Voltage gated sodium channels

河豚毒素是一种具有潜在镇痛活性的神经毒素。河豚毒素与神经细胞膜快速电压门控快速钠通道的孔隙结合，抑制神经动作电位，阻断神经传递。

在缺氧后2分钟加入缬曲定，乳酸合成减少，在使用缬曲定30分钟后完全阻断。河豚毒素(TTX)，在缬草碱之前，阻止了缬草碱诱导的乳酸合成抑制，而（±）-kavain的效果不如TTX（图2(A)）。尽管未经处理的囊泡乳酸生成呈线性（图2(A)），但在缺氧情况下，ATP含量持续下降，半衰期（τ）为14.5 min（表1）。缺氧60min后，ATP降至有氧条件下初始值（2.85±0.16 nmol ATP/mg蛋白，n = 6）的21.0%。verattrine对缺氧囊泡的额外刺激加速了ATP的减少，其下降速度比未处理的囊泡快三倍（表1）。在verattrine之前添加TTX（图2(B)）不仅可以阻止verattrine的作用，而且与未处理的囊泡相比，TTX还降低了ATP下降的速度（表1）并提高了缺氧时ATP的含量（图2(B)）。（±）-Kavain也抑制了veratridine对ATP的作用，但效果不如TTX（图2(B)）。由表1可知，缺氧后60min测定的ATP含量与ATP下降τ的相关顺序为：[缬草碱+ TTX] >[对照]>[缬草碱+（±）-kavain] >[缬草碱]。如果用40 mmol/l KCl代替veratridine刺激囊泡，既没有检测到乳酸合成的抑制（图3(A)），也没有检测到对atp含量的任何影响（图3(B)）。需要强调的是，与缬草碱处理囊泡获得的结果（图2(B)）相比，将TTX应用于kcl去极化囊泡，与对照组相比，未能提高atp含量（图3(B)）。与TTX一样，（±）-kavain对乳酸合成（图3(A)）、ATP下降速度和缺氧结束时测定的最终ATP含量（图3(B)）没有任何影响。[1]

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由于长时间缺氧后只有缬曲啶阻断了乳酸合成，加速了ATP含量的下降，因此我们在短期缺氧和缬曲啶刺激期间连续跟踪[Na+]i、[Ca2+]i和乳酸生成，以确定Na+过载是否直接影响乳酸合成。如图4(A)所示，缺氧后[Na+]i持续升高，缺氧后380秒检测到的基础[Na+]i从18±2 mmol/l增加到41±7 mmol/l。用缬草碱对缺氧囊泡进行额外刺激会立即增加[Na+]i，然后趋于稳定至119±21 mmol/l Na+ （n = 6，图4(A)）。缺氧前应用河豚毒素(TTX)和（±）-kavain分别可减少或阻止缺氧和缬草碱诱导的[Na+]i升高。然而，这两种化合物都未能完全阻断[Na+]i的增加（图4(A)）。为了评估TTX和（±）-kavain对缺氧诱导的[Na+]i升高的抑制作用，计算缺氧前20秒和缺氧后380秒测定的基础[Na+]i与缺氧诱导的[Na+]i的差异，并用Δ[Na+]i表示。未处理的囊泡Δ[Na+]i（22.1±6.1 mmol/l Na+, n = 6）被TTX和（±）-kavain分别抑制至77%（17±5 mmol/l Na+, n = 6）和59%(13±6 mmol/l Na+, n = 6)（图4(A)）。[1]

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关于[Ca2+]i的测量，在缺氧前20秒检测到的基础[Ca2+]i为581±86 nmol/l Ca2+，在缺氧380秒后持续增加到856±87 nmol/l Ca2+ (n = 6)（图4(B)）。与[Na+]i相比，向缺氧小泡中添加缬草碱会引起[Ca2+]i的快速增加（图4(a)），但未能保持平稳，而是线性增加（图4(B)），其速率为355±126 nmol Ca2+/min/mg蛋白（n = 6）。在缺氧前应用河豚毒素(TTX)或（±）-kavain降低了缺氧和缬草碱诱导的[Ca2+]i增强，但是，正如已经观察到的[Na+]i，两种化合物都不能完全阻止[Ca2+]i的增加（图4(B)）。考虑到TTX和（±）-kavain对缺氧增强[Ca2+]i的作用，Δ[Ca2+]i类比于Δ[Na+]i，如上所述。TTX和（±）-kavain使未处理的囊泡（275±35 nmol/l Ca2+, n = 6）的Δ[Ca2+]i分别降低41%（113±13 nmol/l Ca2+, n = 6）和48%（133±35 nmol/l Ca2+, n = 6）。[1]

