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PubMed1994

A structural model of the tetrodotoxin and saxitoxin binding site of the Na+ channel.

Biophysical evidence has placed the binding site for the naturally occurring marine toxins tetrodotoxin (TTX) and saxitoxin (STX) in the external mouth of the Na+ channel ion permeation pathway. We developed a molecular model of the binding pocket for TTX and STX, composed of antiparallel beta-hairpins formed from peptide segments of the four S5-S6 loops of the voltage-gated Na+ channel. For TTX the guanidinium moiety formed salt bridges with three carboxyls, while two toxin hydroxyls (C9-OH and C10-OH) interacted with a fourth carboxyl on repeats I and II. This alignment also resulted in a hydrophobic interaction with an aromatic ring of phenylalanine or tyrosine residues for the brainII and skeletal Na+ channel isoforms, but not with the cysteine found in the cardiac isoform. In comparison to TTX, there was an additional interaction site for STX through its second guanidinium group with a carboxyl on repeat IV. This model satisfactorily reproduced the effects of mutations in the S5-S6 regions and the differences in affinity by various toxin analogs. However, this model differed in important ways from previously published models for the outer vestibule and the selectivity region of the Na+ channel pore. Removal of the toxins from the pocket formed by the four beta-hairpins revealed a structure resembling a funnel that terminated in a narrowed region suitable as a candidate for the selectivity filter of the channel. This region contained two carboxyls (Asp384 and Glu942) that substituted for molecules of water from the hydrated Na+ ion. Simulation of mutations in this region that have produced Ca2+ permeation of the Na+ channel created a site with three carboxyls (Asp384, Glu942, and Glu1714) in proximity.

Amino Acid Sequence↗
PubMed1994

Post-repolarization block of cloned sodium channels by saxitoxin: the contribution of pore-region amino acids.

Sodium channels expressed in oocytes exhibited isoform differences in phasic block by saxitoxin (STX). Neuronal channels (rat IIa co-expressed with beta 1 subunit, Br2a + beta 1) had slower kinetics of phasic block for pulse trains than cardiac channels (RHI). After the membrane was repolarized from a single brief depolarizing step, a test pulse at increasing intervals showed first a decrease in current (post-repolarization block) then eventual recovery in the presence of STX. This block/unblock process for Br2a + beta 1 was 10-fold slower than that for RHI. A model accounting for these results predicts a faster toxin dissociation rate and a slower association rate for the cardiac isoform, and it also predicts a shorter dwell time in a putative high STX affinity conformation for the cardiac isoform. The RHI mutation (Cys374-->Phe), which was previously shown to be neuronal-like with respect to high affinity tonic toxin block, was also neuronal-like with respect to the kinetics of post-repolarization block, suggesting that this single amino acid is important for conferring isoform-specific transition rates determining post-repolarization block. Because the same mutation determines both sensitivity for tonic STX block and the kinetics of phasic STX block, the mechanisms accounting for tonic block and phasic block share the same toxin binding site. We conclude that the residue at position 374, in the putative pore-forming region, confers isoform-specific channel kinetics that underlie phasic toxin block.

Animals↗
PubMed1998

Differences in saxitoxin and tetrodotoxin binding revealed by mutagenesis of the Na+ channel outer vestibule.

The marine guanidinium toxins, saxitoxin (STX) and tetrodotoxin (TTX), have played crucial roles in the study of voltage-gated Na+ channels. Because they have similar actions, sizes, and functional groups, they have been thought to associate with the channel in the same manner, and early mutational studies supported this idea. Recent experiments by. Biophys. J. 67:2305-2315) have suggested that the toxins bind differently to the isoform-specific domain I Phe/Tyr/Cys location. In the adult skeletal muscle Na+ channel isoform (microliter), we compared the effects on both TTX and STX affinities of mutations in eight positions known to influence toxin binding. The results permitted the assignment of energies contributed by each amino acid to the binding reaction. For neutralizing mutations of Asp400, Glu755, and Lys1237, all thought to be part of the selectivity filter of the channel, the loss of binding energy was identical for the two toxins. However, the loss of binding energy was quite different for vestibule residues considered to be more superficial. Specifically, STX affinity was reduced much more by neutralizations of Glu758 and Asp1532. On the other hand, mutation of Tyr401 to Cys reduced TTX binding energy twice as much as it reduced STX binding energy. Kinetic analysis suggested that all outer vestibule residues tested interacted with both toxins early in the binding reaction (consistent with larger changes in the binding than unbinding rates) before the transition state and formation of the final bound complex. We propose a revised model of TTX and STX binding in the Na+ channel outer vestibule in which the toxins have similar interactions at the selectivity filter, TTX has a stronger interaction with Tyr401, and STX interacts more strongly with the more extracellular residues.

