Specific Voltage-gated Sodium Channel Research Articles

Total syntheses of Tetrodotoxin and 9-epiTetrodotoxin

Tetrodotoxin and congeners are specific voltage-gated sodium channel blockers that exhibit remarkable anesthetic and analgesic effects. Here, we present a scalable asymmetric syntheses of Tetrodotoxin and 9-epiTetrodotoxin from the abundant chemical feedstock furfuryl alcohol. The optically pure cyclohexane skeleton is assembled via a stereoselective Diels-Alder reaction. The dense heteroatom substituents are established sequentially by a series of functional group interconversions on highly oxygenated cyclohexane frameworks, including a chemoselective cyclic anhydride opening, and a decarboxylative hydroxylation. An innovative SmI2-mediated concurrent fragmentation, an oxo-bridge ring opening and ester reduction followed by an Upjohn dihydroxylation deliver the highly oxidized skeleton. Ruthenium-catalyzed oxidative alkyne cleavage and formation of the hemiaminal and orthoester under acidic conditions enable the rapid assembly of Tetrodotoxin, anhydro-Tetrodotoxin, 9-epiTetrodotoxin, and 9-epi lactone-Tetrodotoxin.

First Identification of 12β-Deoxygonyautoxin 5 (12α-Gonyautoxinol 5) in the Cyanobacterium Dolichospermum circinale (TA04) and 12β-Deoxysaxitoxin (12α-Saxitoxinol) in D. circinale (TA04) and the Dinoflagellate Alexandrium pacificum (Group IV) (120518KureAC).

Saxitoxin and its analogues, paralytic shellfish toxins (PSTs), are potent and specific voltage-gated sodium channel blockers. These toxins are produced by some species of freshwater cyanobacteria and marine dinoflagellates. We previously identified several biosynthetic intermediates of PSTs, as well as new analogues, from such organisms and proposed the biosynthetic and metabolic pathways of PSTs. In this study, 12β-deoxygonyautoxin 5 (12α-gonyautoxinol 5 = gonyautoxin 5-12(R)-ol) was identified in the freshwater cyanobacterium, Dolichospermum circinale (TA04), and 12β-deoxysaxitoxin (12α-saxitoxinol = saxitoxin-12(R)-ol) was identified in the same cyanobacterium and in the marine dinoflagellate Alexandrium pacificum (Group IV) (120518KureAC) for the first time from natural sources. The authentic standards of these compounds and 12α-deoxygonyautoxin 5 (12β-gonyautoxinol 5 = gonyautoxin 5-12(S)-ol) were prepared by chemical derivatization from the major PSTs, C1/C2, produced in D. circinale (TA04). These standards were used to identify the deoxy analogues by comparing the retention times and MS/MS spectra using high-resolution LC-MS/MS. Biosynthetic or metabolic pathways for these analogues have also been proposed based on their structures. The identification of these compounds supports the α-oriented stereoselective oxidation at C12 in the biosynthetic pathway towards PSTs.

Generation of two induced pluripotent stem cell (iPSC) lines (BBANTWi006-A, BBANTWi007-A) from Brugada syndrome patients carrying an SCN5A mutation

Brugada syndrome (BrS) is an inherited primary electrical disorder of the heart. 25% of BrS patients carry a mutation in the SCN5A gene, encoding the cardiac specific voltage-gated sodium channel Nav1.5. Here we report two iPSC lines (BBANTWi006-A, BBANTWi007-A) of a brother and a sister carrying an SCN5A mutation (c.4813 + 3_4813 + 6dupGGGT) causing BrS. iPSCs were generated from dermal fibroblasts and reprogrammed with the Cytotune®-iPS 2.0 Sendai Reprogramming Kit (Invitrogen). The generated iPSCs showed a normal karyotype, expressed pluripotency markers, were differentiated into cells of the three germ layers and carried the original genotype.

Cardioprotective Effect of Glycyrrhizin on Myocardial Remodeling in Diabetic Rats.