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为了比较[Na+]i和[Ca2+]i随乳酸合成而增加的时间过程，采用荧光法连续监测乳酸生成。如图5所示，在缺氧条件下，无刺激囊泡的基础乳酸合成速率从3.5±1.4 nmol乳酸/min/mg蛋白质提高到14.6±1.5 nmol乳酸/min/mg蛋白质，提高了4.2倍（表1）。通过线性回归计算，这些速率是根据分光光度法测定乳酸的速率的1.2-1.5倍。造成这种差异的原因尚不清楚，可能取决于不同的孵化程序。然而，在缺氧前240秒添加缬草碱使乳酸产量翻倍（图5，痕量4），这被认为是由于Na+内流激活Na+/K+- atp酶而导致能量需求增加的结果，因为河豚毒素(TTX)和（±）-kavain，都在缬草碱前120秒使用，阻止了这种刺激（图5）。在500秒的缺氧条件下，veratridine和预施用TTX和（±）-kavain都没有影响乳酸生成率（图5，表1），这段孵育时间足以使[Na+]i和[Ca2+]i提高到120 mmol/l Na+和3 μmol/l Ca2+（图4(A，B)）。

Spectrophotometric lactate determination [1]
The vesicle pellet was resuspended in 3 ml incubation buffer (125 mmol/l NaCl, 3.5 mmol/l KCl, 1.2 mmol/l CaCl2, 1.2 mmol/l MgCl2, 25 mmol/l NaHCO3, 10 mmol/l glucose) equilibrated with 95% O2 and 5% CO2 to obtain final protein concentrations of 3 mg/ml. The suspension was transferred into a self-made, closed chamber surrounded by a water jacket to maintain an incubation temperature of 37°C. Additionally, the incubation chamber was equipped with two gas taps for in- and out-streaming gas, allowing a surface equilibration. The O2 content of the suspension, expressed as % O2 saturation, was monitored continuously with a built-in, Clark-type, oxygen electrode, which was calibrated with sodium dithionite (Hitchman, 1978). One hundred percent O2 saturation was adjusted by equilibration of incubation buffer with O2 for 45 min at 37°C and 0.0% O2 saturation was attained by the addition of 500 mmol/l sodium dithionite, which traps dissolved O2 according to the reaction: 2Na2S2O4+2H2O+3H2O→4NaHSO4. After bubbling the suspension for 15 min with 95% O2 and 5% CO2 at 37°C, it was equilibrated with 95% N2 and 5% CO2 and the vesicles were allowed to respire until the medium was depleted of oxygen (0.0% O2 saturation). This point was defined as onset of anoxia. Lactate was determined enzymatically by a commercially available test kit according to the instructions of the supplier. Samples of vesicles, taken as indicated, were immediately frozen in liquid nitrogen and stored at −20°C until performance of the lactate assay. l-Lactate was determined spectrophotometrically (Noll, 1984) by the generation of NADH due to the oxidation of lactate to pyruvate, catalysed by lactate dehydrogenase (LDH, EC 1.1.1.27).

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ATP determination [1]
Twenty microlitres of the vesicle suspension with a protein content of about 3 mg/ml were mixed with 200 μl precooled 1.0 mol/l perchloric acid, 50 mmol/l EDTA at 0°C for 60 sec. The suspension was adjusted to pH 7.5–8.0 by the addition of 316 μl 1.0 mol/l KOH and 180 μl 0.1 mol/l HEPES, 10 mmol/l MgCl2 (pH 7.75, 25°C) and centrifuged at 14 000g for 10 min. Five-hundred microlitres of the supernatant was mixed with 500 μl 0.1 mol/l HEPES, 10 mmol/l MgCl2 (pH 7.75, 25°C) and the ATP content was detected by the luciferase-bioluminescence method employing the kit HS II and the luminometer LB 9502 (Berthold GmbH, Bad Wildbad, Germany). The ATP content was calculated according to a calibration curve, using different ATP concentrations as standards.

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Fluorometric lactate determination [1]
Continuous detection of liberated l-lactate during short-term anoxia was performed enzymatically employing LDH (EC 1.1.1.27) and 3-acetylpyridineadeninedinucleotide (APAD) as an analogue of NAD (Kaplan and Ciotti, 1956) according to the reaction: l-lactate+ APAD ↫ pyruvate+APADH. The generation of APADH was fluorometrically detected at excitation and emission wavelengths of 410 nm and 490 nm, respectively, taking bandpass slits of 20 nm for both monochromators. For fluorescence measurement, each vesicle pellet was resuspended in 3 ml incubation buffer to obtain a final protein concentration of 3 mg/ml. The suspension was transferred to a temperature-controlled, stirred cuvette located in a spectrofluorometer. Additionally, LDH and APAD were added to obtain final concentrations of 50 units/ml LDH and 5 mmol/l APAD. Afterwards, the cuvette was sealed with a cap equipped with two gas taps for in- and out-streaming gas and a built-in, Clark-type, oxygen electrode allowing continuous oxygen detection. Anoxic conditions were induced by surface equilibration of the suspension with 95% N2 and 5% CO2. The amount of lactate released from vesicles was calculated according to a calibration curve performed with a vesicle suspension (3 mg/ml protein) and different l-lactate standards.

