Use-dependent block of the voltage-gated Na+ channel by tetrodotoxin and saxitoxin: Effect of pore mutations that change ionic selectivity

Voltage-gated Na⁺ channels (NaV channels) are specifically blocked by guanidinium toxins such as tetrodotoxin (TTX) and saxitoxin (STX) with nanomolar to micromolar affinity depending on key amino acid substitutions in the outer vestibule of the channel that vary with NaV gene isoforms. All NaV channels that have been studied exhibit a use-dependent enhancement of TTX/STX affinity when the channel is stimulated with brief repetitive voltage depolarizations from a hyperpolarized starting voltage. Two models have been proposed to explain the mechanism of TTX/STX use dependence: a conformational mechanism and a trapped ion mechanism. In this study, we used selectivity filter mutations (K1237R, K1237A, and K1237H) of the rat muscle NaV1.4 channel that are known to alter ionic selectivity and Ca²⁺ permeability to test the trapped ion mechanism, which attributes use-dependent enhancement of toxin affinity to electrostatic repulsion between the bound toxin and Ca²⁺ or Na⁺ ions trapped inside the channel vestibule in the closed state. Our results indicate that TTX/STX use dependence is not relieved by mutations that enhance Ca²⁺ permeability, suggesting that ion–toxin repulsion is not the primary factor that determines use dependence. Evidence now favors the idea that TTX/STX use dependence arises from conformational coupling of the voltage sensor domain or domains with residues in the toxin-binding site that are also involved in slow inactivation.     

Verification and quantification of saxitoxin from algal samples using fast and validated hydrophilic interaction liquid chromatography-tandem mass spectrometry method

Hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was validated with algal samples for verification and quantification of saxitoxin (STX), a potent neurotoxin which is listed in the Chemical Weapons Convention (CWC) in Schedule 1A. Isocratic elution, conventional bore HILIC column and high flow rate together with accurate post-column splitter provided detection of STX in 6.5 min with total analysis time of 9 min per sample. STX analogue, gonyautoxin 1 (GTX 1) was used as an internal standard. Sample preparation of freeze-dried algae included liquid extraction and centrifugal filtering with mean recovery of 99.9% at concentration level of 10 ng/ml (n=3). Retention times for STX and GTX 1 were 6.47±0.03 min and 4.44±0.01 min (n=45), respectively. Four diagnostic product ions were used for reliable verification of saxitoxin. Method was found to be precise and linear (R(2)=0.9714 and R(2)=0.9768) in concentration ranges of 5-50 ng/ml and 25-200 ng/ml, respectively. For saxitoxin, calculated LOD was 3 ng/ml and LLOQ 11 ng/ml. Validation was conducted using spiked algal matrix since this method is not only needed for verification analysis for the CWC but also for safety analysis of other environmental samples for presence of STX. Identification criteria for verification of STX with HILIC-MS/MS method are discussed.     

Use-dependent block with tetrodotoxin and saxitoxin at frog Ranvier nodes I. Intrinsic channel and toxin parameters

The use-dependent phasic blockage of sodium channels by tetrodotoxin (TTX) and saxitoxin (STX) was examined in frog nodes of Ranvier using trains of depolarizing pulses. The decline of the peak Na⁺ current from its initial value (I ₀) before the train to a stationary value (I ) after the train was more pronounced at more negative holding potentials. The relationship betweenI /I ₀ and holding potential was fitted by a sigmoid function which yielded values for the steepness of the voltage dependencies of around –15 mV for TTX and – 8 mV for STX. Similar values were obtained at toxin concentrations of 4 and 8 nM. The higher voltage sensitivity of STX versus TTX is interpreted in terms of the higher charge and the faster binding kinetics of STX. These differences also explain the frequency dependence of the decline of Na⁺ currents with STX (between 0.5 and 2 Hz) and the frequency independence with TTX. Variation of the pulse amplitude in a train of conditioning pulses revealed that the magnitude of the use-dependent actions of STX parallels the steady-state Na⁺ inactivation curveh . Inhibition of inactivation, by pre-treatment with chloramine-T, did not, however, abolish the use dependence. Instead, it introduced a change in the time constants of the decline of the Na⁺ currents and the magnitude became independent of the holding potential.     

Development of an ultra high sensitive tissue biosensor for determination of swellfish poisoning, tetrodotoxin

A simple tissue biosensor for measuring Na⁺ channel blockers such as tetrodotoxin (TTX) and saxitoxin (STX) has been developed. The membrane of frog bladder has Na⁺ channels which control the passage of Na⁺. It is well known that TTX blocks Na⁺ channels. The tissue biosensor consists of a Na⁺ electrode integrated within a flow cell. The tip of the electrode was covered with frog bladder membrane sandwiched between two sheets of cellulose acetate membrane, and the electrode was set in a flow cell.