Myocardial fibrosis is one of the major complications of long-term diabetes. Hyperglycemia induced cardiomyocyte atrophy is a frequent pathophysiological indicator of diabetic heart. The objective of this study was to investigate the cardioprotective effect of glycyrrhizin (GLC) on myocardial damage in diabetic rats and assess the anti-inflammatory and anti-fibrotic effect of GLC. Our study demonstrates that hyperglycemia can elevate cardiac atrophy in diabetic animals. Type 2 diabetic fatty and the lean control rats were evaluated for cardiac damage and inflammation at 8–12 weeks after the development of diabetes. Western blot and immunohistochemical studies revealed that gap junction protein connexin-43 (CX43), cardiac injury marker troponin I, cardiac muscle specific voltage gated sodium channel NaV1.5 were significantly altered in the diabetic heart. Furthermore, oxidative stress mediator receptor for advanced glycation end-products (RAGE), as well as inflammatory mediator phospho-p38 MAPK and chemokine receptor CXCR4 were increased in the diabetic heart whereas the expression of nuclear factor erythroid-2-related factor 2 (Nrf2), the antioxidant proteins that protect against oxidative damage was reduced. We also observed an increase in the expression of the pleiotropic cytokine, transforming growth factor beta (TGF-β) in the diabetic heart. GLC treatment exhibited a decrease in the expression of phospho-p38 MAPK, RAGE, NaV1.5 and TGF-β and it also altered the expression of CX43, CXCR4, Nrf2 and troponin I. These observations suggest that GLC possesses cardioprotective effects in diabetic cardiac atrophy and that these effects could be mediated through activation of Nrf2 and inhibition of CXCR4/SDF1 as well as TGF-β/p38MAPK signaling pathway.

Future in regional anesthesia and pain medicine: the pharmacological view.

Local anesthetics are still incompletely understood, and none of the currently available drugs are optimal. The primary target of local anesthetics is the voltage-gated sodium channel (VGSC), where they lead to a temporary interruption of nerve conduction. Unfortunately, local anesthetics are neither specific at blocking a specific VGSC isoform, nor a specific cell type. We realize now that the old classification of A and C fibers according to myelin thickness is outdated, that next to differing myelin configuration, cells differ also by their molecular biology, and that there are close to 20 different neuronal subgroups when function is concerned. The ideal local anesthetic would only block sensory impulses, or even only painful impulses. In the search for that drug, several research avenues have been followed. First, efforts have been undertaken to extend duration of local anesthetics, by additives or extended-release formulation. Second, blockade of specific pain fibers has been attempted by targeting permanently charged anesthetics specifically into nociceptors. Third, blockade of specific isoforms using antibodies, and adaptation of naturally occurring neurotoxins has shown promise. Lastly, combination of local anesthetics with other analgesics may improve their duration of action.

Safety, Tolerability, Pharmacokinetics, and Concentration-QTc Analysis of Tetrodotoxin: A Randomized, Dose Escalation Study in Healthy Adults.

Tetrodotoxin (TTX) is a highly specific voltage-gated sodium channel (VGSC) blocker in clinical evaluation as a peripheral-acting analgesic for chronic pain. This study presents the first published results of the safety including cardiac liability of TTX at therapeutic-relevant concentrations in twenty-five healthy adults. Randomized, double-blind, placebo-, and positive- (moxifloxacin) controlled study evaluated single ascending doses of 15 µg, 30 µg, and 45 µg TTX over 3 periods with a 7-day washout between each period. Subcutaneous injections of TTX were readily absorbed, reaching maximum plasma concentration (Cmax) within 1.5 h. Both extent of exposure (AUC) and Cmax increased in proportion to dose. No QT prolongation was identified by concentration-QTc analysis and the upper bounds of the two-sided 90% confidence interval of predicted maximum baseline and placebo corrected QTcF (ΔΔQTcF) value did not exceed 10 ms for all tetrodotoxin doses, thereby meeting the criteria of a negative QT study. Safety assessments showed no clinically relevant changes with values similar between all groups and no subject withdrawing due to adverse events. Paresthesia, oral-paresthesia, headache, dizziness, nausea, and myalgia were the most common TEAEs (overall occurrence ≥5%) in the TTX treatment groups. TTX doses investigated in this study are safe, well-tolerated, and lack proarrhythmic proclivity.