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Intravesicular NADH ([NADH]i) [1]
Three millilitres of vesicle suspension (3 mg/ml protein) were transferred to a cuvette and incubated as described for fluorometric lactate determination, except for the addition of LDH and APAD. The increase in [NADH]i was followed by its fluorescence, determined at excitation and emission wavelengths of 340 nm and 460 nm, respectively, taking bandpass slits of 20 nm for both monochromators.

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Loading with SBFI-AM and FURA-2-AM [1]
[Na+]i and [Ca2+]i were determined by the ratio fluorescence method employing the acetoxymethyl esters (AM) of SBFI and FURA-2 as Na+- and Ca2+-sensitive fluorophores, respectively (Minta and Tsien, 1989; Grynkiewicz et al., 1985). Considering [Ca2+]i measurements, 80 μl of 1 mmol/l FURA-2-AM, dissolved in dimethylsulphoxide (DMSO), was added to 8 ml suspension with a protein content of about 4.5 mg/ml, to obtain final concentrations of 1% (v/v) DMSO and 10 μmol/l FURA-2-AM. In the case of [Na+]i determination, 66 μl of 2 mmol/l SBFI-AM and 22 μl 20% (w/v) pluronic F127, both dissolved in DMSO, were added to the suspension, to obtain final concentrations of 16.5 μmol/l SBR-AM, 0.055% (w/v) pluronic F127 and 1.1% (v/v) DMSO. The suspensions were incubated for 30 min at room temperature and subsequently washed three times with incubation buffer by centrifugation (5000g, 5 min) to remove unhydrolysed dye. Finally, the last pellet was resuspended in 12 ml incubation buffer, and the suspension was divided into portions of 9 mg protein. After centrifugation (5000g, 5 min) the pellets were stored on ice until measurement of fluorescence.

Absorption, Distribution and Excretion
In 1999, 23 specimens of the genus Polypedates were collected from two sites in Bangladesh (Mymensing and Barisal), and their toxicity scores and toxin components were determined. Of all tissues, only the skin of the Mymensing specimens showed toxicity in mouse tests, with toxicity scores ranging from 31 to 923 μg/g. The toxin isolated from the skin was identified as tetrodotoxin, a toxin component, by high-performance liquid chromatography, electrospray ionization time-of-flight mass spectrometry, and proton nuclear magnetic resonance analysis. Tetrodotoxin (TTX) and its analogues (TTXs) are widely distributed in marine and terrestrial animals and can cause dangerous poisoning. These highly toxic toxins are also pathogenic factors in pufferfish poisoning. Tetrodotoxin (TTX), dehydrated tetrodotoxin, 11-deoxytetrodotoxin, and trimeoxytetrodotoxin were determined in tissues isolated from the Bangladeshi pufferfish (Takifugu oblongus). Tetrodotoxin (TTX) was mainly found in the skin, muscle, and liver, while tripeoxytetrodotoxin was mainly found in the ovaries. The toxicity of each tissue was determined using a mouse bioassay. To investigate the relationship between tetrodotoxin toxicity and the distribution of tetrodotoxin-producing bacteria, we isolated bacteria from various organs (ovaries, liver, intestines, and gallbladder) of the red-finned pufferfish (Fugu rubripes) collected from the Bohai Sea, China, and screened their ability to produce tetrodotoxin (TTX). Of the 36 isolated strains, 20 were able to produce TTX in vitro. The number and toxicity of TTX-producing strains were higher in organs with higher toxicity, such as the ovaries and liver. Based on morphological observation, physiological and biochemical characteristics, and DNA G+C content, most TTX-producing strains were identified as Bacillus (19 strains) and Actinomyces (1 strain). The purified toxin was identified as TTX by high-performance liquid chromatography, thin-layer chromatography, and electrospray ionization mass spectrometry. Our results indicate that TTX-producing bacteria are closely related to pufferfish poisoning. Further research is needed to elucidate the synthetic mechanism of tetrodotoxin (TTX) and its role in bacteria.
Differential centrifugation was used to separate pufferfish liver homogenate into hemocyte, nucleus, mitochondria, microsomes, and cytosol components. High-performance liquid chromatography (HPLC) and liquid chromatography-fast atomic bombardment mass spectrometry (LC-FABMS) analysis showed that tetrodotoxin was the major toxic component in each component. These results reveal that tetrodotoxin is widely distributed in organelles of hepatocytes, but is mainly present in the cytosol component.
More data on the absorption, distribution, and excretion (ADEX) of tetrodotoxins (9 in total) can be found on the HSDB record page.
Metabolism/Metabolites
The metabolic source of tetrodotoxin is unclear. Algal sources have not been identified; until recently, tetrodotoxin was considered a host metabolite. However, recent reports indicate that various bacteria, including Vibrionaceae, Pseudomonas sp., and Photobacterium phosphoreum, can produce tetrodotoxin/dehydrated tetrodotoxin, suggesting that this toxin family may originate from bacteria. To investigate genes related to tetrodotoxin (TTX) biosynthesis or accumulation in pufferfish, we compared mRNA expression patterns in the livers of pufferfish (including Takifugu chrysops and Takifugu niphobles) carrying different concentrations of TTX and its derivatives using RAP RT-PCR. RAP RT-PCR yielded a 383 bp cDNA fragment whose transcript was expressed at higher levels in the livers of toxic pufferfish than in non-toxic pufferfish. Its deduced amino acid sequence is similar to fibrinogen-like proteins reported in other vertebrates. Northern blot analysis and rapid cDNA end amplification (RACE) revealed that the 383 bp cDNA fragment was composed of at least three fibrinogen-like protein (flp) genes, namely flp-1, flp-2, and flp-3. The relative mRNA levels of flp-1, flp-2, and flp-3 were linearly correlated with the toxicity of the livers of the two pufferfish species.

