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1.  Confirmation and Distribution of Tetrodotoxin for the First Time in Terrestrial Invertebrates: Two Terrestrial Flatworm Species (Bipalium adventitium and Bipalium kewense) 
PLoS ONE  2014;9(6):e100718.
The potent neurotoxin tetrodotoxin (TTX) is known from a diverse array of taxa, but is unknown in terrestrial invertebrates. Tetrodotoxin is a low molecular weight compound that acts by blocking voltage-gated sodium channels, inducing paralysis. However, the origins and ecological functions of TTX in most taxa remain mysterious. Here, we show that TTX is present in two species of terrestrial flatworm (Bipalium adventitium and Bipalium kewense) using a competitive inhibition enzymatic immunoassay to quantify the toxin and high phase liquid chromatography to confirm the presence. We also investigated the distribution of TTX throughout the bodies of the flatworms and provide evidence suggesting that TTX is used during predation to subdue large prey items. We also show that the egg capsules of B. adventitium have TTX, indicating a further role in defense. These data suggest a potential route for TTX bioaccumulation in terrestrial systems.
PMCID: PMC4070999  PMID: 24963791
2.  Batrachotoxin-activated Na+ channels in planar lipid bilayers. Competition of tetrodotoxin block by Na+ 
The Journal of General Physiology  1984;84(5):665-686.
Single Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers formed from neutral phospholipids and were observed in the presence of batrachotoxin. The batrachotoxin-modified channel activates in the voltage range -120 to - 80 mV and remains open almost all the time at voltages positive to -60 mV. Low levels of tetrodotoxin (TTX) induce slow fluctuations of channel current, which represent the binding and dissociation of single TTX molecules to single channels. The rates of association and dissociation of TTX are both voltage dependent, and the association rate is competitively inhibited by Na+. This inhibition is observed only when Na+ is increased on the TTX binding side of the channel. The results suggest that the TTX receptor site is located at the channel's outer mouth, and that the Na+ competition site is not located deeply within the channel's conduction pathway.
PMCID: PMC2228758  PMID: 6096478
3.  Fluidic Force Discrimination Assays: A New Technology for Tetrodotoxin Detection 
Marine Drugs  2010;8(3):565-576.
Tetrodotoxin (TTX) is a low molecular weight (~319 Da) neurotoxin found in a number of animal species, including pufferfish. Protection from toxin tainted food stuffs requires rapid, sensitive, and specific diagnostic tests. An emerging technique for the detection of both proteins and nucleic acids is Fluidic Force Discrimination (FFD) assays. This simple and rapid method typically uses a sandwich immunoassay format labeled with micrometer-diameter beads and has the novel capability of removing nonspecifically attached beads under controlled, fluidic conditions. This technique allows for near real-time, multiplexed analysis at levels of detection that exceed many of the conventional transduction methods (e.g., ELISAs). In addition, the large linear dynamic range afforded by FFD should decrease the need to perform multiple sample dilutions, a common challenge for food testing. By applying FFD assays to an inhibition immunoassay platform specific for TTX and transduction via low magnification microscopy, levels of detection of ~15 ng/mL and linear dynamic ranges of 4 to 5 orders of magnitude were achieved. The results from these studies on the first small molecule FFD assay, along with the impact to detection of seafood toxins, will be discussed in this manuscript.
PMCID: PMC2857369  PMID: 20411115
tetrodotoxin; antibody inhibition assay; bioassay; Fluidic Force Discrimination; microbead labels
4.  The Chemical and Evolutionary Ecology of Tetrodotoxin (TTX) Toxicity in Terrestrial Vertebrates 
Marine Drugs  2010;8(3):577-593.
Tetrodotoxin (TTX) is widely distributed in marine taxa, however in terrestrial taxa it is limited to a single class of vertebrates (Amphibia). Tetrodotoxin present in the skin and eggs of TTX-bearing amphibians primarily serves as an antipredator defense and these taxa have provided excellent models for the study of the evolution and chemical ecology of TTX toxicity. The origin of TTX present in terrestrial vertebrates is controversial. In marine organisms the accepted hypothesis is that the TTX present in metazoans results from either dietary uptake of bacterially produced TTX or symbiosis with TTX producing bacteria, but this hypothesis may not be applicable to TTX-bearing amphibians. Here I review the taxonomic distribution and evolutionary ecology of TTX in amphibians with some attention to the origin of TTX present in these taxa.