The voltage-gated sodium channel inhibitor, 4,9-anhydrotetrodotoxin, blocks human Nav1.1 in addition to Nav1.6

Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in neurons. The human genome includes ten human VGSC α-subunit genes, SCN(X)A, encoding Nav1.1–1.9 plus Nax. To understand the unique role that each VGSC plays in normal and pathophysiological function in neural networks, compounds with high affinity and selectivity for specific VGSC subtypes are required. Toward that goal, a structural analog of the VGSC pore blocker tetrodotoxin, 4,9-anhydrotetrodotoxin (4,9-ah-TTX), has been reported to be more selective in blocking Na+ current mediated by Nav1.6 than other TTX-sensitive VGSCs, including Nav1.2, Nav1.3, Nav1.4, and Nav1.7. While SCN1A, encoding Nav1.1, has been implicated in several neurological diseases, the effects of 4,9-ah-TTX on Nav1.1-mediated Na+ current have not been tested. Here, we compared the binding of 4,9-ah-TTX for human and mouse brain preparations, and the effects of 4,9-ah-TTX on human Nav1.1-, Nav1.3- and Nav1.6-mediated Na+ currents using the whole-cell patch clamp technique in heterologous cells. We show that, while 4,9-ah-TTX administration results in significant blockade of Nav1.6-mediated Na+ current in the nanomolar range, it also has significant effects on Nav1.1-mediated Na+ current. Thus, 4,9-ah-TTX is not a useful tool in identifying Nav1.6-specific effects in human brain networks.

Assessment of the TRPM8 inhibitor AMTB in breast cancer cells and its identification as an inhibitor of voltage gated sodium channels

AimsTo assess levels of the calcium permeable transient receptor potential cation channel, subfamily melastatin, member 8 (TRPM8) in breast cancer molecular subtypes and to assess the consequences of TRPM8 pharmacological inhibition with AMTB (an inhibitor of TRPM8) on breast cancer cell lines. Materials and methodsCell viability and migration of breast cancer cells was determined using MTS assays and wound healing assays, respectively. RNA-Seq analysis of breast tumours and qPCR in breast cancer cell lines were used to assess mRNA levels of ion channels. Membrane potential assays were employed to assess the effects of AMTB against specific voltage gated sodium channels (NaV). Key findingsTRPM8 levels were significantly higher in breast cancers of the basal molecular subtype. AMTB decreased viable cell number in MDA-MB-231 and SK-BR-3 breast cancer cell lines (30 and 100 μM), and also reduced the migration of MDA-MB-231 cells (30 μM). However, these effects were independent of TRPM8, as no TRPM8 mRNA was detected in MDA-MB-231 cells. AMTB was identified as an inhibitor of NaV isoforms. NaV1.1–1.9 were expressed in a number of breast cancer cell lines, with NaV1.5 mRNA highest in MDA-MB-231 cells compared to the other breast cancer cell lines assessed. SignificanceTRPM8 levels may be elevated in basal breast cancers, however, TRPM8 expression appears to be lost in many breast cancer cell lines. Some of the effects of AMTB attributed to TRPM8 may be due to effects on NaV channels.

Reduced Nav1.6 Sodium Channel Activity in Mice Increases In Vivo Sensitivity to Volatile Anesthetics

Nav1.6 is a major voltage-gated sodium channel in the central and peripheral nervous systems. Within neurons, the channel protein is concentrated at the axon initial segment and nodes of Ranvier, where it functions in initiation and propagation of action potentials. We examined the role of Nav1.6 in general anesthesia using two mouse mutants with reduced activity of Nav1.6, Scn8a medJ/medJ and Scn8a 9J/9J. The mice were exposed to the general anesthetics isoflurane and sevoflurane in step-wise increments; the concentration required to produce loss of righting reflex, a surrogate for anesthetic-induced unconsciousness in rodents, was determined. Mice homozygous for these mutations exhibited increased sensitivity to both isoflurane and sevoflurane. The increased sensitivity was observed during induction of unconsciousness but not during the recovery phase, suggesting that the effect is not attributable to compromised systemic physiology. Electroencephalographic theta power during baseline waking was lower in mutants, suggesting decreased arousal and reduced neuronal excitability. This is the first report linking reduced activity of a specific voltage-gated sodium channel to increased sensitivity to general anesthetics in vivo.

