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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.  Combined Liquid and Solid-Phase Extraction Improves Quantification of Brain Estrogen Content 
Accuracy in quantifying brain-derived steroid hormones (“neurosteroids”) has become increasingly important for understanding the modulation of neuronal activity, development, and physiology. Relative to other neuroactive compounds and classical neurotransmitters, steroids pose particular challenges with regard to isolation and analysis, owing to their lipid solubility. Consequently, anatomical studies of the distribution of neurosteroids have relied primarily on the expression of neurosteroid synthesis enzymes. To evaluate the distribution of synthesis enzymes vis-à-vis the actual steroids themselves, traditional steroid quantification assays, including radioimmunoassays, have successfully employed liquid extraction methods (e.g., ether, dichloromethane, or methanol) to isolate steroids from microdissected brain tissue. Due to their sensitivity, safety, and reliability, the use of commercial enzyme-immunoassays (EIA) for laboratory quantification of steroids in plasma and brain has become increasingly widespread. However, EIAs rely on enzymatic reactions in vitro, making them sensitive to interfering substances in brain tissue and thus producing unreliable results. Here, we evaluate the effectiveness of a protocol for combined, two-stage liquid/solid-phase extraction (SPE) as compared to conventional liquid extraction alone for the isolation of estradiol (E2) from brain tissue. We employ the songbird model system, in which brain steroid production is pronounced and linked to neural mechanisms of learning and plasticity. This study outlines a combined liquid–SPE protocol that improves the performance of a commercial EIA for the quantification of brain E2 content. We demonstrate the effectiveness of our optimized method for evaluating the region specificity of brain E2 content, compare these results to established anatomy of the estrogen synthesis enzyme and estrogen receptor, and discuss the nature of potential EIA interfering substances.
PMCID: PMC3164112  PMID: 21909323
estrogen; neurosteroid; ELISA; protocol
3.  Tetrodotoxin-Resistant Sodium Channels in Sensory Neurons Generate Slow Resurgent Currents That Are Enhanced by Inflammatory Mediators 
The Journal of Neuroscience  2014;34(21):7190-7197.
Resurgent sodium currents contribute to the regeneration of action potentials and enhanced neuronal excitability. Tetrodotoxin-sensitive (TTX-S) resurgent currents have been described in many different neuron populations, including cerebellar and dorsal root ganglia (DRG) neurons. In most cases, sodium channel Nav1.6 is the major contributor to these TTX-S resurgent currents. Here we report a novel TTX-resistant (TTX-R) resurgent current recorded from rat DRG neurons. The TTX-R resurgent currents are similar to classic TTX-S resurgent currents in many respects, but not all. As with TTX-S resurgent currents, they are activated by membrane repolarization, inhibited by lidocaine, and enhanced by a peptide-mimetic of the β4 sodium channel subunit intracellular domain. However, the TTX-R resurgent currents exhibit much slower kinetics, occur at more depolarized voltages, and are sensitive to the Nav1.8 blocker A803467. Moreover, coimmunoprecipitation experiments from rat DRG lysates indicate the endogenous sodium channel β4 subunits associate with Nav1.8 in DRG neurons. These results suggest that slow TTX-R resurgent currents in DRG neurons are mediated by Nav1.8 and are generated by the same mechanism underlying TTX-S resurgent currents. We also show that both TTX-S and TTX-R resurgent currents in DRG neurons are enhanced by inflammatory mediators. Furthermore, the β4 peptide increased excitability of small DRG neurons in the presence of TTX. We propose that these slow TTX-R resurgent currents contribute to the membrane excitability of nociceptive DRG neurons under normal conditions and that enhancement of both types of resurgent currents by inflammatory mediators could contribute to sensory neuronal hyperexcitability associated with inflammatory pain.