PMCID: PMC2857372  PMID: 20411116
tetrodotoxin; TTX; Amphibia; Caudata; Anura; Salamandridae; Taricha; Notophthalmus; Cynops; Atelopus
5.  Tetrodotoxin —A brief history— 
Tetrodotoxin (TTX), contained in puffer, has become an extremely popular chemical tool in the physiological and pharmacological laboratories since our discovery of its channel blocking action in the early 1960s. This brief review describes the history of discovery of TTX action on sodium channels, and represents a story primarily of my own work. TTX inhibits voltage-gated sodium channels in a highly potent and selective manner without effects on any other receptor and ion channel systems. TTX blocks the sodium channel only from outside of the nerve membrane, and is due to binding to the selectivity filter resulting in prevention of sodium ion flow. It does not impairs the channel gating mechanism. More recently, the TTX-resistant sodium channels have been discovered in the nervous system and received much attention because of their role in pain sensation. TTX is now known to be produced not by puffer but by bacteria, and reaches various species of animals via food chain.
PMCID: PMC2858367  PMID: 18941294
tetrodotoxin; saxitoxin; sodium channels; sodium currents; puffer; selectivity filter
6.  Analytical Challenges: Determination of Tetrodotoxin in Human Urine and Plasma by LC-MS/MS 
Marine Drugs  2011;9(11):2291-2303.
Tetrodotoxin (TTX) is a powerful sodium channel blocker found in puffer fish and some marine animals. Cases of TTX poisoning most often result from puffer fish ingestion. Diagnosis is mainly from patient’s signs and symptoms or the detection of TTX in the leftover food. If leftover food is unavailable, the determination of TTX in the patient’s urine and/or plasma is essential to confirm the diagnosis. Although various methods for the determination of TTX have been published, most of them are for food tissue samples. Dealing with human urine and blood samples is much more challenging. Unlike in food, the amount of toxin in the urine and blood of a patient is generally extremely low; therefore a very sensitive method is required to detect it. In this regard, mass spectrometry (MS) methods are the best choice. Since TTX is a very polar compound, there will be lack of retention on conventional reverse-phase columns; use of ion pair reagent or hydrophilic interaction liquid chromatography (HILIC) can help solve this problem. The problem of ion suppression is another challenge in analyzing polar compound in biological samples. This review will discuss different MS methods and their pros and cons.
PMCID: PMC3229236  PMID: 22163187
TTX poisoning; biological samples; ion suppression; LC-MS/MS
7.  Native Pyroglutamation of Huwentoxin-IV: A Post-Translational Modification that Increases the Trapping Ability to the Sodium Channel 
PLoS ONE  2013;8(6):e65984.
Huwentoxin-IV (HWTX-IV), a tetrodotoxin-sensitive (TTX-s) sodium channel antagonist, is found in the venom of the Chinese spider Ornithoctonus huwena. A naturally modified HWTX-IV (mHWTX-IV), having a molecular mass 18 Da lower than HWTX-IV, has also been isolated from the venom of the same spider. By a combination of enzymatic fragmentation and MS/MS de novo sequencing, mHWTX-IV has been shown to have the same amino acid sequence as that of HWTX-IV, except that the N-terminal glutamic acid replaced by pyroglutamic acid. mHWTX-IV inhibited tetrodotoxin-sensitive voltage-gated sodium channels of dorsal root ganglion neurons with an IC50 nearly equal to native HWTX-IV. mHWTX-IV showed the same activation and inactivation kinetics seen for native HWTX-IV. In contrast with HWTX-IV, which dissociates at moderate voltage depolarization voltages (+50 mV, 180000 ms), mHWTX-IV inhibition of TTX-sensitive sodium channels is not reversed by strong depolarization voltages (+200 mV, 500 ms). Recovery of Nav1.7current was voltage-dependent and was induced by extreme depolarization in the presence of HWTX-IV, but no obvious current was elicited after application of mHWTX-IV. Our data indicate that the N-terminal modification of HWTX-IV gives the peptide toxin a greater ability to trap the voltage sensor in the sodium channel. Loss of a negative charge, caused by cyclization at the N-terminus, is a possible reason why the modified toxin binds much stronger. To our knowledge, this is the first report of a pyroglutamic acid residue in a spider toxin; this modification seems to increase the trapping ability of the voltage sensor in the sodium channel.
PMCID: PMC3691182  PMID: 23826086
8.  Tetrodotoxin Blockade on Canine Cardiac L-Type Ca2+ Channels Depends on pH and Redox Potential 
Marine Drugs  2013;11(6):2140-2153.