Toxicity Overview
Identification and Uses: Tetrodotoxin (TTX) is a solid. Since its channel-blocking effect was discovered in the early 1960s, TTX in inhaled aerosols has become an extremely common chemical tool in physiological and pharmacological laboratories. In recent years, TTX-tolerant sodium channels have been discovered in the nervous system and have attracted much attention due to their role in pain perception. It is now known that TTX is not produced by inhaled aerosols, but by bacteria and is distributed to various animals through the food chain. Human Studies: TTX is a lethal neurotoxin that selectively inhibits the Na(+) activation mechanism of nerve impulses without affecting K(+) ion permeability. TTX interferes with the transmission of nerve signals to muscles by blocking sodium channels. This leads to rapid weakness and paralysis of muscles (including respiratory muscles), which may eventually lead to respiratory arrest and death. Tetrodotoxin (TTX) poisoning can have a rapid onset (10 to 45 minutes) or a delayed onset (usually within 3 to 6 hours, but rarely longer than 3 to 6 hours). Death can occur within 20 minutes of exposure or up to 24 hours later; however, it usually occurs within the first 4 to 8 hours. Patients/victims who survive the acute poisoning phase within the first 24 hours usually recover without sequelae. Symptoms may last for several days, and recovery also takes several days. The first stage after ingestion of tetrodotoxin presents with numbness, tingling, and tingling of the lips and tongue (paresthesia), followed by paresthesia and numbness of the face and extremities, headache, lightheadedness, excessive sweating (hyperhidrosis), dizziness, drooling (salivation), nausea, vomiting, diarrhea, abdominal pain (upper abdominal pain), motor dysfunction, weakness (malaise), and difficulty speaking. The second stage presents with progressive paralysis, initially in the extremities, then in other parts of the body, and finally in the respiratory muscles; difficulty breathing or shortness of breath (tachypnea); cardiac arrhythmias; abnormally low blood pressure (hypotension); dilated pupils (fixed pupils); coma; seizures; respiratory arrest; and death. Several case reports describe food poisoning caused by tetrodotoxin (TTX). Most poisoning incidents occurred after consuming homemade pufferfish products, rather than commercially sourced pufferfish. In vitro experiments showed that TTX was not genotoxic in human lymphocytes, regardless of metabolic activation. Animal studies: Clinical symptoms and signs in dogs treated with intravenous TTX were similar to those of anticholinesterase poisoning. Studies found that oral TTX was approximately 50 times less toxic to mice than intraperitoneal injection, with later onset of death. In rats, serotonin levels in the brain significantly increased and peaked 4 hours after TTX administration. Levels of acetylcholine, histamine, and norepinephrine in the brain also significantly increased, but peaked after 6 hours. The effects of pufferfish gonadal extract were more pronounced and longer-lasting than those of skin extract. On the other hand, no significant changes in brain adrenaline levels were observed during the experiments. Intravenous injection of sublethal doses of tetrodotoxin (TTX) into male rabbits leads to perfusion failure, accompanied by lactic acidosis, hypoproteinemia, prolonged bleeding time, decreased red blood cell count, and decreased platelet count, resulting in shock. The severity of poisoning is directly proportional to the dose of tetrodotoxin. Autopsy studies revealed hemorrhage in the brain, liver, lungs, and diaphragm. Significant differences in susceptibility to TTX were observed among the five mouse strains tested. Both in vitro and in vivo experiments demonstrated that TTX is not genotoxic. Ecotoxicity studies: TTX and its analogues (TTXs) are widely distributed in marine and terrestrial animals and can cause dangerous poisoning. In addition to helping defend against predators, pufferfish's resistance to TTX allows them to selectively prey on organisms containing TTX. However, tetrodotoxin (TTX) does not protect Guam's flatworms from predators; instead, it is used to capture moving prey.