Adult↗
PubMedDate not supplied

Expression and characterization of Flag-epitope- and hexahistidine-tagged derivatives of saxiphilin for use in detection and assay of saxitoxin.

Saxiphilin is a plasma protein from the bullfrog (Rana catesbiana) that binds saxitoxin (STX), a causative agent of paralytic shellfish poisoning. Saxiphilin is homologous to transferrin and consists of two internally homologous domains called the N-lobe and the C-lobe. STX binds to a single site in the C-lobe of saxiphilin. In this study, cloned genes coding for recombinant saxiphilin and C-lobe saxiphilin were modified to contain two tandemly located affinity tags, Flag epitope (DYKDDDDK) and His(6) (HHHHHH), at the protein C-terminus and were expressed in cultured insect cells using baculovirus vectors. Both tagged proteins are readily detected on immunoblots by anti-Flag monoclonal antibody. Flag-His(6)-tagged saxiphilin was purified to homogeneity using Ni(2+)-chelate affinity chromatography and Heparin Sepharose chromatography. Equilibrium analysis of [3H]STX binding to tagged saxiphilin and tagged C-lobe saxiphilin gave K(D) values of 0.75 and 2.7 nM, respectively. Flag-His(6)-tagged saxiphilin was also utilized in a microtiter well solid-phase assay with Reacti-bind metal chelate plates to measure [3H]STX binding and binding competition by unlabeled STX. Such Flag-His(6)-tagged derivatives of saxiphilin have many possible applications in the assay of STX and related toxinological research.

Amphibian Proteins↗
PubMed2003

Identification of Enterobacter bacteria as saxitoxin producers in cattle's rumen and surface water from Venezuelan Savannahs.

We have previously shown that a paralytic toxin able to block sodium channels in nerve is associated with a cattle disease known as bovine paraplegic syndrome (BPS) [Toxicon. 31 (1993) 1581]. We have now identified this as saxitoxin (STX) using HPLC by either the methods of [Toxicon. 31 (1993) 1581], or [Toxicon. 25 (1987) 1105]. In recent experiments we were able to collect and cultivate facultative anaerobic bacteria growing on rumen, grass and ponds of corrals with high incidence of BPS; the cultured bacteria produce compounds indistinguishable from STX under both HPLC procedures described above. Two species of the Enterobacter genus (E. asburiae and E. cloacae) and a strain of Klebsiella pneumoniae, were identified using standard biochemical criteria as well as gas chromatography of bacterial lipids. All these bacteria produced STX in aerobic cultures.

Animal Feed↗
PubMed1999

Toxic effects, pharmacokinetics and clearance of saxitoxin, a component of paralytic shellfish poison (PSP), in cats.