Tetrodotoxin (TTX) is believed to be one of the most selective inhibitors of voltage-gated fast Na+ channels in excitable tissues. Recently, however, TTX has been shown to block L-type Ca2+ current (ICa) in canine cardiac cells. In the present study, the TTX-sensitivity of ICa was studied in isolated canine ventricular myocytes as a function of (1) channel phosphorylation, (2) extracellular pH and (3) the redox potential of the bathing medium using the whole cell voltage clamp technique. Fifty-five micromoles of TTX (IC50 value obtained under physiological conditions) caused 60% ± 2% inhibition of ICa in acidic (pH = 6.4), while only a 26% ± 2% block in alkaline (pH = 8.4) milieu. Similarly, the same concentration of TTX induced 62% ± 6% suppression of ICa in a reductant milieu (containing glutathione + ascorbic acid + dithiothreitol, 1 mM each), in contrast to the 31% ± 3% blockade obtained in the presence of a strong oxidant (100 μM H2O2). Phosphorylation of the channel protein (induced by 3 μM forskolin) failed to modify the inhibiting potency of TTX; an IC50 value of 50 ± 4 μM was found in forskolin. The results are in a good accordance with the predictions of our model, indicating that TTX binds, in fact, to the selectivity filter of cardiac L-type Ca channels.
PMCID: PMC3721225  PMID: 23771047
tetrodotoxin; calcium current; dog heart; pH dependence; redox potential
9.  Thermal sensitivity of voltage-gated Na+ channels and A-type K+ channels contributes to somatosensory neuron excitability at cooling temperatures 
Journal of neurochemistry  2012;122(6):1145-1154.
Cooling temperatures may modify action potential firing properties to alter sensory modalities. Here we investigated how cooling temperatures modify action potential firing properties in two groups of rat dorsal root ganglion (DRG) neurons, tetrodotoxin-sensitive (TTXs) Na+ channel-expressing neurons and tetrodotoxin-resistant (TTXr) Na+ channel-expressing neurons. We found that multiple action potential firing in response to membrane depolarization was suppressed in TTXs neurons but maintained or facilitated in TTXr neurons at cooling temperatures. We showed that cooling temperatures strongly inhibited A-type K+ currents (IA) and TTXs Na+ channels but had fewer inhibitory effects on TTXr Na+ channels and non-inactivating K+ currents (IK). We demonstrated that the sensitivity of A-type K+ channels and voltage-gated Na+ channels to cooling temperatures and their interplay determine somatosensory neuron excitability at cooling temperatures. Our results provide a putative mechanism by which cooling temperatures modify different sensory modalities including pain.
PMCID: PMC3433580  PMID: 22712529
Cold; voltage-gated Na+ channels; voltage-gated K+ channels; dorsal root ganglions; pain
10.  First Identification of 5,11-Dideoxytetrodotoxin in Marine Animals, and Characterization of Major Fragment Ions of Tetrodotoxin and Its Analogs by High Resolution ESI-MS/MS 
Marine Drugs  2013;11(8):2799-2813.
Even though tetrodotoxin (TTX) is a widespread toxin in marine and terrestrial organisms, very little is known about the biosynthetic pathway used to produce it. By describing chemical structures of natural analogs of TTX, we can start to identify some of the precursors that might be important for TTX biosynthesis. In the present study, an analog of TTX, 5,11-dideoxyTTX, was identified for the first time in natural sources, the ovary of the pufferfish and the pharynx of a flatworm (planocerid sp. 1), by comparison with totally synthesized (−)-5,11-dideoxyTTX, using high resolution ESI-LC-MS. Based on the presence of 5,11-dideoxyTTX together with a series of known deoxy analogs, 5,6,11-trideoxyTTX, 6,11-dideoxyTTX, 11-deoxyTTX, and 5-deoxyTTX, in these animals, we predicted two routes of stepwise oxidation pathways in the late stages of biosynthesis of TTX. Furthermore, high resolution masses of the major fragment ions of TTX, 6,11-dideoxyTTX, and 5,6,11-trideoxyTTX were also measured, and their molecular formulas and structures were predicted to compare them with each other. Although both TTX and 5,6,11-trideoxyTTX give major fragment ions that are very close, m/z 162.0660 and 162.1020, respectively, they are distinguishable and predicted to be different molecular formulas. These data will be useful for identification of TTXs using high resolution LC-MS/MS.
PMCID: PMC3766866  PMID: 23924959
tetrodotoxin; LC-MS/MS; 5,11-dideoxytetrodotoxin; biosynthesis
11.  Tetrodotoxin – Distribution and Accumulation in Aquatic Organisms, and Cases of Human Intoxication 
Marine Drugs  2008;6(2):220-242.