PMCID: PMC4028496  PMID: 24849353
action potential; hyperexcitability; nociceptor; resurgent sodium current; sodium current; voltage clamp
4.  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
5.  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
6.  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
7.  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
8.  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
9.  Update on Controls for Isolation and Quantification Methodology of Extracellular Vesicles Derived from Adipose Tissue Mesenchymal Stem Cells 
The research field on extracellular vesicles (EV) has rapidly expanded in recent years due to the therapeutic potential of EV. Adipose tissue human mesenchymal stem cells (ASC) may be a suitable source for therapeutic EV. A major limitation in the field is the lack of standardization of the challenging techniques to isolate and characterize EV. The aim of our study was to incorporate new controls for the detection and quantification of EV derived from ASC and to analyze the applicability and limitations of the available techniques. ASC were cultured in medium supplemented with 5% of vesicles-free fetal bovine serum. The EV were isolated from conditioned medium by differential centrifugation with size filtration (0.2 μm). As a control, non-conditioned culture medium was used (control medium). To detect EV, electron microscopy, conventional flow cytometry, and western blot were used. The quantification of the EV was by total protein quantification, ExoELISA immunoassay, and Nanosight. Cytokines and growth factors in the EV samples were measured by multiplex bead array kit. The EV were detected by electron microscope. Total protein measurement was not useful to quantify EV as the control medium showed similar protein contents as the EV samples. The ExoELISA kits had technical troubles and it was not possible to quantify the concentration of exosomes in the samples. The use of Nanosight enabled quantification and size determination of the EV. It is, however, not possible to distinguish protein aggregates from EV with this method. The technologies for quantification and characterization of the EV need to be improved. In addition, we detected protein contaminants in the EV samples, which make it difficult to determine the real effect of EV in experimental models. It will be crucial in the future to optimize design novel methods for purification and characterization of EV.
PMCID: PMC4204528  PMID: 25374572
extracellular vesicles; mesenchymal stem cells; differential centrifugation; flow cytometry; electron microscopy; nanosight
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.  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
12.  Sensory discrimination between innocuous and noxious cold by TRPM8-expressing DRG neurons of rats 
Molecular Pain  2012;8:79.
The TRPM8 channel is a principal cold transducer that is expressed on some primary afferents of the somatic and cranial sensory systems. However, it is uncertain whether TRPM8-expressing afferent neurons have the ability to convey innocuous and noxious cold stimuli with sensory discrimination between the two sub-modalities. Using rat dorsal root ganglion (DRG) neurons and the patch-clamp recording technique, we characterized membrane and action potential properties of TRPM8-expressing DRG neurons at 24°C and 10°C. TRPM8-expressing neurons could be classified into TTX-sensitive (TTXs/TRPM8) and TTX-resistant (TTXr/TRPM8) subtypes based on the sensitivity to tetrodotoxin (TTX) block of their action potentials. These two subtypes of cold-sensing cells displayed different membrane and action potential properties. Voltage-activated inward Na+ currents were highly susceptible to cooling temperature and abolished by ~95% at 10°C in TTXs/TRPM8 DRG neurons, but remained substantially large at 10°C in TTXr/TRPM8 cells. In both TTXs/TRPM8 and TTXr/TRPM8 cells, voltage-activated outward K+ currents were substantially inhibited at 10°C, and the cooling-sensitive outward currents resembled A-type K+ currents. TTXs/TRPM8 neurons and TTXr/TRPM8 neurons were shown to fire action potentials at innocuous and noxious cold temperatures respectively, demonstrating sensory discrimination between innocuous and noxious cold by the two subpopulations of cold-sensing DRG neurons. The effects of cooling temperatures on voltage-gated Na+ channels and A-type K+ currents are likely to be contributing factors to sensory discrimination of cold by TTXs/TRPM8 and TTXr/TRPM8 afferent neurons.
PMCID: PMC3495675  PMID: 23092296
13.  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
14.  Suppression of Neurotoxic Lesion-Induced Seizure Activity: Evidence for a Permanent Role for the Hippocampus in Contextual Memory 
PLoS ONE  2011;6(11):e27426.
Damage to the hippocampus (HPC) using the excitotoxin N-methyl-D-aspartate (NMDA) can cause retrograde amnesia for contextual fear memory. This amnesia is typically attributed to loss of cells in the HPC. However, NMDA is also known to cause intense neuronal discharge (seizure activity) during the hours that follow its injection. These seizures may have detrimental effects on retrieval of memories. Here we evaluate the possibility that retrograde amnesia is due to NMDA-induced seizure activity or cell damage per se. To assess the effects of NMDA induced activity on contextual memory, we developed a lesion technique that utilizes the neurotoxic effects of NMDA while at the same time suppressing possible associated seizure activity. NMDA and tetrodotoxin (TTX), a sodium channel blocker, are simultaneously infused into the rat HPC, resulting in extensive bilateral damage to the HPC. TTX, co-infused with NMDA, suppresses propagation of seizure activity. Rats received pairings of a novel context with foot shock, after which they received NMDA-induced, TTX+NMDA-induced, or no damage to the HPC at a recent (24 hours) or remote (5 weeks) time point. After recovery, the rats were placed into the shock context and freezing was scored as an index of fear memory. Rats with an intact HPC exhibited robust memory for the aversive context at both time points, whereas rats that received NMDA or NMDA+TTX lesions showed a significant reduction in learned fear of equal magnitude at both the recent and remote time points. Therefore, it is unlikely that observed retrograde amnesia in contextual fear conditioning are due to disruption of non-HPC networks by propagated seizure activity. Moreover, the memory deficit observed at both time points offers additional evidence supporting the proposition that the HPC has a continuing role in maintaining contextual memories.