Saxitoxin (STX) was the first known and most studied toxic component of paralytic shellfish poisoning (PSP). This toxin blocks neuronal transmission by binding to the voltage-gated Na+ channel. Although the toxin's mechanism of action is well known at the molecular level, there are still many unresolved questions about its pharmacokinetics and the PSP intoxication syndrome in mammals. Some of these questions are addressed in the present paper, which describes an experimental design which allowed us to follow the dynamics of STX poisoning in vivo. Adult cats were anaesthetized and permanently coupled to artificial ventilation, they were then intravenously injected with Low (2.7 microg of STX/kg) and high doses (10 microg of STX/kg) of toxin. Cardiovascular parameters such as blood pressure and electrocardiograms were recorded, urine and blood samples were collected during the four hours of experimental time. In order to quantify mass amount of STX, we used the post-column derivatization HPLC method. Urine and blood samples were cleansed using a C-18 Sep-Pack cartridge and ultrafree microcentrifuge filters. At the end of each experiment, the animals were killed and tissue samples from brain, liver, spleen and medulla oblongata were extracted to measure the amount of STX. As compared to control period, Low doses of STX made no difference in hemodynamics parameters. In contrast, high doses drastically reduced blood pressure, produced myocardial failure and finally cardiac arrest. Administration of 2.5 microg/kg x min of dobutamine restored hemodynamics parameters and allowed the animal to overcome the shock. With high doses, the calculated STX renal clearance in cats is 0.81 ml/min x kg(-1). This valued corresponds to 20.25% of the reported inulin renal clearance. Nevertheless with Low doses the STX renal clearance is 3.99 ml/min x kg(-1). This data suggest that in cats with normal cardiovascular parameters and diuresis, the STX excretion mainly involves glomerular filtration. During experimental time, no PSP toxins other than STX was detected in the body fluids and tissue samples analyzed, indicating that the mammals can not metabolize this molecule. STX was found in intensely irrigated organs such as the liver and spleen but also in the central nervous system (brain and medulla oblongata), showing that STX was capable of crossing the blood brain barrier.

Anesthesia↗
PubMed1995

Expression of saxiphilin in insect cells and localization of the saxitoxin-binding site to the C-terminal domain homologous to the C-lobe of transferrins.

Saxiphilin is a plasma protein from the bullfrog (Rana catesbeiana) that is homologous to transferrin. Most known transferrins contain two binding sites for Fe3+/HCO3-, one in each of two homologous domains called the N-lobe and C-lobe. However, native saxiphilin does not bind Fe3+ but stoichiometrically binds one molecule of the neurotoxin saxitoxin (STX) with a dissociation constant (KD) of approximately 0.2 nM. To pursue structural analysis of the STX binding sites, cDNA encoding saxiphilin was used to construct a baculovirus expression vector that directs synthesis and secretion of a approximately 92-kDa recombinant saxiphilin protein (R-sax) in cultured insect cells. Culture medium harvested from infected cells contained 25-67 pmol of [3H]STX binding sites/mL with a KD of 0.22 nM. The kinetics and pH dependence (pK0.5 = 5.4) of [3H]STX binding to R-sax are similar to native saxiphilin, implying proper folding and functional activity. Another baculovirus expression vector was constructed to encode a deletion mutant of saxiphilin consisting of the first 20 N-terminal residues containing the secretory signal sequence spliced to the C-terminal, 361-residue fragment homologous to the C-lobe domain of transferrins. This vector directed the secretion of a approximately 38-kDa derivative of saxiphilin (C-sax) that was recognized by antisaxiphilin antibody. C-sax also exhibited [3H]STX binding activity with a lower affinity KD of approximately 0.9 nM, a 4-fold faster dissociation rate for [3H]STX than native saxiphilin, and a pH dependence (pK0.5 = 5.7) similar to R-sax (pK0.5 = 5.4).(ABSTRACT TRUNCATED AT 250 WORDS)

Amphibian Proteins↗
PubMed1987

Kinetic basis for insensitivity to tetrodotoxin and saxitoxin in sodium channels of canine heart and denervated rat skeletal muscle.

The single-channel blocking kinetics of tetrodotoxin (TTX), saxitoxin (STX), and several STX derivatives were measured for various Na-channel subtypes incorporated into planar lipid bilayers in the presence of batrachotoxin. The subtypes studied include Na channels from rat skeletal muscle and rat brain, which have high affinity for TTX/STX, and Na channels from denervated rat skeletal muscle and canine heart, which have about 20-60-fold lower affinity for these toxins at 22 degrees C. The equilibrium dissociation constant of toxin binding is an exponential function of voltage (e-fold per 40 mV) in the range of -60 to +60 mV. This voltage dependence is similar for all channel subtypes and toxins, indicating that this property is a conserved feature of channel function for batrachotoxin-activated channels. The decrease in binding affinity for TTX and STX in low-affinity subtypes is due to a 3-9-fold decrease in the association rate constant and a 4-8-fold increase in the dissociation rate constant. For a series of STX derivatives, the association rate constant for toxin binding is approximately an exponential function of net toxin charge in membranes of neutral lipids, implying that there is a negative surface potential due to fixed negative charges in the vicinity of the toxin receptor. The magnitude of this surface potential (-35 to -43 mV at 0.2 M NaCl) is similar for both high- and low-affinity subtypes, suggesting that the lower association rate of toxin binding to toxin-insensitive subtypes is not due to decreased surface charge but rather to a slower protein conformational step. The increased rates of toxin dissociation from insensitive subtypes can be attributed to the loss of a few specific bonding interactions in the binding site such as loss of a hydrogen bond with the N-1 hydroxyl group of neosaxitoxin, which contributes about 1 kcal/mol of intrinsic binding energy.

