaths of increasing concentrations of PbTx-3 (30-300 pg/mL) for 4 consecutive days. Airway responsiveness to carbachol (1 and 8 days after) and airway inflammation as assessed by bronchoalveolar lavage (0 and 7 days after) were measured. RESULTS: Both groups developed airway hyperresponsiveness (AHR) 1 day after challenge; the severity was concentration dependent and more severe in the allergic group. AHR remained after 8 days, but the difference in the severity between the groups was lost. Both groups developed an inflammatory response after exposure to 300 pg/mL PbTx-3. Immediately after exposure, lung neutrophilia was prominent. This neutrophilia persisted for 7 days in addition to increases in total cells and macrophages. CONCLUSION: Repeated exposures to PbTx-3 result in prolonged AHR and lung inflammation. These pathophysiologic responses could be underlying contributors to the prolonged respiratory symptoms in humans after red tides.

Aerosolized Red Tide Toxins (Brevetoxins) and Asthma: Continued health effects after 1 hour beach exposure.

Blooms of the toxic dinoflagellate, Karenia brevis, produce potent neurotoxins in marine aerosols. Recent studies have demonstrated acute changes in both symptoms and pulmonary function in asthmatics after only 1 hour of beach exposure to these aerosols. This study investigated if there were latent and/or sustained effects in asthmatics in the days following the initial beach exposure during periods with and without an active Florida red tide.Symptom data and spirometry data were collected before and after 1 hour of beach exposure. Subjects kept daily symptom diaries and measured their peak flow each morning for 5 days following beach exposure. During non-exposure periods, there were no significant changes in symptoms or pulmonary function either acutely or over 5 days of follow-up. After the beach exposure during an active Florida red tide, subjects had elevated mean symptoms which did not return to the pre-exposure baseline for at least 4 days. The peak flow measurements decreased after the initial beach exposure, decreased further within 24 hours, and continued to be suppressed even after 5 days. Asthmatics may continue to have increased symptoms and delayed respiratory function suppression for several days after 1 hour of exposure to the Florida red tide toxin aerosols.

Florida Red Tide Toxins (Brevetoxins) and Longitudinal Respiratory Effects in Asthmatics.

Having demonstrated significant and persistent adverse changes in pulmonary function for asthmatics after 1 hour exposure to brevetoxins in Florida red tide (Karenia brevis bloom) aerosols, we assessed the possible longer term health effects in asthmatics from intermittent environmental exposure to brevetoxins over 7 years. 125 asthmatic subjects were assessed for their pulmonary function and reported symptoms before and after 1 hour of environmental exposure to Florida red tide aerosols for upto 11 studies over seven years. As a group, the asthmatics came to the studies with normal standardized percent predicted pulmonary function values. The 38 asthmatics who participated in only one exposure study were more reactive compared to the 36 asthmatics who participated in ≥4 exposure studies. The 36 asthmatics participating in ≥4 exposure studies demonstrated no significant change in their standardized percent predicted pre-exposure pulmonary function over the 7 years of the study. These results indicate that stable asthmatics living in areas with intermittent Florida red tides do not exhibit chronic respiratory effects from intermittent environmental exposure to aerosolized brevetoxins over a 7 year period.

Review of Florida Red Tide and Human Health Effects.

This paper reviews the literature describing research performed over the past decade on the known and possible exposures and human health effects associated with Florida red tides. These harmful algal blooms are caused by the dinoflagellate, Karenia brevis, and similar organisms, all of which produce a suite of natural toxins known as brevetoxins. Florida red tide research has benefited from a consistently funded, long term research program, that has allowed an interdisciplinary team of researchers to focus their attention on this specific environmental issue-one that is critically important to Gulf of Mexico and other coastal communities. This long-term interdisciplinary approach has allowed the team to engage the local community, identify measures to protect public health, take emerging technologies into the field, forge advances in natural products chemistry, and develop a valuable pharmaceutical product. The Review includes a brief discussion of the Florida red tide organisms and their toxins, and then focuses on the effects of these toxins on animals and humans, including how these effects predict what we might expect to see in exposed people.