PMCID: PMC3215748  PMID: 22110648
15.  Genetic architecture of a feeding adaptation: garter snake (Thamnophis) resistance to tetrodotoxin bearing prey 
Detailing the genetic basis of adaptive variation in natural populations is a first step towards understanding the process of adaptive evolution, yet few ecologically relevant traits have been characterized at the genetic level in wild populations. Traits that mediate coevolutionary interactions between species are ideal for studying adaptation because of the intensity of selection and the well-characterized ecological context. We have previously described the ecological context, evolutionary history and partial genetic basis of tetrodotoxin (TTX) resistance in garter snakes (Thamnophis). Derived mutations in a voltage-gated sodium channel gene (Nav1.4) in three garter snake species are associated with resistance to TTX, the lethal neurotoxin found in their newt prey (Taricha). Here we evaluate the contribution of Nav1.4 alleles to TTX resistance in two of those species from central coastal California. We measured the phenotypes (TTX resistance) and genotypes (Nav1.4 and microsatellites) in a local sample of Thamnophis atratus and Thamnophis sirtalis. Allelic variation in Nav1.4 explains 23 per cent of the variation in TTX resistance in T. atratus while variation in a haphazard sample of the genome (neutral microsatellite markers) shows no association with the phenotype. Similarly, allelic variation in Nav1.4 correlates almost perfectly with TTX resistance in T. sirtalis, but neutral variation does not. These strong correlations suggest that Nav1.4 is a major effect locus. The simple genetic architecture of TTX resistance in garter snakes may significantly impact the dynamics of phenotypic coevolution. Fixation of a few alleles of major effect in some garter snake populations may have led to the evolution of extreme phenotypes and an ‘escape’ from the arms race with newts.
PMCID: PMC2981930  PMID: 20522513
adaptation; gene of major effect; coevolution; Thamnophis; tetrodotoxin; sodium channel
16.  Differential Interactions of Na+ Channel Toxins with T-type Ca2+ Channels 
The Journal of General Physiology  2008;132(1):101-113.
Two types of voltage-dependent Ca2+ channels have been identified in heart: high (ICaL) and low (ICaT) voltage-activated Ca2+ channels. In guinea pig ventricular myocytes, low voltage–activated inward current consists of ICaT and a tetrodotoxin (TTX)-sensitive ICa component (ICa(TTX)). In this study, we reexamined the nature of low-threshold ICa in dog atrium, as well as whether it is affected by Na+ channel toxins. Ca2+ currents were recorded using the whole-cell patch clamp technique. In the absence of external Na+, a transient inward current activated near −50 mV, peaked at −30 mV, and reversed around +40 mV (HP = −90 mV). It was unaffected by 30 μM TTX or micromolar concentrations of external Na+, but was inhibited by 50 μM Ni2+ (by ∼90%) or 5 μM mibefradil (by ∼50%), consistent with the reported properties of ICaT. Addition of 30 μM TTX in the presence of Ni2+ increased the current approximately fourfold (41% of control), and shifted the dose–response curve of Ni2+ block to the right (IC50 from 7.6 to 30 μM). Saxitoxin (STX) at 1 μM abolished the current left in 50 μM Ni2+. In the absence of Ni2+, STX potently blocked ICaT (EC50 = 185 nM) and modestly reduced ICaL (EC50 = 1.6 μM). While TTX produced no direct effect on ICaT elicited by expression of hCaV3.1 and hCaV3.2 in HEK-293 cells, it significantly attenuated the block of this current by Ni2+ (IC50 increased to 550 μM Ni2+ for CaV3.1 and 15 μM Ni2+ for CaV3.2); in contrast, 30 μM TTX directly inhibited hCaV3.3-induced ICaT and the addition of 750 μM Ni2+ to the TTX-containing medium led to greater block of the current that was not significantly different than that produced by Ni2+ alone. 1 μM STX directly inhibited CaV3.1-, CaV3.2-, and CaV3.3-mediated ICaT but did not enhance the ability of Ni2+ to block these currents. These findings provide important new implications for our understanding of structure–function relationships of ICaT in heart, and further extend the hypothesis of a parallel evolution of Na+ and Ca2+ channels from an ancestor with common structural motifs.
PMCID: PMC2442173  PMID: 18591418
17.  Activity-dependent regulation of inhibition via GAD67 
The Journal of Neuroscience  2012;32(25):8521-8531.