Animals↗
PubMed2001

Radioreceptor assays for sensitive detection and quantitation of saxitoxin and its analogues from strains of the freshwater cyanobacterium, Anabaena circinalis.

Toxic freshwater cyanobacteria can contaminate water supplies and adversely effect humans, agricultural livestock, and wildlife. Toxicity is strain-specific so morphological observations alone cannot predict the hazard level. Two microtiter plate based bioassays have emerged for measuring saxitoxin (STX) and its derivatives, commonly found in the freshwater cyanobacteria Anabaena and Aphanizomenon. They use radioactively labeled STX binding by sodium channels, STX's pharmacological target, or an unrelated protein, saxiphilin. These bioassays were challenged with extracts of toxic and nontoxic strains of Anabaena circinalis, and the results were compared with HPLC analysis. Both radioreceptor assays had detection limits of 2 microg STX equivalents (STXeq)/L, which is belowthe concentration proposed for a health alert, namely 3 microg STXeq/L. In all cases, statistically significant correlations existed between all toxicity measurements of the same extracts with the methods used herein. Sodium channel and saxiphilin assays however predicted less toxicity relative to HPLC analysis. The only exception to this was the equivalency observed between saxiphilin measurement and HPLC quantitation corrected for mammalian toxicity. Saxiphilin assay predicted toxicity in one strain was 3 orders of magnitude more than by sodium channel assay, and no STX was detected by HPLC. Lack of acetylcholinesterase inhibition showed this bioactivity was not anatoxin-a(S), a toxin also produced by this A. circinalis with some resemblance to the region of STX bound by saxiphilin. Presence of anatoxin-a(S) was predicted for another strain by this same acetylcholinesterase assay that, if confirmed by chemical analysis, would be the first report of anatoxin-a(S) in an Australian cyanobacterium.

Acetylcholinesterase↗
PubMed2006

Detection of marine toxins, brevetoxin-3 and saxitoxin, in seawater using neuronal networks.

There is a need for assay systems that can detect known and unanticipated neurotoxins associated with harmful algal blooms. The present work describes our attempt to monitor the presence of brevetoxin-3 (PbTx-3) and saxitoxin (STX) in a seawater matrix using the neuronal network biosensor (NNB). The NNB relies on cultured mammalian neurons grown over microelectrode arrays, where the inherent bioelectrical activity of the network manifested as extracellular action potentials can be monitored noninvasively. Spinal cord neuronal networks were prepared from embryonic mice and the mean spike rate across the network was analyzed before and during exposure to the toxins. Extracellular action potentials from the network are highly sensitive not only to purified STX and PbTx-3, but also when in combination with matrixes such as natural seawater and algal growth medium. Detection limits for STX and PbTx-3, respectively, are 0.031 and 0.33 nM in recording buffer and 0.076 and 0.48 nM in the presence of 25-fold-diluted seawater. Our results demonstrated that neuronal networks could be used for analysis of Alexandrium fundyense (STX-producer) and Karenia brevis (PbTx-producer) algal samples lysed directly in the seawater-based growth medium and appropriately diluted with HEPES-buffered recording medium. The cultured network responded by changes in mean spike rate to the presence of STX-or PbTx-producing algae but not to the samples of two non-STX and non-PbTx isolates of the same algal genera. This work provides evidence that the NNB has the capacity to rapidly detect toxins associated with cells of toxic algal species or as dissolved forms present in seawater and hasthe potential for monitoring toxin levels during harmful algal blooms.

Animals↗
PubMed2006

A synthesis of (+)-saxitoxin.