Plasticity at axon initial segment of hippocampal CA3 neurons in rat after status epilepticus induced by lithium–pilocarpine

The axon initial segment (AIS) is a specialized membrane region in the axon of neurons wherein numerous specific voltage-gated sodium channels (VGSCs) are clustered and action potentials are initiated. The AIS is currently considered as a new plastic hotspot. We investigated the alterations in Nav1.6 (SCN8A) and its adapter protein ankyrin G in the AIS of the hippocampal cornu ammonis 3 (CA3) pyramidal cells of rat after status epilepticus induced by lithium-pilocarpine (PISE). Nav1.6 and ankyrin G were colocalized in the AIS of hippocampal CA3 pyramidal neurons. Compared with the control group, the protein and mRNA expression of Nav1.6 increased within 24 h and 60 days after PISE. By contrast, ankyrin G protein expression decreased slightly within 24 h but increased within 60 days, whereas ankyrin G mRNA increased within 24 h and 60 days after PISE. However, the protein and mRNA expression levels of Nav1.6 and ankyrin G within 7 days after PISE did not differ significantly with those of the control. Nav1.6 and ankyrin G may participate in the plastic changes in the AIS of hippocampus CA3 neurons after PISE and play potential roles in epileptogenesis by regulating neuronal excitability.

Designing a C84 fullerene as a specific voltage-gated sodium channel blocker

Fullerene derivatives demonstrate considerable potential for numerous biological applications, such as the effective inhibition of HIV protease. Recently, they were identified for their ability to indiscriminately block biological ion channels. A fullerene derivative which specifically blocks a particular ion channel could lead to a new set of drug leads for the treatment of various ion channel-related diseases. Here, we demonstrate their extraordinary potential by designing a fullerene which mimics some of the functions of μ-conotoxin, a peptide derived from cone snail venom which potently binds to the bacterial voltage-gated sodium channel (NavAb). We show, using molecular dynamics simulations, that the C84 fullerene with six lysine derivatives uniformly attached to its surface is selective to NavAb over a voltage-gated potassium channel (Kv1.3). The side chain of one of the lysine residues protrudes into the selectivity filter of the channel, while the methionine residues located just outside of the channel form hydrophobic contacts with the carbon atoms of the fullerene. The modified C84 fullerene strongly binds to the NavAb channel with an affinity of 46 nM but binds weakly to Kv1.3 with an affinity of 3 mM. This potent blocker of NavAb may serve as a structural template from which potent compounds can be designed for the targeting of mammalian Nav channels. There is a genuine need to target mammalian Nav channels as a form of treatment of various diseases which have been linked to their malfunction, such as epilepsy and chronic pain.

Identification of Novel Interaction Sites that Determine Specificity between Fibroblast Growth Factor Homologous Factors and Voltage-gated Sodium Channels

Fibroblast growth factor homologous factors (FHFs, FGF11-14) bind to the C termini (CTs) of specific voltage-gated sodium channels (VGSC) and thereby regulate their function. The effect of an individual FHF on a specific VGSC varies greatly depending upon the individual FHF isoform. How individual FHFs impart distinctive effects on specific VGSCs is not known and the specificity of these pairwise interactions is not understood. Using several biochemical approaches combined with functional analysis, we mapped the interaction site for FGF12B on the Na(V)1.5 C terminus and discovered previously unknown determinants necessary for FGF12 interaction. Also, we demonstrated that FGF12B binds to some, but not all Na(V)1 CTs, suggesting specificity of interaction. Exploiting a human single nucleotide polymorphism in the core domain of FGF12 (P149Q), we identified a surface proline that contributes a part of this pairwise specificity. This proline is conserved among all FHFs, and mutation of the homologous residue in FGF13 also leads to loss of interaction with a specific VGSC CT (Na(V)1.1) and loss of modulation of the resultant Na(+) channel function. We hypothesized that some of the specificity mediated by this proline may result from differences in the affinity of the binding partners. Consistent with this hypothesis, surface plasmon resonance data showed that the P149Q mutation decreased the binding affinity between FHFs and VGSC CTs. Moreover, immunocytochemistry revealed that the mutation prevented proper subcellular targeting of FGF12 to the axon initial segment in neurons. Together, these results give new insights into details of the interactions between FHFs and Na(V)1.x CTs, and the consequent regulation of Na(+) channels.