Many pufferfish of the family Tetraodontidae possess a potent neurotoxin, tetrodotoxin (TTX). In marine pufferfish species, toxicity is generally high in the liver and ovary, whereas in brackish water and freshwater species, toxicity is higher in the skin. In 1964, the toxin of the California newt was identified as TTX as well, and since then TTX has been detected in a variety of other organisms. TTX is produced primarily by marine bacteria, and pufferfish accumulate TTX via the food chain that begins with these bacteria. Consequently, pufferfish become non-toxic when they are fed TTX-free diets in an environment in which the invasion of TTX-bearing organisms is completely shut off. Although some researchers claim that the TTX of amphibians is endogenous, we believe that it also has an exogenous origin, i.e., from organisms consumed as food. TTX-bearing animals are equipped with a high tolerance to TTX, and thus retain or accumulate TTX possibly as a biologic defense substance. There have been many cases of human intoxication due to the ingestion of TTX-bearing pufferfish, mainly in Japan, China, and Taiwan, and several victims have died. Several cases of TTX intoxication due to the ingestion of small gastropods, including some lethal cases, were recently reported in China and Taiwan, revealing a serious public health issue.
PMCID: PMC2525488  PMID: 18728726
tetrodotoxin; pufferfish; marine bacteria; newt; gastropod; human intoxication
12.  Equilibrium and Kinetic Properties of the Interaction between Tetrodotoxin and the Excitable Membrane of the Squid Giant Axon 
The Journal of General Physiology  1970;55(3):309-335.
Squid giant axons were treated with tetrodotoxin (TTX) in concentrations ranging from 1 nM to 25 nM and the resulting decrease in sodium current was followed in time using the voltage clamp technique. The removal of TTX from the bathing solution produced only partial recovery of the sodium current. This suggests that the over-all interaction is more complex than just a reversible reaction. By correcting for the partial irreversibility of the decrease in sodium current, a dissociation constant of 3.31 x 10-9 M was calculated for the reaction between TTX and the reactive site of the membrane. The data obtained fit a dose-response curve modified to incorporate the correction for partial irreversibility when calculated for a one-to-one stoichiometry. The fit disagreed with that calculated for a reaction between two molecules of TTX with a single membrane-reactive site, but neither supported nor disproved the possibility of a complex formed by two reactive sites with one molecule of TTX. Values of the rate constants for the formation and dissociation of the TTX-membrane complex, k1 and k2, respectively, were obtained from the kinetic data. The values are: k1 = 0.202 x 108 M-1, and k2 = 0.116 min-1. The magnitude of the dissociation constant derived from these values is 5.74 x 10-9 M, which has the same order of magnitude as that obtained from equilibrium measurements. Arrhenius plots of the rate constants gave values for the thermodynamic quantities of activation.
PMCID: PMC2203005  PMID: 5520505
13.  Sodium currents during differentiation in a human neuroblastoma cell line 
The Journal of General Physiology  1991;97(3):521-539.
The electrophysiological properties of a human neuroblastoma cell line, LA-N-5, were studied with the whole-cell configuration of the patch clamp technique before and after the induction of differentiation by retinoic acid, a vitamin A metabolite. Action potentials could be elicited from current clamped cells before the induction of differentiation, suggesting that some neuroblasts of the developing sympathetic nervous system are excitable. The action potential upstroke was carried by a sodium conductance, which was composed of two types of sodium currents, described by their sensitivity to tetrodotoxin (TTX) as TTX sensitive and TTX resistant. TTX-sensitive and TTX-resistant sodium currents were blocked by nanomolar and micromolar concentrations of TTX, respectively. The voltage sensitivity of activation and inactivation of TTX-resistant sodium current is shifted -10 to -30 mV relative to TTX-sensitive sodium current, suggesting that TTX-resistant sodium current could play a role in the initiation of action potentials. TTX-sensitive current comprised greater than 80% of the total sodium current in undifferentiated LA-N-5 cells. The surface density of total sodium current increased from 24.9 to 57.8 microA/microF after cells were induced to differentiate. The increase in total sodium current density was significant (P less than 0.05). The surface density of TTX-resistant sodium current did not change significantly during differentiation, from which we conclude that an increase in TTX-sensitive sodium current underlies the increase in total current.
PMCID: PMC2216486  PMID: 1645394
14.  The properties of batrachotoxin-modified cardiac Na channels, including state-dependent block by tetrodotoxin 
The Journal of General Physiology  1987;90(3):341-360.