Sodium channel activation augments NMDA receptor function and promotes neurite outgrowth in immature cerebrocortical neurons.

A range of extrinsic signals, including afferent activity, affect neuronal growth and plasticity. Neuronal activity regulates intracellular Ca(2+), and activity-dependent calcium signaling has been shown to regulate dendritic growth and branching (Konur and Ghosh, 2005). NMDA receptor (NMDAR) stimulation of Ca(2+)/calmodulin-dependent protein kinase signaling cascades has, moreover, been demonstrated to regulate neurite/axonal outgrowth (Wayman et al., 2004). We used a sodium channel activator, brevetoxin (PbTx-2), to explore the relationship between intracellular [Na(+)] and NMDAR-dependent development. PbTx-2 alone, at a concentration of 30 nM, did not affect Ca(2+) dynamics in 2 d in vitro cerebrocortical neurons; however, this treatment robustly potentiated NMDA-induced Ca(2+) influx. The 30 nM PbTx-2 treatment produced a maximum [Na(+)](i) of 16.9 +/- 1.5 mM, representing an increment of 8.8 +/- 1.8 mM over basal. The corresponding membrane potential change produced by 30 nM PbTx-2 was modest and, therefore, insufficient to relieve the voltage-dependent Mg(2+) block of NMDARs. To unambiguously demonstrate the enhancement of NMDA receptor function by PbTx-2, we recorded single-channel currents from cell-attached patches. PbTx-2 treatment was found to increase both the mean open time and open probability of NMDA receptors. These effects of PbTx-2 on NMDA receptor function were dependent on extracellular Na(+) and activation of Src kinase. The functional consequences of PbTx-2-induced enhancement of NMDAR function were evaluated in immature cerebrocortical neurons. PbTx-2 concentrations between 3 and 300 nM enhanced neurite outgrowth. Voltage-gated sodium channel activators may accordingly represent a novel pharmacologic strategy to regulate neuronal plasticity through an NMDA receptor and Src family kinase-dependent mechanism.

Characterization of tamulamides A and B, polyethers isolated from the marine dinoflagellate Karenia brevis.

Florida red tides occur as the result of blooms of the marine dinoflagellate Karenia brevis. K. brevis is known to produce several families of fused polyether ladder compounds. The most notable compounds are the brevetoxins, potent neurotoxins that activate mammalian sodium channels. Additional fused polyether ladder compounds produced by K. brevis include brevenal, brevisin, and hemibrevetoxin B, all with varying affinities for the same binding site on voltage-sensitive sodium channels. The structure elucidation and biological activity of two additional fused polyether ladder compounds containing seven fused ether rings will be described in this paper. Tamulamide A (MW = 638.30) and tamulamide B (MW = 624.29) were isolated from K. brevis cultures, and their structures elucidated using a combination of NMR spectroscopy and high-resolution mass spectrometry. Tamulamides A and B were both found to compete with tritiated brevetoxin-3 ([(3)H]-PbTx-3) for its binding site on rat brain synaptosomes. However, unlike the brevetoxins, tamulamides A and B showed no toxicity to fish at doses up to 200 nM and did not cause significant bronchoconstriction in sheep pulmonary assays.

Absolute configuration of brevisamide and brevisin: confirmation of a universal biosynthetic process for Karenia brevis polyethers.

The discovery of brevisin, the first example of an "interrupted" polycyclic ether, obtained from the dinoflagellate Karenia brevis, posed some important questions regarding the mechanism of the cyclization process. Consequently, we have established absolute configurations of brevisin and its related metabolite brevisamide using a modified Mosher's esterification method. For brevisin, analysis was carried out on both the 31-monokis- and the 10,31-bis-MTPA esters. The results suggest that both metabolites, like other polyethers from K. brevis, result from polyepoxide precursors with uniform (S, S) configurations for all epoxides and provide further support for a universal stereochemical model for dinoflagellate polyether formation.