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Interactions
This study investigated the passive protective effect of a tetrodotoxin (TTX)-specific monoclonal antibody against lethal TTX challenge. The monoclonal antibody, T20G10, has an estimated affinity for TTX of approximately 10⁻⁹ M, exhibits approximately 50-fold lower reactivity to dehydrated tetrodotoxin, and is unresponsive to tetrodotoxin in a competitive immunoassay. T20G10 specifically inhibited TTX binding in an in vitro radioligand receptor binding assay but had no effect on the binding of tetrodotoxin to rat meningeal sodium channels. In a prophylactic study, mice were administered T20G10 via tail vein injection 30 minutes prior to challenge with intraperitoneal injection of TTX (10 μg/kg). Under these conditions, 100 μg of T20G10 protected 6/6 mice, while 50 μg of T20G10 protected 3/6 mice. The nonspecific control monoclonal antibody failed to prevent death. In a study simulating oral poisoning, a lethal dose of TTX was dissolved in phosphate buffer and skim milk powder via gavage. Six out of six mice that did not receive T20G10 treatment died within 25-35 minutes. However, intravenous injection of 500 μg of T20G10 via the tail vein 10-15 minutes after oral TTX prevented death in all six mice. Lower doses of monoclonal antibodies provided weaker protection. PMID: 8585093
In dogs 24 hours after coronary artery occlusion, lidocaine (4 mg/kg, intravenous) and tetrodotoxin (2 μg/kg, intravenous) both showed significant antiarrhythmic activity. When the dose was reduced by half, neither substance alone had any effect on arrhythmias, but when used in combination, cardiac rhythm was almost completely restored.
Frog toxin increases sodium uptake by synaptosomes. Veratrine also increases sodium uptake. Tetrodotoxin blocks the effects of the above toxins.
Denervated muscles in mice were removed after 5-6 days and incubated in 0.5% papain solution at 28-9°C for 5-8 minutes. This proteolytic treatment, by partially removing unstable surface proteins, eliminated the shielding effect of sodium ion channels, thereby restoring the sensitivity of receptors to tetrodotoxin after denervation injury.

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Non-human toxicity values
Oral LD50 in mice: 0.435 mg/kg Lewis, RJ Sr. (ed) Sax's Dangerous Properties of Industrial Materials. 11th Edition. Wiley-Interscience, Wiley & Sons, Inc. Hoboken, NJ. 2004., p. 1827
Intraperitoneal LD50 in mice: 0.008 mg/kg Lewis, RJ Sr. (ed) Sax's Dangerous Properties of Industrial Materials. 11th Edition. Wiley-Interscience, Wiley & Sons, Inc. Hoboken, NJ. 2004., p. 1827
Subcutaneous LD50 in mice: 0.008 mg/kg. Lewis, RJ Sr. (ed.) Sax's Dangerous Properties of Industrial Materials, 11th Edition. Wiley-Interscience, Wiley & Sons, Inc., Hoboken, NJ, 2004, p. 1827.
Intravenous LD50 in mice: 0.009 mg/kg. Lewis, RJ Sr. (ed.), Hazardous Properties of Materials for Saxe Industries, 11th ed. Wiley-Interscience, Wiley & Sons, Inc., Hoboken, NJ, 2004, p. 1827.

[1]. The protective action of tetrodotoxin and (±)-kavain on anaerobic glycolysis, ATP content and intracellular Na+and Ca2+of anoxic brain vesicles.Neuropharmacology, 1996, 35,1743.