An asymmetric synthesis of the bis-guanidinium poison, (+)-saxitoxin (STX), is described. Commencing from an N,O-acetal starting material made readily available through sulfamate ester C-H amination, the completed route to STX showcases the utility of oxathiazinane dioxide heterocycles for the assembly of polyfunctionalized amine derivatives. In the final preparative stages, an unusual nine-membered ring guanidine intermediate is oxidized selectively and made to undergo dehydrative cyclization to afford the tricyclic core of the natural product. Access to STX and related structures will provide unique pharmacological tools for the study of voltage-regulated Na+ ion channel proteins.

Saxitoxin↗
PubMed2003

Three novel hydroxybenzoate saxitoxin analogues isolated from the dinoflagellate Gymnodinium catenatum.

In a recent survey of paralytic shellfish poisoning (PSP) toxins in Gymnodinium catenatum Graham extracts, using LC with postcolumn oxidation and fluorescence detection, three novel saxitoxin analogues were revealed in isolates from several locations, including Australian waters. We have named them as G. catenatum toxins, GC1 (1), GC2 (2), and GC3 (3). The compounds were isolated from a culture of the Australian strain by LC-MS-guided fractionation employing a C18-silica column and hydrophilic interaction chromatography. The unusual structures of these novel compounds were characterized by low- and high-resolution MS, MS/MS, and NMR spectroscopy. GC3 (3) was found to be the 4-hydroxybenzoate ester derivative of decarbamoylsaxitoxin, while GC1 (1) and GC2 (2) are the epimeric 11-hydroxysulfate derivatives of GC3 (3).

Animals↗
PubMed2005

Sodium channel mutation leading to saxitoxin resistance in clams increases risk of PSP.

Bivalve molluscs, the primary vectors of paralytic shellfish poisoning (PSP) in humans, show marked inter-species variation in their capacity to accumulate PSP toxins (PSTs) which has a neural basis. PSTs cause human fatalities by blocking sodium conductance in nerve fibres. Here we identify a molecular basis for inter-population variation in PSP resistance within a species, consistent with genetic adaptation to PSTs. Softshell clams (Mya arenaria) from areas exposed to 'red tides' are more resistant to PSTs, as demonstrated by whole-nerve assays, and accumulate toxins at greater rates than sensitive clams from unexposed areas. PSTs lead to selective mortality of sensitive clams. Resistance is caused by natural mutation of a single amino acid residue, which causes a 1,000-fold decrease in affinity at the saxitoxin-binding site in the sodium channel pore of resistant, but not sensitive, clams. Thus PSTs might act as potent natural selection agents, leading to greater toxin resistance in clam populations and increased risk of PSP in humans. Furthermore, global expansion of PSP to previously unaffected coastal areas might result in long-term changes to communities and ecosystems.

Amino Acid Sequence↗
PubMed2006

Saxitoxin, a toxic marine natural product that targets a multitude of receptors.

Saxitoxin (STX) was discovered early last century and can contaminate seafood and drinking water, and over time has become an invaluable research tool and an internationally regulated chemical weapon. Among natural products, toxins obtain a unique reputation from their high affinity and selectivity for their target pharmacological receptor, which for STX has long been considered to only be the voltage gated sodium channel. In recent times however, STX has been discovered to also bind to calcium and potassium channels, neuronal nitric oxide synthase, STX metabolizing enzymes and two circulatory fluid proteins, namely a transferrin-like family of proteins and a unique protein found in the blood of pufferfish.

Amino Acid Sequence↗
PubMed1974

Evidence that tetrodotoxin and saxitoxin act at a metal cation binding site in the sodium channels of nerve membrane.

The effects of monovalent, divalent, and trivalent cations on the binding of tetrodotoxin and saxitoxin to intact nerves and to a preparation of solubilized nerve membranes have been examined. All eight divalent and trivalent cations tested, and the monovalent ions Li(+), Tl(+), and H(+) appear to compete reversibly with the toxins for their binding site. The ability of lithium to reduce toxin binding is paralleled by its ability to reduce tetrodotoxin-sensitive ion fluxes through the nerve membrane. We conclude that the toxins act at a metal cation binding site in the sodium channel and suggest that this site is the principal coordination site for cations (normally Na(+) ions) as they pass through the membrane during an action potential. The dissociation constant for Li(+) is 0.1-0.2 M and for Na(+) > 0.6 M, reflecting the weak binding necessary for rapid passage of sodium ions through the channel.