Personal exposure to aerosolized red tide toxins (brevetoxins).

Florida red tides occur annually in the Gulf of Mexico from blooms of the marine dinoflagellate, Karenia brevis, which produces highly potent natural polyether toxins, brevetoxins. Several epidemiologic studies have demonstrated that human exposure to red tide aerosol could result in increased respiratory symptoms. Environmental monitoring of aerosolized brevetoxins was performed using a high-volume sampler taken hourly at fixed locations on Siesta Beach, Florida. Personal exposure was monitored using personal air samplers and taking nasal swab samples from the subjects who were instructed to spend 1 hr on Sarasota Beach during two sampling periods of an active Florida red tide event in March 2005, and in May 2008 when there was no red tide. Results showed that the aerosolized brevetoxins from the personal sampler were in modest agreement with the environmental concentration taken from a high-volume sampler. Analysis of nasal swab samples for brevetoxins demonstrated 68% positive samples in the March 2005 sampling period when air concentrations of brevetoxins were between 50 to 120 ng/m(3) measured with the high-volume sampler. No swab samples showed detectable levels of brevetoxins in the May 2008 study, when all personal samples were below the limit of detection. However, there were no statistical correlations between the amounts of brevetoxins detected in the swab samples with either the environmental or personal concentration. Results showed that the personal sample might provide an estimate of individual exposure level. Nasal swab samples showed that brevetoxins indeed were inhaled and deposited in the nasal passage during the March 2005 red tide event.

Brevisin: an aberrant polycyclic ether structure from the dinoflagellate Karenia brevis and its implications for polyether assembly.

Brevisin is an unprecedented polycyclic ether isolated from the dinoflagellate Karenia brevis, an organism well-known to produce complex polycyclic ethers. The structure of brevisin was determined by detailed analyses of MS and 2D NMR spectra and is remarkable in that it consists of two separate fused polyether ring assemblies linked by a methylene group. One of the polycyclic moieties contains a conjugated aldehyde side chain similar to that recently observed in other K. brevis metabolites, though the "interrupted" polyether structure of brevisin is novel and provides further insight into the biogenesis of such fused-ring polyether systems. On the basis of the unusual structure of brevisin, principles underlying the initiation of polyether assemblies are proposed. Brevisin was found to inhibit the binding of [(3)H]-PbTx-3 to its binding site on the voltage-sensitive sodium channels in rat brain synaptosomes.

Molecular Determinants of Brevetoxin Binding to Voltage-Gated Sodium Channels.

Brevetoxins are produced by dinoflagellates such as Karenia brevis in warm-water red tides and cause neurotoxic shellfish poisoning. They bind to voltage-gated sodium channels at neurotoxin receptor 5, making the channels more active by shifting the voltage-dependence of activation to more negative potentials and by slowing the inactivation process. Previous work using photoaffinity labeling identified binding to the IS6 and IVS5 transmembrane segments of the channel α subunit. We used alanine-scanning mutagenesis to identify molecular determinants for brevetoxin binding in these regions as well as adjacent regions IVS5-SS1 and IVS6. Most of the mutant channels containing single alanine substitutions expressed functional protein in tsA-201 cells and bound to the radioligand [42-3H]-PbTx3. Binding affinity for the great majority of mutant channels was indistinguishable from wild type. However, transmembrane segments IS6, IVS5 and IVS6 each contained 2 to 4 amino acid positions where alanine substitution resulted in a 2-3-fold reduction in brevetoxin affinity, and additional mutations caused a similar increase in brevetoxin affinity. These findings are consistent with a model in which brevetoxin binds to a protein cleft comprising transmembrane segments IS6, IVS5 and IVS6 and makes multiple distributed interactions with these α helices. Determination of brevetoxin affinity for Nav1.2, Nav1.4 and Nav1.5 channels showed that Nav1.5 channels had a characteristic 5-fold reduction in affinity for brevetoxin relative to the other channel isoforms, suggesting the interaction with sodium channels is specific despite the distributed binding determinants.