Batrachotoxin (BTX) modification and tetrodotoxin (TTX) block of BTX- modified Na channels were studied in single cardiac cells of neonatal rats using the whole-cell patch-clamp recording technique. The properties of BTX-modified Na channels in heart are qualitatively similar to those in nerve. However, quantitative differences do exist between the modified channels of these two tissues. In the heart, the shift of the conductance-voltage curve for the modified channel was less pronounced, the maximal activation rate constant, (tau m)max, of modified channels was considerably slower, and the slow inactivation of the BTX-modified cardiac Na channels was only partially abolished. TTX blocked BTX-modified mammalian cardiac Na channels and the block decreased over the potential range of -80 to -40 mV. The apparent dissociation constant of TTX changed from 0.23 microM at -50 mV to 0.69 microM at 0 mV. No further reduction of block was observed at potentials greater than -40 mV. This is the potential range over which gating from closed to open states occurred. These results were explained by assuming that TTX has a higher affinity for closed BTX- modified channels than for open modified channels. Hence, the TTX- binding rate constants are considered to be state dependent rather than voltage dependent. This differs from the voltage dependence of TTX block reported for BTX-modified Na channels from membrane vesicles incorporated into lipid bilayers and from amphibian node of Ranvier.
PMCID: PMC2228841  PMID: 2443605
15.  LC/MS Analysis of Tetrodotoxin and Its Deoxy Analogs in the Marine Puffer Fish Fugu niphobles from the Southern Coast of Korea, and in the Brackishwater Puffer Fishes Tetraodon nigroviridis and Tetraodon biocellatus from Southeast Asia 
Marine Drugs  2010;8(4):1049-1058.
Tetrodotoxin (TTX) and its deoxy analogs, 5-deoxyTTX, 11-deoxyTTX, 6,11-dideoxyTTX, and 5,6,11-trideoxyTTX, were quantified in the tissues of three female and three male specimens of the marine puffer fish, Fugu niphobles, from the southern coast of Korea, and in the whole body of the brackishwater puffer fishes, Tetraodon nigroviridis (12 specimens) and Tetrodon biocellatus (three specimens) from Southeast Asia using LC/MS in single ion mode (SIM). Identification of these four deoxy analogs in the ovarian tissue of F. niphobles were further confirmed by LC/MS/MS. TTX and 5,6,11-trideoxyTTX were detected in all three puffer fish species as the major TTX analogs, similar to Japanese Fugu pardalis. While 6,11-dideoxyTTX was also found to be a major analog in almost all tissues of Korean F. niphobles, this analog was minor in the two Tetraodon species and Japanese F. pardalis. Among the tissues of F. niphobles, the concentrations of TTXs were highest in the ovaries (female) and skin (female and male).
PMCID: PMC2866474  PMID: 20479966
tetrodotoxin; 6,11-dideoxytetrodotoxin; puffer fish; LC/MS; LC/MS/MS
16.  New Gastropod Vectors and Tetrodotoxin Potential Expansion in Temperate Waters of the Atlantic Ocean 
Marine Drugs  2012;10(4):712-726.
Tetrodotoxin is a potent low weight marine toxin found in warm waters, especially of the Indian and Pacific Oceans. Intoxications are usually linked to the consumption of the puffer fish, although TTX was already detected in several different edible taxa. Benthic organisms such as mollusks and echinoderms, with different feeding habits, were collected monthly along the Portuguese coast from the summer of 2009 until the end of 2010. The extraction and analysis techniques were optimized and TTX and some analogues were detected for the first time in two intertidal gastropod species—Gibbula umbilicalis and Monodonta lineata by LC-MS/MS and UPLC-MS/MS. Although the levels are low, these findings suggest that monitoring of TTX and analogues in North Atlantic species should be implemented so as to detect potentially new toxin vectors and seasonal and/or geographical patterns.
PMCID: PMC3366671  PMID: 22690139
tetrodotoxin; new vectors; gastropods; North Atlantic Waters
17.  Quantitative Monitoring of Insulin Secretion from Single Islets of Langerhans in Parallel on a Microfluidic Chip 
Analytical chemistry  2009;81(8):3119-3127.