The Roles of Sodium Channels in Nociception: Implications for Mechanisms of Neuropathic Pain

Animal models have provided useful insights into the development and treatment of neuropathic pain. New genetic data from both human studies and transgenic mouse models suggest that specific voltage-gated sodium channel subtypes are associated with specific types of pain and, as such, may be useful analgesic drug targets for a variety of pain types including neuropathic pain. Global voltage-gated sodium channel blockers such as lidocaine have proven efficacy in treating pain but can be limited by adverse effects when administered systemically. Selective sodium channel blockers targeting channels at the periphery (Nav1.7, Nav1.8, and Nav1.9) could potentially reduce the side effect profile. Individual isoforms of voltage-gated sodium channels have been linked to particular types of pain. Nav1.7 is a useful target for ameliorating acute mechanical pain and inflammatory pain, and strong evidence also suggests that Nav1.9 could be targeted for treating inflammatory pain. Selective blockers of Nav1.8 could also have clinical benefit for visceral pain. Although there is no association between a single sodium channel isoform and neuropathic pain, combined blockade of peripherally expressed isoforms Nav1.7, Nav1.8, and Nav1.9 may prove useful.

An in vivo tethered toxin approach for the cell-autonomous inactivation of voltage-gated sodium channel currents in nociceptors.

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.

Synthesis, Solution Structure, and Phylum Selectivity of a Spider δ-Toxin That Slows Inactivation of Specific Voltage-gated Sodium Channel Subtypes

Magi 4, now renamed delta-hexatoxin-Mg1a, is a 43-residue neurotoxic peptide from the venom of the hexathelid Japanese funnel-web spider (Macrothele gigas) with homology to delta-hexatoxins from Australian funnel-web spiders. It binds with high affinity to receptor site 3 on insect voltage-gated sodium (Na(V)) channels but, unlike delta-hexatoxins, does not compete for the related site 3 in rat brain despite being previously shown to be lethal by intracranial injection. To elucidate differences in Na(V) channel selectivity, we have undertaken the first characterization of a peptide toxin on a broad range of mammalian and insect Na(V) channel subtypes showing that delta-hexatoxin-Mg1a selectively slows channel inactivation of mammalian Na(V)1.1, Na(V)1.3, and Na(V)1.6 but more importantly shows higher affinity for insect Na(V)1 (para) channels. Consequently, delta-hexatoxin-Mg1a induces tonic repetitive firing of nerve impulses in insect neurons accompanied by plateau potentials. In addition, we have chemically synthesized and folded delta-hexatoxin-Mg1a, ascertained the bonding pattern of the four disulfides, and determined its three-dimensional solution structure using NMR spectroscopy. Despite modest sequence homology, we show that key residues important for the activity of scorpion alpha-toxins and delta-hexatoxins are distributed in a topologically similar manner in delta-hexatoxin-Mg1a. However, subtle differences in the toxin surfaces are important for the novel selectivity of delta-hexatoxin-Mg1a for certain mammalian and insect Na(V) channel subtypes. As such, delta-hexatoxin-Mg1a provides us with a specific tool with which to study channel structure and function and determinants for phylum- and tissue-specific activity.