A solution of 8% NaCl was carried in the cell and the output of the electrode allowed to stabilize. TTX was injected into the sensor system and measured from the inhibition ratio of the sensor peak output. One assay took approximately 5 min. The lower limit of detection was 86 fg. The continuous determination of TTX was feasible for 250 h in the presence of 0·003% NaN₃. A Linear correlation was obtained between TTX activities of F-niphobles and F-parudale determined by the methods of TTX sensor and mouse assay.     

Periodate treatment reduces the tetrodotoxin-sensitivity of voltage-gated Na+ channels

(1) Voltage-clamped nerve fibres of the frog Rana esculenta were treated with periodate in the extracellular solution. (2) Periodate treatment irreversibly reduced the effect of tetrodotoxin (TTX) on the Na+ currents. (3) The effect of saxitoxin (STX) was also reduced but less than that of TTX. (4) The presence of STX during the application of periodate to the nerve fibre almost completely prevented the effect of the chemical reagent on the TTX sensitivity of the Na+ channels. (5) The reduction of the TTX effect is not due to the reaction of small amounts of periodate with the diol group of this toxin, because the effect was seen after prolonged washing with reagent-free Ringer solution with or without high amounts of ribose. (6) Carboxyl groups present in the Na+ channel seem to be quite important for the binding of TTX and STX. Periodate modifies several amino acid side chains, however, it does not attack carboxyl groups in a peptide chain. Thus, these results suggest that periodate modifies a further group critically involved in the binding of TTX and STX.     

Effects of deuterium oxide on the rate and dissociation constants for saxitoxin and tetrodotoxin action. Voltage-clamp studies on frog myelinated nerve

The actions of tetrodotoxin (TTX) and saxitoxin (STX) in normal water and in deuterium oxide (D2O) have been studied in frog myelinated nerve. Substitution of D2O for H2O in normal Ringer's solution has no effect on the potency of TTX in blocking action potentials but increases the potency of STX by approximately 50%. Under voltage clamp, the steady-state inhibition of sodium currents by 1 nM STX is doubled in D2O as a result of a halving of the rate of dissociation of STX from the sodium channel; the rate of block by STX is not measurably changed by D2O. Neither steady-state inhibition nor the on- or off-rate constants of TTX are changed by D2O substitution. The isotopic effects on STX binding are observed less than 10 min after the toxin has been added to D2O, thus eliminating the possibility that slow-exchange (t 1/2 greater than 10 h) hydrogen-binding sites on STX are involved. The results are consistent with a hypothesis that attributes receptor-toxin stabilization to isotopic changes of hydrogen bonding; this interpretation suggests that hydrogen bonds contribute more to the binding of STX than to that of TTX at the sodium channel.     

Differential effects of sulfhydryl reagents on saxitoxin and tetrodotoxin block of voltage-dependent Na channels.

We have probed a cysteine residue that confers resistance to tetrodotoxin (TTX) block in heart Na channels, with membrane-impermeant, cysteine-specific, methanethiosulfonate (MTS) analogs. Covalent addition of a positively charged group to the cysteinyl sulfhydryl reduced pore conductance by 87%. The effect was selectively prevented by treatment with TTX, but not saxitoxin (STX). Addition of a negatively charged group selectively inhibited STX block without affecting TTX block. These results agree with models that place an exposed cysteinyl sulfhydryl in the TTX site adjacent to the mouth of the pore, but do not support the contention that STX and TTX are interchangeable. The surprising differences between the two toxins are consistent with the hypothesis that the toxin-receptor complex can assume different conformations when STX or TTX bound.     

Effect of heterologous paralytic shellfish poisoning toxin-enzyme conjugates on the cross-reactivity of a saxitoxin enzyme immunoassay

A polyclonal antiserum against saxitoxin (STX) was used in a competitive enzyme immunoassay for the detection of paralytic shellfish poisoning toxins. The extent of cross-reactions was determined from the amounts of neoSTX, decarbamoylSTX and gonyautoxin 2/3 (GTX2/3) that gave 50% inhibition in the assay. Horseradish peroxidase (HRP) conjugates of the toxins and a bovine serum albumin conjugate of STX (STX-BSA) were used. When compared with STX-BSA and STX as standard, the extent varied to which heterologous conjugates affected the binding values of the other toxins to the antibodies. The antibodies did not bind GTX2/3-HRP. By use of neoSTX-HRP or decarbamoylSTX-HRP as the labelled antigen instead of STX-HRP, the detection limit for neoSTX was improved to 100 pg ml^-1.     