Quantification of insulin release from pancreatic islets of Langerhans is of interest for diabetes research. Typical insulin secretion experiments are performed using offline techniques that are expensive, slow, have low-throughput, and require multiple islets. We have developed a microfluidic device for high-throughput, automated, and online monitoring of insulin secretion from individual islets in parallel. This chip consists of 15 channel networks each capable of superfusing a single islet and mixing superfusate from each islet online with fluorescein isothiocyanate-labeled insulin and anti-insulin antibody for a competitive immunoassay. The resulting continuous reaction streams are periodically injected onto parallel electrophoresis channels where the mixtures are separated. The resulting traces are used to quantify relative insulin released from islets. Serial immunoassays were performed at 10 s intervals on all 15 channels, corresponding to 5400 immunoassays per hour, to create temporally resolved insulin release profiles that captured single islet secretion dynamics. The chip was used to demonstrate that free fatty acid induced lipotoxicity in islets eliminates pulsatile insulin secretion.
PMCID: PMC2679996  PMID: 19364142
18.  Batrachotoxin-modified sodium channels in planar lipid bilayers. Characterization of saxitoxin- and tetrodotoxin-induced channel closures 
The Journal of General Physiology  1987;89(6):873-903.
The guanidinium toxin-induced inhibition of the current through voltage- dependent sodium channels was examined for batrachotoxin-modified channels incorporated into planar lipid bilayers that carry no net charge. To ascertain whether a net negative charge exists in the vicinity of the toxin-binding site, we studied the channel closures induced by tetrodotoxin (TTX) and saxitoxin (STX) over a wide range of [Na+]. These toxins carry charges of +1 and +2, respectively. The frequency and duration of the toxin-induced closures are voltage dependent. The voltage dependence was similar for STX and TTX, independent of [Na+], which indicates that the binding site is located superficially at the extracellular surface of the sodium channel. The toxin dissociation constant, KD, and the rate constant for the toxin- induced closures, kc, varied as a function of [Na+]. The Na+ dependence was larger for STX than for TTX. Similarly, the addition of tetraethylammonium (TEA+) or Zn++ increased KD and decreased kc more for STX than for TTX. These differential effects are interpreted to arise from changes in the electrostatic potential near the toxin- binding site. The charges giving rise to this potential must reside on the channel since the bilayers had no net charge. The Na+ dependence of the ratios KDSTX/KDTTX and kcSTX/kcTTX was used to estimate an apparent charge density near the toxin-binding site of about -0.33 e X nm-2. Zn++ causes a voltage-dependent block of the single-channel current, as if Zn++ bound at a site within the permeation path, thereby blocking Na+ movement. There was no measurable interaction between Zn++ at its blocking site and STX or TTX at their binding site, which suggests that the toxin-binding site is separate from the channel entrance. The separation between the toxin-binding site and the Zn++ blocking site was estimated to be at least 1.5 nm. A model for toxin-induced channel closures is proposed, based on conformational changes in the channel subsequent to toxin binding.
PMCID: PMC2215969  PMID: 2440978
19.  Expression of skeletal muscle NaV1.4 Na channel isoform in canine cardiac Purkinje myocytes 
Background and Aim
The action potential plateau of Purkinje fibers is particularly sensitive to tetrodotoxin (TTX) and this could be due to a TXX-sensitive Na+ current. The expression of TTX-sensitive neuronal NaV1.1 and NaV1.2 isoforms has been reported in canine Purkinje myocytes. Our aim was to investigate by means of biochemical and functional techniques whether the TTX-sensitive skeletal NaV1.4 isoform is also expressed in canine cardiac Purkinje myocytes.
Methods and Results
Using NaV1.4 specific primers, a PCR product corresponding to NaV1.4 was amplified from canine Purkinje fibers RNA and confirmed by sequencing and megablast of the gene bank. Confocal indirect immunostaining using anti-NaV1.4 antibody demonstrates distinct sarcolemmal staining pattern compared to that of the cardiac isoform NaV1.5. Expression of NaV1.4 in tsA201 cells yielded a TTX-sensitive Na+ current with an IC50 of 10 nM.
These results demonstrate the expression of the TTX-sensitive NaV1.4 channel in canine cardiac Purkinje myocytes. This novel finding suggests a role of NaV1.4 channel in Purkinje myocytes and thus has important clinical implications for the mechanisms and management of ventricular arrhythmias originating in the Purkinje network.
PMCID: PMC1849952  PMID: 17286959
Na+ channel isoforms; cardiac tissues; Purkinje myocytes; tetrodotoxin
20.  Na+-activated K+ channels express a large delayed outward current in neurons during normal physiology 
Nature neuroscience  2009;12(6):745-750.