Tetrodotoxin is an aminoperhydroquinazoline toxin primarily found in the liver and ovaries of tetradontidae fish, which are edible. The toxin causes paresthesia and paralysis by interfering with neuromuscular transmission. Wex Pharmaceuticals is investigating tetrodotoxin for the treatment of chronic and explosive pain in patients with advanced cancer, as well as for opioid dependence. Tetrodotoxin has also been reported in yellowfin pufferfish (Takifugu flavidus), redfin pufferfish (Takifugu rubripes), and several other organisms with relevant data. Tetrodotoxin is a neurotoxin with potential analgesic activity. It binds to the pores of rapidly voltage-gated sodium channels on nerve cell membranes, inhibiting nerve action potentials and blocking nerve conduction. Although tetrodotoxin is present in a variety of fish (e.g., pufferfish), salamanders, frogs, flatworms, and crabs, there is currently no known antidote. It is actually produced by Vibrio alginolyticus, Pseudomonas tetradontidae, and other Vibrio and Pseudomonas bacteria. Tetrodotoxin is an aminoperhydroquinazoline toxin found primarily in the liver and ovaries of edible tetrodotoxin fish. This toxin causes paresthesia and paralysis by interfering with neuromuscular transmission. Drug Indications: Used to treat chronic and explosive pain in patients with advanced cancer, and to treat opioid dependence. Mechanism of Action: Tetrodotoxin binds to site 1 of a fast-voltage-gated sodium channel located at the opening of extracellular pores. Binding of any molecule to this site temporarily inhibits the function of the ion channel. Tetrodotoxin and several cone snail toxins also bind to this site. The sodium current (I(Na)) of the mammalian heart is resistant to tetrodotoxin (TTX) due to the low affinity of the cardiac sodium channel (Na(v)) subtype Na(v)1.5 for TTX. To test whether this finding applies to other vertebrates, we examined the sensitivity of fish cardiac I(Na) to TTX and its molecular composition. Methods: We used molecular cloning and whole-cell patch-clamp techniques to investigate the composition of the α subunit of Na(v) in the heart of rainbow trout (Oncorhynchus mykiss) and the inhibitory effect of tetrodotoxin (TTX). The sodium ion current (I(Na)) in the rainbow trout heart was approximately 1000 times more sensitive to tetrodotoxin (TTX) than that in the mammalian heart (IC50 = 1.8–2 nM). It was generated by three sodium ion channel (Na(v)) α subunits, which were homologous to Na(v)1.4 in mammalian skeletal muscle, Na(v)1.5 in the heart, and Na(v)1.6 in the peripheral nervous system, respectively. In rainbow trout (Oncorhynchus mykiss), omNa(v)1.4a is the dominant isoform in its heart, accounting for over 80% of the Na(v) transcripts, while omNa(v)1.5a accounts for approximately 18%, and omNa(v)1.6a accounts for only 0.1%. Both omNa(v)1.4a and omNa(v)1.6a contain the aromatic amino acids phenylalanine and tyrosine, respectively, at the critical position 401 of the TTX-binding site in the I domain, conferring them high sensitivity to TTX. Even more surprisingly, omNa(v)1.5a also contains an aromatic tyrosine at this position, instead of the cysteine found in the mammalian TTX-resistant Na(v)1.5. Conclusion: The ortholog of mammalian skeletal muscle isoform omNa(v)1.4a is the major Na(v)α subunit in trout heart. All trout heart isoforms contain an aromatic residue at position 401, making fish heart I(Na) highly sensitive to TTX. TTX inhibits voltage-gated sodium channels in a highly efficient and selective manner without affecting any other receptor and ion channel systems. Tetrodotoxin (TTX) blocks sodium channels only from the outside of the nerve membrane; its mechanism of action is by binding to selective filters to prevent sodium ion flow. It does not impair the channel gating mechanism. In recent years, sodium channels tolerant to TTX have been discovered in the nervous system and have attracted much attention due to their role in pain perception. It is now known that TTX is not produced by inhalers but by bacteria and spreads to various animals through the food chain. Therapeutic Uses: Corneal damage can lead to photophobia, i.e., an aversion to light. We evaluated whether increased photophobia induced by corneal injury in rats was due to enhanced corneal afferent nerve activity through topical application of the local anesthetic lidocaine and the selective voltage-gated sodium channel blocker tetrodotoxin (TTX). Twenty-four hours after corneal ablation-induced corneal injury, exposure to strong light (460–485 nm) for 30 seconds induced an enhanced eye-closing response. While topical lidocaine did not affect the baseline eye-closing response to strong light in control rats, it eliminated the enhanced photophobia following corneal injury. Similarly, topical TTX did not affect the eye-closing response to strong light in rats with intact corneas but significantly reduced photophobia in rats with corneal injury. Given the known corneal toxicity of local anesthetics, we recommend TTX as a treatment option for photophobia and other clinical symptoms that may be associated with corneal nociceptor sensitization. Outlook: We found that both lidocaine and TTX can alleviate photophobia induced by corneal injury. Although corneal toxicity limits the use of local anesthetics, TTX may be a safer treatment option to alleviate photophobia associated with corneal injury. PMID: 26086898 Full text: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4664153 Green PG et al.; J Pain 16 (9): 881-6 (2015)
/EXPL THER/ Burns are considered the leading cause of injury in 5% of U.S. service members withdrawn from Operation Iraqi Freedom and Operation Enduring Freedom. Severe burn-related pain is often treated with opioids such as fentanyl, morphine, and methadone. Side effects of opioids include respiratory depression, cardiac depression, decreased motor and cognitive function, and the development of hyperalgesia, tolerance, and dependence. These effects have prompted the search for novel analgesics to treat burn-related pain in wounded combatants. Tetrodotoxin (TTX) is a selective voltage-gated sodium channel blocker and is currently undergoing clinical trials as an analgesic. A Phase III clinical trial for cancer-related pain has been completed, and a Phase III clinical trial for chemotherapy-induced neuropathic pain is planned. In mouse studies, TTX has also been shown to inhibit the development of chemotherapy-induced neuropathic pain. TTX, originally a neurotoxin discovered in marine animals, has now been shown to be safe for humans at therapeutic doses. The analgesic effect of TTX is believed to be achieved by inhibiting the influx of Na(+) ions required to initiate and conduct nociceptive impulses. A TTX-sensitive sodium channel, Nav1.7, has been shown to be crucial in reducing the thermal pain threshold after burns. To date, the analgesic effect of TTX has not been tested in burn-related pain. Male Sprague-Dawley rats suffered full-thickness burns to their right hind paw. TTX (8 μg/kg) was administered subcutaneously once daily, starting on day 3 post-burn and continuing until day 7 post-burn. Thermal hyperalgesia and mechanodynia were assessed at 60 and 120 minutes after each TTX treatment. TTX significantly reduced thermal hyperalgesia at all test time points, with a weaker but still statistically significant inhibitory effect on mechanical analgesia. These results suggest that systemic TTX may be an effective and rapidly acting battlefield burn analgesic, and could potentially replace or reduce the need for opioid analgesics. PMID:26424077 Salas MM et al.; Neuroscience Letters 607: 108-113 (2015)
/Exploring Treatments/ Persistent muscle pain is a common and disabling symptom with limited efficacy of existing treatments. Given the significant analgesic effect of tetrodotoxin (TTX) in a persistent skin pain model, we tested its local analgesic effect in a rat model of muscle pain induced by inflammation, ergonomic injury, and chemotherapy-induced neuropathy. Although local injection of TTX (0.03–1 μg) into the gastrocnemius muscle did not affect the mechanoresonance threshold in untreated rats, exposure to the inflammagenic carrageenan resulted in significant muscle mechanoresonance, which TTX dose-dependently inhibited. This anti-hyperalsonance effect remained significant after 24 hours. TTX also demonstrated significant analgesic effects on mechanoresonance induced by eccentric movement of the gastrocnemius muscle (an ergonomic pain model). Furthermore, TTX produced a small but significant inhibitory effect on neuromuscular pain induced by systemic injection of the anticancer chemotherapy drug oxaliplatin. These results suggest that TTX-sensitive sodium currents in nociceptors play a central role in various states of skeletal muscle nociceptive sensitization, supporting the idea that TTX-based therapeutic interventions may be effective for treating muscle pain. PMID:26548414 Full text: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4679288 Alvarez P, Levine JD; Neuroscience 311: 499-507 (2015)
/Exploring Treatment/ Objective: This study evaluated the efficacy of subcutaneous tetrodotoxin (TTX) for moderate to severe, poorly controlled cancer-related pain. Methods: Eligible patients were randomly assigned to receive TTX (30 μg) or placebo, administered subcutaneously twice daily for four consecutive days. Efficacy was assessed using pain and composite endpoints (including pain and quality of life indicators), and safety was assessed using standard methods. Results: A total of 165 patients were recruited from 19 research centers in Canada, Australia, and New Zealand, of whom 149 were included in the “intent-to-treat” population of the primary analysis. The primary analysis results support a clinical benefit of TTX compared to placebo, with a clinically significant effect size estimate of 16.2% (p = 0.0460) based solely on the pain endpoint. After pre-specified (Bonferroni-Holm) adjustment for both primary endpoints, the p-values were nominally statistically significant but not significant at the pre-specified two-sided 5% level. The mean duration of the analgesic response was 56.7 days (TTX group) and 9.9 days (placebo group). The most common adverse events were nausea, dizziness, and numbness or tingling in the mouth, which were generally mild to moderate and transient. Conclusion: Despite the small sample size, this study demonstrates a clinically meaningful analgesic signal. TTX may provide clinically meaningful analgesia for patients with persistent moderate to severe cancer pain despite optimal analgesia. PMID:28555092