Animals↗
PubMed1980

Molecular characterization, reconstitution, and "transport-specific fractionation" of the saxitoxin binding protein/Na+ gate of mammalian brain.

The saxitoxin (STX) binding protein has been solubilized by sodium cholate, both from axolemma and from synaptosomal membranes of mammalian brain. On the basis of agarose gel filtration and sedimentation properties in H2O and 2H2O, the solubilized particle has the following molecular properties: Stokes radius, 120 A; partial specific volume, 0.85 cm3/g; mass, 1,020,000 daltons; frictional ratio f/fo, 1.6. The solubilized STX binding protein was incorporated into unilamellar (approximately 550-A) artificial phosphatidylcholine vesicles. Based on the expectation that the STX binding protein contains functional monovalent cation gating activity ("action potential Na+ gate") that can be activated by veratridine and inhibited by tetrodotoxin, a strategy was devised for partial purification of the reconstituted sodium gate/STX binding protein by "transport-specific fractionation." When the entire vesicle population was preloaded with 0.4 M cesium ion, addition of veratridine allowed Cs+ efflux from specifically those vesicles containing the ion gate; the concomitant reduction in intravesicular density permitted the ion gate/STX binding protein to be fractionated on density gradients. These observations demonstrate functional reconstitution and partial (30- to 50-fold) purification of the STX binding protein/Na+ gate of mammalian brain.

Amphibian Proteins↗
PubMed1981

Purification of the saxitoxin receptor of the sodium channel from rat brain.

The saxitoxin (STX) receptor has been purified 740-fold from rat brain by a combination of ion exchange chromatography, wheat germ agglutinin chromatography, and sedimentation on sucrose gradients to a specific activity of 1488 pmol/mg of protein. The best fractions were estimated to be 47% pure from their specific activity or 66% pure on the basis of NaDodSO4 gel electrophoresis. Two polypeptides, alpha (Mr approximately equal to 270,000 +/- 10,000) and beta (Mr approximately equal to 38,300 +/- 2000) (mean +/- SD) copurify with STX binding activity. Two polypeptides of the same apparent Mr are specifically covalently labeled by photoreactive derivatives of 125I-labeled scorpion toxin in rat brain synaptosomes and are likely to be identical to alpha and beta. The solubilized STX receptor has a Mr of 316,000 +/- 63,000, limiting its composition to one alpha polypeptide and one or more beta polypeptides per soluble receptor. Our results suggest that the alpha and beta polypeptides contain both the STX binding site and the scorpion toxin binding site of the mammalian sodium channel.

Amphibian Proteins↗
PubMed1983

Extraneuronal saxitoxin binding sites in rabbit myelinated nerve.

The changes in binding of 3H-labeled saxitoxin (STX) to rabbit sciatic nerve during axonal regeneration (after nerve crush) and during axonal degeneration (after nerve section) were measured and compared with the corresponding changes in the sciatic nerves of other mammals (rat, guinea pig, and cat). In the rabbit and rat, regeneration after nerve crush is associated with a 2- to 4-fold increase in STX binding capacity, consistent with the known corresponding increase in the number of nodes of Ranvier in regenerating nerve. Furthermore, consistent with the disappearance of nodes that occurs with Wallerian degeneration, nerve section leads to a disappearance of all, or most, of the STX binding in rat and guinea pig nerve, similar to that previously found for cat nerve. However, in the rabbit, nerve section leads to a large maintained increase in STX binding. Intraneural injection of diphtheria toxin, which is known to damage Schwann cells and which causes an increase in STX binding in intact nerves, abolishes the binding in cut nerves. It is suggested that the increased binding in cut nerves is to nonneuronal sites situated on the surface membrane of the Schwann cells, which have greatly proliferated in number as axonal degeneration has progressed. The reason for the difference between rabbits and other species and the possibility that the binding sites of rabbit Schwann cells represent functional sodium channels remain to be investigated.

Amphibian Proteins↗