Alterations in network activity trigger compensatory changes in excitation and inhibition that restore neuronal firing rate to an optimal range. One example of such synaptic homeostasis is the downregulation of inhibitory transmission by chronic inactivity, in part, through the reduction of vesicular transmitter content. The enzyme glutamic acid decarboxylase 67 (GAD67) is critical for GABA synthesis, but its involvement in homeostatic plasticity is unclear. We explored the role of GAD67 in activity-dependent synaptic plasticity using a mouse line (Gad1−/−) in which GAD67 expression is disrupted by genomic insertion of the green fluorescent protein (GFP). Homozygous deletion of Gad1 significantly reduced miniature inhibitory postsynaptic current (mIPSC) amplitudes and GABA levels in cultured hippocampal neurons. The fractional block of mIPSC amplitude by a low affinity, competitive GABAA receptor antagonist was higher in GAD67-lacking neurons, suggesting that GABA concentration in the synaptic cleft is lower in knockout animals. Chronic suppression of activity by the application of tetrodotoxin (TTX) reduced mIPSC amplitudes and the levels of GAD67 and GABA. Moreover, TTX reduced GFP levels in interneurons, suggesting that GAD67 gene expression is a key regulatory target of activity. These in vitro experiments were corroborated by in vivo studies in which olfactory deprivation reduced mIPSC amplitudes and GFP levels in glomerular neurons in the olfactory bulb. Importantly, TTX-induced downregulation of mIPSC was attenuated in Gad1−/− neurons. Altogether, these findings indicate that activity-driven expression of GAD67 critically controls GABA synthesis and, thus, vesicular filling of the transmitter.
PMCID: PMC3388776  PMID: 22723692
18.  Parallel Evolution of Tetrodotoxin Resistance in Three Voltage-Gated Sodium Channel Genes in the Garter Snake Thamnophis sirtalis 
Molecular Biology and Evolution  2014;31(11):2836-2846.
Members of a gene family expressed in a single species often experience common selection pressures. Consequently, the molecular basis of complex adaptations may be expected to involve parallel evolutionary changes in multiple paralogs. Here, we use bacterial artificial chromosome library scans to investigate the evolution of the voltage-gated sodium channel (Nav) family in the garter snake Thamnophis sirtalis, a predator of highly toxic Taricha newts. Newts possess tetrodotoxin (TTX), which blocks Nav’s, arresting action potentials in nerves and muscle. Some Thamnophis populations have evolved resistance to extremely high levels of TTX. Previous work has identified amino acid sites in the skeletal muscle sodium channel Nav1.4 that confer resistance to TTX and vary across populations. We identify parallel evolution of TTX resistance in two additional Nav paralogs, Nav1.6 and 1.7, which are known to be expressed in the peripheral nervous system and should thus be exposed to ingested TTX. Each paralog contains at least one TTX-resistant substitution identical to a substitution previously identified in Nav1.4. These sites are fixed across populations, suggesting that the resistant peripheral nerves antedate resistant muscle. In contrast, three sodium channels expressed solely in the central nervous system (Nav1.1–1.3) showed no evidence of TTX resistance, consistent with protection from toxins by the blood–brain barrier. We also report the exon–intron structure of six Nav paralogs, the first such analysis for snake genes. Our results demonstrate that the molecular basis of adaptation may be both repeatable across members of a gene family and predictable based on functional considerations.
PMCID: PMC4209135  PMID: 25135948
adaptation; coevolution; gene families; molecular evolution; predator–prey interactions; toxins
19.  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
20.  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
21.  An Efficient Strategy for Heterologous Expression and Purification of Active Peptide Hainantoxin-IV 
PLoS ONE  2015;10(2):e0117099.
Hainantoxin-IV (HNTX-IV) from the venom of the spider Selenocosmia hainana is a potent antagonist that specifically inhibits the tetrodotoxin-sensitive (TTX-S) sodium channels. The toxin peptide consists of 35 amino acids and adopts a typical inhibitory cystine knot (ICK) motif. To obtain adequate HNTX-IV peptides for further insight into the structure-activity relationships of the toxin, a novel strategy including cloning, expression and purification was developed in an E. coli expression system. For this purpose, a seamless restriction-free (RF) cloning method was employed for the construction of an expression vector to avoid introducing unwanted sequences into the target gene. Furthermore, the solubility of recombinant HNTX-IV could be promoted efficiently by the combination of a glutathione S-transferase (GST) tag and a small ubiquitin-related modifier (SUMO) tag. Finally, an affinity-chromatography-free purification strategy was developed by cut-off dialysis tubing combined with trichloroacetic acid (TCA) extraction. Further HPLC purification yielded recombinant, tag-free HNTX-IV with high yield and purity. The molecular weight of recombinant HNTX-IV (rHNTX-IV) is identical to its theoretical value according to Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) analysis. The recombinant toxin has similar activity (IC50 value of 120 nM) on the tetrodotoxin-sensitive (TTX-S) sodium channels in adult rat dorsal root ganglion (DRG) neurons to native toxins. In the report, an efficient and cost-effective strategy for producing rHNTX-IV was developed, which paved the way for the further study of structure-activity relationships of rHNTX-IV and its pharmaceutical applications.