Determination Of The Specificity For FHF/Na Channel Interactions

Specific FHFs (Fibroblast growth factor homologous factors) regulate voltage-gated sodium channel (VGSC) function by modulating activation, inactivation and/or subcellular distribution through binding to the C-terminus (CT) of specific VGSC alpha subunits. The effect upon VGSC function varies greatly depending upon the particular FHF isoform and its VGSC binding partner, but the specificity of these pair wise interactions are not understood. To identify the determinants for interaction between specific FHFs and VGSC pairs, we studied binding in a recombinant bacterial co-expression system. We show that FHF1b (aa 1- 144), which contains the conserved FHF β-trefoil core domain and is 60-70% homologous to the other members of the FHF family, bound to a NaV1.5 CT containing aa 1773-1940, but not to a NaV1.5 CT comprised of aa 1773-1878. Since the part of the CT necessary for binding contains the calmodulin (CaM) interaction site, we tested whether CaM and FHF1b competed for binding and found that all three components could form a complex; thus, CaM and FHF do not compete for interaction with the NaV1.5 CT. A full length FHF1b and FHF2b also bound to the NaV1.5 CT (aa 1773-1940) but not NaV1.5 CT (aa 1773-1878), suggesting that the region between aa 1878-1940 is necessary for FHF interaction. FHF1b also bound to NaV1.6 (aa 1767- 1926), but did not bind NaV1.1 (aa 1787-1948) nor NaV1.2 (aa 1772-1937). Since the distal regions among the tested NaV1.x CTs are less similar than the proximal regions, we predict that the specificity for interaction and the consequent particular effects upon VGSC function derives at least in part from this less-well conserved NaV1.x region.

Expression, Purification and Biophysical Characterization of a Superfamily of Prokaryotic Voltage-gated Sodium Channels

Eukaryotic voltage-gated sodium channels are monomeric membrane proteins comprised of four pseudo-repeats of domains containing six transmembrane segments, and have a molecular weight of >200 KDa. Their size and complexity makes them an arduous target for production in heterologous expression systems, a necessary step in acquiring the amounts of protein needed for biophysical and structural characterization. The simplified, single domain bacterial sodium channel containing six transmembrane segments isolated from Bacillus halodurans, NaChBac, can be expressed in E. coli in yields suitable for biophysical characterization and may enable successful crystallization and 3-D structure determination. Upon purification in 0.1% DDM, NaChBac is functional and associates to form a stable homotetramer (Nurani et al. (2008) Biochemistry 31:8114-8121). Seven different bacterial sodium channels with significant homology to NaChBac have been expressed in E. coli, purified in high-yield and characterized for secondary structure, thermal stability and drug binding. The experimental ease of obtaining a pure and homogeneous sample varies amongst the superfamily of sodium channels studied. The bacterial sodium channels are extremely thermal stable but individual members differ in their long-term stability when stored at room temperature, 4°C and −80°C, and their ability to bind the drug mibefradil. These channels also differ in their ability to form stable tetramers upon purification in different detergents. The differences and similarities found in this superfamily of sodium channels may prove valuable for determining general structural features important for specific voltage-gated sodium channel functions. The ability to express, purify and reconstitute multiple active bacterial sodium channels in membrane-mimic environments provides an arsenal of resources for elucidating structural features and identifying residues important for sustaining function in voltage-gated sodium channels. (Supported by a grants from the BBSRC to the MPSI consortium and BAW)

Epidermal growth factor potentiates in vitro metastatic behaviour of human prostate cancer PC-3M cells: involvement of voltage-gated sodium channel

BackgroundAlthough a high level of functional voltage-gated sodium channel (VGSC) expression has been found in strongly metastatic human and rat prostate cancer (PCa) cells, the mechanism(s) responsible for the upregulation is unknown. The concentration of epidermal growth factor (EGF), a modulator of ion channels, in the body is highest in prostatic fluid. Thus, EGF could be involved in the VGSC upregulation in PCa. The effects of EGF on VGSC expression in the highly metastatic human PCa PC-3M cell line, which was shown previously to express both functional VGSCs and EGF receptors, were investigated. A quantitative approach, from gene level to cell behaviour, was used. mRNA levels were determined by real-time PCR. Protein expression was studied by Western blots and immunocytochemistry and digital image analysis. Functional assays involved measurements of transverse migration, endocytic membrane activity and Matrigel invasion.ResultsExogenous EGF enhanced the cells' in vitro metastatic behaviours (migration, endocytosis and invasion). Endogenous EGF had a similar involvement. EGF increased VGSC Nav1.7 (predominant isoform in PCa) mRNA and protein expressions. Co-application of the highly specific VGSC blocker tetrodotoxin (TTX) suppressed the effect of EGF on all three metastatic cell behaviours studied.Conclusion1) EGF has a major involvement in the upregulation of functional VGSC expression in human PCa PC-3M cells. (2) VGSC activity has a significant intermediary role in potentiating effect of EGF in human PCa.