One of the largest components of the delayed outward current active during normal physiology in many mammalian neurons such as medium spiny neurons of the striatum and tufted–mitral cells of the olfactory bulb, has gone unnoticed and is due to a Na+-activated-K+-current. Previous studies of K+ currents in mammalian neurons may have overlooked this large outward component because the sodium channel blocker tetrodotoxin (TTX) is typically used in such studies; we find that TTX also eliminates this delayed outward component as a secondary consequence. Unexpectedly we found that the activity of a persistent inward sodium current (persistent INa) is highly effective in activating this large Na+-dependent (TTX-sensitive) delayed outward current. Using siRNA techniques we identified SLO2.2 (Slack) channels as carriers of this delayed outward current. These findings have far reaching implications for many aspects of cellular and systems neuroscience, as well as clinical neurology and pharmacology.
PMCID: PMC2712728  PMID: 19412167
21.  An in vivo tethered toxin approach for the cell-autonomous inactivation of voltage-gated sodium channel currents in nociceptors 
The Journal of Physiology  2010;588(10):1695-1707.
Understanding information flow in sensory pathways requires cell-selective approaches to manipulate the activity of defined neurones. Primary afferent nociceptors, which detect painful stimuli, are enriched in specific voltage-gated sodium channel (VGSC) subtypes. Toxins derived from venomous animals can be used to dissect the contributions of particular ion currents to cell physiology. Here we have used a transgenic approach to target a membrane-tethered isoform of the conotoxin MrVIa (t-MrVIa) only to nociceptive neurones in mice. T-MrVIa transgenic mice show a 44 ± 7% reduction of tetrodotoxin-resistant (TTX-R) VGSC current densities. This inhibition is permanent, reversible and does not result in functional upregulation of TTX-sensitive (TTX-S) VGSCs, voltage-gated calcium channels (VGCCs) or transient receptor potential (TRP) channels present in nociceptive neurones. As a consequence of the reduction of TTX-R VGSC currents, t-MrVIa transgenic mice display decreased inflammatory mechanical hypersensitivity, cold pain insensitivity and reduced firing of cutaneous C-fibres sensitive to noxious cold temperatures. These data validate the use of genetically encoded t-toxins as a powerful tool to manipulate VGSCs in specific cell types within the mammalian nervous system. This novel genetic methodology can be used for circuit mapping and has the key advantage that it enables the dissection of the contribution of specific ionic currents to neuronal function and to behaviour.
PMCID: PMC2887988  PMID: 20308253
22.  An in vivo tethered toxin approach for the cell-autonomous inactivation of voltage-gated sodium channel currents in nociceptors 
The Journal of Physiology  2010;588(Pt 10):1695-1707.
Understanding information flow in sensory pathways requires cell-selective approaches to manipulate the activity of defined neurones. Primary afferent nociceptors, which detect painful stimuli, are enriched in specific voltage-gated sodium channel (VGSC) subtypes. Toxins derived from venomous animals can be used to dissect the contributions of particular ion currents to cell physiology. Here we have used a transgenic approach to target a membrane-tethered isoform of the conotoxin MrVIa (t-MrVIa) only to nociceptive neurones in mice. T-MrVIa transgenic mice show a 44 ± 7% reduction of tetrodotoxin-resistant (TTX-R) VGSC current densities. This inhibition is permanent, reversible and does not result in functional upregulation of TTX-sensitive (TTX-S) VGSCs, voltage-gated calcium channels (VGCCs) or transient receptor potential (TRP) channels present in nociceptive neurones. As a consequence of the reduction of TTX-R VGSC currents, t-MrVIa transgenic mice display decreased inflammatory mechanical hypersensitivity, cold pain insensitivity and reduced firing of cutaneous C-fibres sensitive to noxious cold temperatures. These data validate the use of genetically encoded t-toxins as a powerful tool to manipulate VGSCs in specific cell types within the mammalian nervous system. This novel genetic methodology can be used for circuit mapping and has the key advantage that it enables the dissection of the contribution of specific ionic currents to neuronal function and to behaviour.
PMCID: PMC2887988  PMID: 20308253
23.  Tetrodotoxin Poisoning Due to Pufferfish and Gastropods, and Their Intoxication Mechanism 
ISRN Toxicology  2011;2011:276939.