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Recent reports indicate that sodium channel blockers can act as protective agents against hypoxia-induced neuronal damage, including protecting against anaerobic glycolysis. Therefore, this study investigated the effects of tetrodotoxin (TTX) and (±)-carvaline on brain vesicles in hypoxic rats, specifically lactate synthesis, vesicle ATP content, and cytoplasmic free sodium ion ([Na+]i) and calcium ion ([Ca2+]i) concentrations. Cytoplasmic free sodium ion and calcium ion concentrations were measured using SBFI and FURA-2 fluorescence methods, respectively. After hypoxia, basal lactate production increased from 2.9 nmol lactate/min/mg protein to 9.8 nmol lactate/min/mg protein. Although lactate synthesis appeared to remain stable for at least 45 minutes under hypoxia (inferred from the linear change in lactate production), ATP content continued to decline with a half-life (τ) of 14.5 minutes, indicating that anaerobic glycolysis was insufficient to meet the energy requirements of hypoxic vesicles. Correspondingly, after 6.3 minutes of hypoxia, [Na+]i and [Ca2+]i continued to increase, rising by 22.1 mmol/L Na+ and 274.9 nmol/L Ca2+, respectively. Further stimulation of the vesicles with veratrine accelerated the rate of ATP decline (τ = 5.1 min) and triggered a significant Na+ overload, with the Na+ concentration stabilizing at 119 mmol/L within minutes. Simultaneously, [Ca2+]i increased linearly at a rate of 355 nmol Ca2+/L/min. Despite the severe disturbance of ion homeostasis, lactate production was unaffected during the first 8 minutes of veratrine stimulation. However, after 30 minutes of veratrine addition, lactate synthesis was completely inhibited. If sodium channel blockers TTX and (±)-carvaline are used before hypoxia, vesicle ATP levels can be maintained, hypoxia-induced increases in [Na+]i and [Ca2+]i can be reduced, and veratrine-induced increases in [Na+]i and [Ca2+]i and inhibition of lactate production can be prevented. Data show that during hypoxia, voltage-dependent sodium channel-mediated massive sodium influx accelerates the decrease in ATP and triggers an increase in [Ca2+]i. Veratrine-induced massive sodium and calcium overload does not directly affect lactate synthesis, but it initiates the inhibition of lactate synthesis. © 1997 Elsevier Science Ltd. All rights reserved. [1]