PMCID: PMC4315428  PMID: 25647561
22.  Functional expression of “cardiac-type” Nav1.5 sodium channel in canine intracardiac ganglia 
The autonomic nervous system has been implicated in several arrhythmogenic diseases, including long QT syndrome type 3 (LQT3) and Brugada syndrome. Scarce information on the cellular components of the intrinsic cardiac ganglia from higher mammals has limited our understanding of the role of the autonomic nervous system in such diseases.
The purpose of this study was to isolate and characterize the electrophysiologic properties of canine intracardiac neurons.
Action potentials (APs) and ionic currents were studied in enzymatically dissociated canine intracardiac neurons under current and voltage clamp conditions. Immunohistochemical and reverse transcription-polymerase chain reaction analysis was performed using freshly isolated intracardiac ganglia.
APs recorded from intracardiac neurons displayed a tetrodotoxin-resistant (TTX-R) component. TTX-R APs were abolished in the absence of sodium but persisted in the absence of external calcium. Immunohistochemical studies showed the presence of TTX-R sodium channels in these ganglia. Sodium currents were characterized by two components with different affinities for TTX: a tetrodotoxin-sensitive (TTX-S) component and a TTX-R component. TTX-S current inactivation was characteristic of neuronal sodium currents, whereas TTX-R current inactivation time constants were similar to those previously reported for Nav1.5 channels. TTX sensitivity (IC50 = 1.17 μM) of the TTX-R component was in the range reported for Nav1.5 channels. Expression of Nav1.5 channels in intracardiac ganglia was confirmed by PCR analysis and sequencing.
Our results suggest that canine intracardiac neurons functionally express Nav1.5 channels. These findings open an exciting new door to our understanding of autonomically modulated arrhythmogenic diseases linked to mutations in Nav1.5 channels, including Brugada syndrome and LQT3.
PMCID: PMC1989775  PMID: 16818219
SCN5A; Tetrodotoxin; Autonomic dysfunction; Cardiac arrhythmia; Sudden cardiac death; long QT syndrome; Brugada syndrome
23.  Pacemaking in dopaminergic ventral tegmental area neurons: depolarizing drive from background and voltage-dependent sodium conductances 
Dopaminergic neurons in the ventral tegmental area (VTA) fire spontaneously in a pacemaker-like manner. We analyzed the ionic currents that drive pacemaking in dopaminergic VTA neurons, studied in mouse brain slices. Pacemaking was not inhibited by blocking hyperpolarization-activated cation current (Ih) or blocking all calcium current by Mg2+ replacement of Ca2+. Tetrodotoxin (TTX) stopped spontaneous activity and usually resulted in stable resting potentials near −60 mV to −55 mV, 10–15 mV below the action potential threshold. When external sodium was replaced by N-methyl-D-glucamine (NMDG) with TTX present, cells hyperpolarized by an average of −11 mV, suggesting a significant resting sodium conductance not sensitive to TTX. Voltage-clamp experiments using slow (10 mV/s) ramps showed a steady-state, steeply voltage-dependent current blocked by TTX that activates near −60 mV, as well as a sodium “background” current with little voltage-sensitivity, revealed by NMDG replacement for sodium with TTX present. We quantified these two components of sodium current during the pacemaking trajectory using action potential clamp. The initial phase of depolarization, up to about −55 mV, is driven mainly by non-voltage-dependent sodium background current. Above −55 mV, TTX-sensitive voltage-dependent “persistent” Na current helps drive the final phase of depolarization to the spike threshold. Voltage-dependent calcium current is small at all subthreshold voltages. The pacemaking mechanism in VTA neurons differs from that in substantia nigra pars compacta (SNc) neurons, where subthreshold calcium current plays a dominant role. In addition, we found that non-voltage-dependent background sodium current is much smaller in SNc neurons than VTA neurons.
PMCID: PMC2892804  PMID: 20505107
spontaneous firing; dopaminergic neurons; VTA
24.  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
25.  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

Results 1-25 (1067948)