Nerve growth factor and fibroblast growth factor influence post-fusion expression of Na-channels in cultured rat skeletal muscle

We have examined effects of nerve growth factor (NGF) and fibroblast growth factor (FGF) on the density of tetrodotoxin (TTX)-sensitive Na-channels in cultured rat skeletal muscle. Measurements were made of specific binding of [3H]saxitoxin (STX) and the frequency and rate of rise of spontaneously occurring action potentials, the physiological expression of Na-channel density. Cells were transferred to various growth conditions at 6 days in vitro, and measurements were made beginning 24 hr later. Both growth factors (GF) caused dose-related increases in Na-channels compared with myotubes maintained in normal, serum-supplemented growth medium. Maximum effects occurred with a concentration of NGF of 50 ng/ml and FGF of 15 ng/ml. Scatchard analysis of specific STX binding showed an increase in Bmax with no significant change in Kd. Similar increases occurred on rate of rise and frequency spontaneous action potential. Treatment of cultures with cycloheximide or actinomycin D, inhibitors of protein and RNA synthesis, completely prevented the increase in STX-binding induced by GF treatment. The results indicate that NGF and FGF have important effects on regulation of excitable cell gene products after differentiation.     

Ultraviolet Irradiation Produces Loss of Saxitoxin Binding to Sodium Channels in Rat Synaptosomes

Ultraviolet irradiation (UV) has been shown to cause an electrophysiologically measured inactivation of the rapid, transient sodium conductance system in nerve. Tritiated saxitoxin ([³H]STX) was used as a structural probe to assess the possibility of a corresponding perturbation in the conformation of the STX binding site. UV irradiation caused an irreversible decrease in the total number of high-affinity [³H]STX binding sites in rat synaptosomes, while the dissociation constant of the remaining sites did not change. The receptor loss followed first-order kinetics, and the rate of loss was independent of temperature. The action spectrum for binding loss indicated a peak in spectral sensitivity near 280 nm. A²²Na flux assay in irradiated synaptosomes directly demonstrated that [³H]STX binding sites and veratridinestimulated, STX-blocked ²²Na efflux had similar sensitivities to UV radiation. We conclude that the UV inactivation of functional channels includes a modification of the STX binding-site structure.     

Direct enzyme immunoassay in microtitration plate and test strip format for the detection of saxitoxin in shellfish

Saxitoxin was coupled to horseradish peroxidase via a novel adaptation of the periodate reaction. Based on polyclonal antibodies against saxitoxin, this conjugate was used for the development of two formats of direct enzyme immunoassay (EIA)–a microtitration enzyme-linked immunosorbent assay (ELISA) and a test strip EIA. The detection of saxitoxin without instrumentation by visual evaluation of the test strip EIA is described. The detection limits for saxitoxin were 7 pg/ml (0·35 pg/assay) in the ELISA and 200 pg/ml in the test strip EIA using visual evaluation. Employing a simple procedure of sample preparation, both ELISA and test strip EIA were applied to the analysis of shellfish. The detection limits for saxitoxin in shellfish tissue of the ELISA and the test strip assay were 3 and 4 ng/g, respectively.     

The μI skeletal muscle sodium channel: Mutation E403Q eliminates sensitivity to tetrodotoxin but not to μ-conotoxins GIIIA and GIIIB

Voltage-sensitive Na channels from nerve and muscle are blocked by the guanidinium toxins tetrodotoxin (TTX) and saxitoxin (STX). Mutagenesis studies of brain RII channels have shown that glutamate 387 (E387) is essential for current block by these toxins. We demonstrate here that mutation of glutamate 403 (E403) of the adult skeletal muscle I channel (corresponding to E387 of RII) also prevents current blockade by TTX and STX, and by neo-saxitoxin. However, the mutation fails to prevent blockade by the peptide neurotoxins, -conotoxin GIIIA and GIIIB; these toxins are thought to bind to the same or overlapping sites with TTX and STX. The E403Q mutation may have utility as a marker for exogenous Na channels in transgenic expression studies, since there are no known native channels with the same pharmacological profile.     