Marine pufferfish generally contain a large amount of tetrodotoxin (TTX) in their skin and viscera, and have caused many incidences of food poisoning, especially in Japan. Edible species and body tissues of pufferfish, as well as their allowable fishing areas, are therefore clearly stipulated in Japan, but still 2 to 3 people die every year due to pufferfish poisoning. TTX is originally produced by marine bacteria, and pufferfish are intoxicated through the food chain that starts with the bacteria. Pufferfish become nontoxic when fed TTX-free diets in a closed environment in which there is no possible invasion of TTX-bearing organisms. On the other hand, TTX poisoning due to marine snails has recently spread through Japan, China, Taiwan, and Europe. In addition, TTX poisoning of dogs due to the ingestion of sea slugs was recently reported in New Zealand. TTX in these gastropods also seems to be exogenous; carnivorous large snails are intoxicated by eating toxic starfish, and necrophagous small-to-medium snails, the viscera of dead pufferfish after spawning. Close attention must be paid to the geographic expansion and/or diversification of TTX-bearing organisms, and to the sudden occurrence of other forms of TTX poisoning due to their ingestion.
PMCID: PMC3658506  PMID: 23724281
24.  Isoflurane Inhibits the Tetrodotoxin-resistant Voltagegated Sodium Channel Nav1.8 
Anesthesiology  2009;111(3):591-599.
Voltage-gated sodium channels (Nav) mediate neuronal action potentials. Tetrodotoxin inhibits all Nav isoforms, but Nav1.8 and Nav1.9 are relatively tetrodotoxin-resistant (TTX-r) compared to other isoforms. Nav1.8 is highly expressed in dorsal root ganglion neurons and is functionally linked to nociception, but the sensitivity of TTX-r isoforms to inhaled anesthetics is unclear.
The sensitivities of heterologously expressed rat TTX-r Nav1.8 and endogenous tetrodotoxin-sensitive (TTX-s) Nav to the prototypic inhaled anesthetic isoflurane were tested in mammalian ND7/23 cells using patch-clamp electrophysiology.
From a holding potential of −70 mV, isoflurane (0.53±0.06 mM, ~1.8 MAC at 24°C) reduced normalized peak Na+ current (INa) of Nav1.8 to 0.55±0.03 and of endogenous TTX-s Nav to 0.56±0.06. Isoflurane minimally inhibited INa from a holding potential of −140 mV. Isoflurane did not affect voltage-dependence of activation, but significantly shifted voltage-dependence of steady-state inactivation by −6 mV for Nav1.8 and by −7 mV for TTX-s Nav. IC50 values for inhibition of peak INa were 0.67±0.06 mM for Nav1.8 and 0.66±0.09 mM for TTX-s Nav; significant inhibition occurred at clinically relevant concentrations as low as 0.58 MAC. Isoflurane produced use-dependent block of Nav1.8; at a stimulation frequency of 10 Hz, 0.56±0.08 mM isoflurane reduced INa to 0.64±0.01 vs. 0.78±0.01 for control.
Isoflurane inhibited the tetrodotoxin-resistant isoform Nav1.8 with potency comparable to that for endogenous tetrodotoxin-sensitive Nav isoforms, indicating that sensitivity to inhaled anesthetics is conserved across diverse Nav family members. Block of Nav1.8 in dorsal root ganglion neurons could contribute to the effects of inhaled anesthetics on peripheral nociceptive mechanisms.
PMCID: PMC2756082  PMID: 19672182
25.  Investigating Diet as the Source ofTetrodotoxin in Pleurobranchaea maculata 
Marine Drugs  2013;12(1):1-16.
The origin of tetrodotoxin (TTX) is highly debated; researchers have postulated either an endogenous or exogenous source with the host accumulating TTX symbiotically or via food chain transmission. The aim of this study was to determine whether the grey side-gilled sea slug (Pleurobranchaea maculata) could obtain TTX from a dietary source, and to attempt to identify this source through environmental surveys. Eighteen non-toxic P. maculata were maintained in aquariums and twelve were fed a TTX-containing diet. Three P. maculata were harvested after 1 h, 24 h, 17 days and 39 days and TTX concentrations in their stomach, gonad, mantle and remaining tissue/fluids determined using liquid chromatography-mass spectrometry. Tetrodotoxin was detected in all organs/tissue after 1 h with an average uptake of 32%. This decreased throughout the experiment (21%, 15% and 9%, respectively). Benthic surveys at sites with dense populations of toxic P. maculata detected very low or no TTX in other organisms. This study demonstrates that P. maculata can accumulate TTX through their diet. However, based on the absence of an identifiable TTX source in the environment, in concert with the extremely high TTX concentrations and short life spans of P. maculata, it is unlikely to be the sole TTX source for this species.
PMCID: PMC3917257  PMID: 24368566
diet; Pleurobranchaea maculata; tetrodotoxin

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