C11H17N3O8

319.27

319.101

C, 41.38; H, 5.37; N, 13.16; O, 40.09

4368-28-9

Citrate-buffered Tetrodotoxin (TTX); 4368-28-9

11174599

Crystals

2.8±0.1 g/cm3

702.6±70.0 °C at 760 mmHg

225ºC dec

378.7±35.7 °C

0.0±5.0 mmHg at 25°C

2.087

2.16

187.75

8

9

1

22

562

9

C([C@@]1([C@@H]2[C@@H]3[C@H](NC(=N)N[C@]34[C@@H]([C@H]1O[C@@]([C@H]4O)(O)O2)O)O)O)O

CFMYXEVWODSLAX-QOZOJKKESA-N

InChI=1S/C11H17N3O8/c12-8-13-6(17)2-4-9(19,1-15)5-3(16)10(2,14-8)7(18)11(20,21-4)22-5/h2-7,15-20H,1H2,(H3,12,13,14)/t2-,3-,4-,5+,6-,7+,9+,10-,11+/m1/s1

(1R,5R,6R,7R,9S,11S,12S,13S,14S)-3-amino-14-(hydroxymethyl)-8,10-dioxa-2,4-diazatetracyclo[7.3.1.17,11.01,6]tetradec-3-ene-5,9,12,13,14-pentol

TETRODOTOXIN; Spheroidine; Tarichatoxin; Fugu poison; Maculotoxin; TTX; Tetrodotoxine; 4368-28-9; Babylonia japonica toxin 1; Tetrodoxin; Tectin;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: ~1 mg/mL
Stable Solution: Prepare 1 mg/mL in a dilute citrate or acetate buffer (pH 4-5).
Storage: Aqueous solutions at pH 4-5 are stable when frozen.
Instability: The compound is unstable in strong acid or alkaline solutions and is destroyed by boiling at pH 2.

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。