Ionic channels and nerve membrane constituents. Tetrodotoxin-like interaction of saxitoxin with cholesterol monolayers

Saxitoxin (STX) and tetrodotoxin (TTX) have the same striking property of blocking the Na⁺ channels in the axolemma. Experiments with nerve plasma membrane components of the squid Dosidicus gigas have shown that TTX interacts with cholesterol monolayers. Similar experiments were carried out with STX. The effect of STX on the surface pressure-area diagrams of lipid monolayers and on the fluorescence emission spectra of sonicated nerve membranes was studied. The results indicate a TTX-like interaction of STX with cholesterol monolayers. The expansion of the monolayers caused by 10^-6 M STX was 2.2 A²/cholesterol molecule at 25°C. From surface pressure measurements at constant cholesterol area (39 A²/molecule) in media with various STX concentrations, it was calculated that the STX/cholesterol surface concentration ratio is 0.54. The apparent dissociation constant of the STX-cholesterol monolayer complex is 4.0 x 10^-7 M. The STX/cholesterol ratio and the apparent dissociation constant are similar to those determined for TTX. The presence of other lipids in the monolayers affects the STX-cholesterol association. The interactions of STX and TTX with cholesterol monolayers suggest (a) that cholesterol molecules may be part of the nerve membrane Na⁺ channels, or (b) that the toxin receptor at the nerve membrane shares similar chemical features with the cholesterol monolayers.     

Development of a direct competitive enzyme-linked immunosorbent assay for parathion residue in food samples

A heterologous direct competitive enzyme-linked immunosorbent assay (ELISA) for parathion residue determination is described based on a monoclonal antibody and a new competitor. The effects of several physicochemical factors, such as methanol concentration, ionic strength, pH value, and sample matrix, on the performance of the ELISA were optimized for the sake of obtaining a satisfactory assay sensitivity. Results showed that when the assay medium was in the optimized condition (phosphate buffer solution [PBS] containing 10% [v/v] methanol and 0.2 mol/L NaCl at a pH value of 5.0), the sensitivity (estimated as the IC₅₀ value) and the limit of detection (LOD, estimated as the IC₁₀ value) were 1.19 and 0.08 ng/ml, respectively. The precision investigation indicated that the intraassay precision values all were below 10% and that the interassay precision values ranged from 4.89 to 19.12%. In addition, the developed ELISA showed a good linear correlation (r² = 0.9962) to gas chromatography within the analyte’s concentration range of 0.1 to 16 ng/ml. When applied to the fortified samples (parathion adding level: 5-15 μg/kg), the developed ELISA presented mean recoveries of 127.46, 122.52, 91.92, 124.01, 129.72, 99.37, and 87.17% for tomato, cucumber, banana, apple, orange, pear, and sugarcane, respectively. Results indicated that the established ELISA is a potential tool for parathion residue determination.     

Development and assessment of radioreceptor binding assays for the detection of saxitoxin binding proteins in biological extracts

Several radioreceptor assays using tritiated saxitoxin ([(3)H]STX) were developed to identify a suitable primary screening method for the detection and characterization of soluble saxitoxin binding proteins from biological extracts. Assays using anion and cation exchange, protein binding, and traditional charcoal radioreceptor methods were compared with two previously reported formats. A protein binding assay incorporating filters of mixed cellulose esters (MCE) outperformed all other assay strategies with maximal signal, low background, exceptional reproducibility, minimal matrix effects, and high throughput. Binding site titrations verified that an increase in total protein in the assay led to a concomitant linear increase in the amount of specifically bound [(3)H]STX within the range of 1-90microg total protein. Saturation binding experiments demonstrated that the binding sites were saturable and that nonspecific binding was linear. The MCE assay was unaffected by 600mM NaCl and 500mM KCl. Likewise, minimal variation of specific binding was observed between pH 5 and pH 9, but inhibition was observed below pH 5.     

Development of a noncompetitive phage anti-immunocomplex assay for brominated diphenyl ether 47

We present a new application of the noncompetitive phage anti-immunocomplex assay (PHAIA) by converting an existing competitive assay to a versatile noncompetitive sandwich-type format using immunocomplex binding phage-borne peptides to detect the brominated flame retardant, brominated diphenyl ether 47 (BDE 47). Three phage-displayed 9-mer disulfide-constrained peptides that recognize the BDE 47-polyclonal antibody immunocomplex were isolated. The resulting PHAIAs showed variable sensitivities, and the most sensitive peptide had a dose-response curve with an SC₅₀ (concentration of analyte producing 50% saturation of the signal) of 0.7 ng/ml BDE 47 and a linear range of 0.3-2 ng/ml, which was nearly identical to the best heterologous competitive format (IC₅₀ of 1.8 ng/ml, linear range of 0.4-8.5/ml). However, the PHAIA was 1400-fold better than homologous competitive assay. The validation of the PHAIA with extracts of house furniture foam as well as human and calf sera spiked with BDE 47 showed overall recovery of 80-113%. The PHAIA was adapted to a dipstick format (limit of detection of 3.0 ng/ml), and a blind test with six random extracts of local house furniture foams showed that the results of the PHAIA and dipstick assay were consistent, giving the same positive and negative detection.     