Identification of 4-S-Cysteinyltetrodotoxin from the Liver of the Puffer Fish, Fugu pardalis, and Formation of Thiol Adducts of Tetrodotoxin from 4,9-Anhydrotetrodotoxin

The metabolic pathway of tetrodotoxin (TTX), a powerful and specific voltage-gated sodium channel blocker, has not been well-clarified either in TTX-poisoned patients or in puffer fish. 4-S-CysteinylTTX (4-CysTTX) was isolated from the liver of the puffer fish, Fugu pardalis, as the first adduct of TTX with thiol. The structure was fully characterized by electrospray ionization-MS and two-dimensional NMR spectroscopy. The configuration of Cys in this compound was confirmed to be S (L-Cys) by application of the Marfey's method to cystine obtained from 4-CysTTX by iodine oxidation. We also found that 4-CysTTX was derived from 4,9-anhydroTTX by incubation with a large excess of Cys in aqueous buffer (pH 8.0) for 90 min at 40 degrees C in 33% yield by HPLC. GSH also reacted with 4,9-anhydroTTX to form 4-S-glutathionylTTX (4-GSTTX) in 39% yield under the same conditions, whereas TTX scarcely reacted with Cys and GSH. These reactions were strictly pH-dependent, giving the highest yield at pH 8.0. 4-GSTTX was converted to 4,9-anhydroTTX in 0.8 M sodium phosphate buffer (pH 8.0) at 25 degrees C. Its half-life was approximately 4 h. The minimum lethal doses of 4-CysTTX and 4-GSTTX to mice by ip injection were more than 140 and 860 microg/kg (n = 2), which were 14- and 86-fold larger than the LD(50) of TTX, respectively. 4-GSTTX was hydrolyzed to 4-CysTTX by gamma-glutamyltranspeptidase (Sigma, catalog no. G9270), which was supposed to contain cysteinylglycine dipeptidase. We also examined the effect of Cys or GSH coinjection (ip) with TTX to mice for detoxification of TTX and concluded that these coinjections did not reduce the toxicity of TTX.

Accumulation of PN1 and PN3 sodium channels in painful human neuroma-evidence from immunocytochemistry.

The axolemmal distribution and density of voltage-gated sodium channels largely determines the electrical excitability of sprouting neurites. Recent evidence suggests that accumulation of sodium channels at injured axonal tips may be responsible for ectopic axonal hyperexcitability and the resulting abnormal sensory phenomena of pain and paresthesias. For future improvement in pain management it is necessary to identify structurally significant generators of autorhythmicity. A first step in this regard will be to determine the predominant types of sodium channels in injured axons. The opportunity to test human specimens from painful and non-painful neuroma is of great value. We employed immunocytochemical methods to investigate if two types of highly specific voltage-gated sodium channel subtypes could be detected in sections of human neuroma. Both subtypes of sodium channels PN1 and PN3 accumulated abnormally in human neuromas. The immunoreactive pattern was more pronounced in painful neuromas. This is in contrast to previous reports that focused either on PN1 or PN3 as main generators of hyperexcitability induced pain. Both, PN1 and PN3 seem to be involved in hyperexcitability induced pain. It can be expected that a variety of other highly specific voltage gated sodium channel subtypes will be detected in regenerating peripheral nerve in the near future, which contribute to the development of neuropathic pain states. Thus, in order to therapeutically control hyperexcitability induced neuropathic pain, it might be worthwhile to develop pharmaceuticals that can selectively block different sodium channel subtypes and subunits.A review of the role of sodium channels in neuropathic pain is implemented in the discussion.