Examination of the seasonal dynamics of the toxic dinoflagellate Alexandrium catenella at Redondo Beach, California, by quantitative PCR

The presence of neurotoxic species within the genus Alexandrium along the U.S. coastline has raised concern of potential poisoning through the consumption of contaminated seafood. Paralytic shellfish toxins (PSTs) detected in shellfish provide evidence that these harmful events have increased in frequency and severity along the California coast during the past 25 years, but the timing and location of these occurrences have been highly variable. We conducted a 4-year survey in King Harbor, CA, to investigate the seasonal dynamics of Alexandrium catenella and the presence of a particulate saxitoxin (STX), the parent compound of the PSTs. A quantitative PCR (qPCR) assay was developed for quantifying A. catenella in environmental microbial assemblages. This approach allowed for the detection of abundances as low as 12 cells liter⁻¹, 2 orders of magnitude below threshold abundances that can impact food webs. A. catenella was found repeatedly during the study, particularly in spring, when cells were detected in 38% of the samples (27 to 5,680 cells liter⁻¹). This peak in cell abundances was observed in 2006 and corresponded to a particulate STX concentration of 12 ng liter⁻¹, whereas the maximum STX concentration of 26 ng liter⁻¹ occurred in April 2008. Total cell abundances and toxin levels varied strongly throughout each year, but A. catenella was less abundant during summer, fall, and winter, when only 2 to 11% of the samples yielded positive qPCR results. The qPCR method developed here provides a useful tool for investigating the ecology of A. catenella at subbloom and bloom abundances.     

The sodium channel from rat brain. Role of the beta 1 and beta 2 subunits in saxitoxin binding

Procedures are described for the selective removal of the beta 1 or the beta 2 subunits from the detergent-solubilized channel from rat brain, and the functional integrity of the resulting protein complex is examined. Treatment of the channel with 1.0 M MgCl2 followed by sedimentation through sucrose gradients results in complete separation of beta 1 from the alpha-beta 2 complex and complete loss of [3H]saxitoxin (STX) binding activity. At intermediate MgCl2 concentrations, the loss of beta 1 and the loss of [3H]STX binding activity are closely correlated. Tetrodotoxin (TTX) quantitatively stabilizes the solubilized complex against both the loss of beta 1 and the loss of [3H]STX binding activity. This indicates that association of the alpha and beta 1 subunits is required to maintain the STX/TTX binding site in a conformation with high affinity for STX and TTX in the detergent-solubilized state. Treatment of the solubilized sodium channel with dithiothreitol in the presence of TTX causes specific release of the beta 2 subunit, without significantly removing beta 1. There is little or no correlation between the amount of beta 2 in the sodium channel complex and the ability of the preparation to bind [3H]STX. We conclude from these studies that the presence of beta 1, but not beta 2, is required for the integrity of the STX/TTX binding site of the solubilized and purified rat brain sodium channel.     

A direct competitive enzyme-linked immunosorbent assay by antibody coated for diethyl phthalate analysis

A direct competitive enzyme-linked immunosorbent assay (ELISA) has been developed for detection of diethyl phthalate (DEP). Protein-hapten conjugate was synthesized to produce polyclonal antibodies against DEP. Experimental parameters were optimized, including immunoreaction conditions, the dilution ratio of horseradish peroxidase (HRP)-antigen conjugate, time of the antibody coated, effect of pH, and ionic strength. The limit of detection was 0.096 ng/ml, and the linear range was 0.1-3500 ng/ml with a regression coefficient (R²) of 0.9957. Recoveries were between 96.4 and 106.2%. The cross-reactivities of the anti-DEP antibody to six structurally related phthalate esters were less than 9%. The method was successfully applied to the determination of DEP in tap water, river water (Yangtze River), and leachate from plastic drinking bottles. This immunoassay was highly specific, sensitive, rapid, simple, and suitable for DEP monitoring. The results obtained were compared with those obtained using the high-performance liquid chromatography method.     

己烯雌酚特异性抗体的制备及鉴定

利用4-溴丁酸乙酯对小分子半抗原己烯雌酚(DES)进行活化,引入羧基活性基团,应用活泼酯法将其与牛血清白蛋白(BSA)偶联,合成DES-CP-BSA完全抗原,免疫新西兰长耳白兔,制备特异性抗体.结果显示:成功制备了DES完全抗原,且由此获得了特异性的DES抗体,效价达1.28×105,与己烷雌酚、双烯雌酚的交叉反应分别...