Marked difference in saxitoxin and tetrodotoxin affinity for the human nociceptive voltage-gated sodium channel (Nav1.7) [corrected].
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ABSTRACT:
SUBMITTER: Walker JR
PROVIDER: S-EPMC3497785 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Tetrodotoxin (TTX) is used to distinguish between two classes of voltage-gated sodium channel (VGSC)--TTX sensitive (TTXS) and TTX resistant (TTXR). The resistance of TTXR VGSCs is thought to result from a low binding affinity of TTX, although at high TTX concentrations channel block does occur. Here, we show that, at concentrations below those which produce block, TTX can bind to TTXR VGSCs.Whole-cell voltage clamp recordings were made from dissociated rat dorsal root ganglion neurones that expressed both TTXS and TTXR sodium currents. Voltage-gated calcium currents were blocked by 10 microM extracellular lanthanum chloride. TTXS, but not TTXR, current was suppressed by using a holding potential of -50 mV, and the effect of TTX on the isolated TTXR current was explored.Extracellular application of 0.5 microM TTX produced a 40% increase in TTXR current amplitude, a negative shift in the voltage-dependence of current activation (approximately -8 mV) and inactivation (approximately -10 mV) and increased rates of current activation and inactivation. The effect of TTX on current amplitude was dose-dependent (EC50 = 364 nM). Removal of lanthanum prevented the effect of TTX on TTXR current amplitude, whereas reducing extracellular calcium did not.The findings are consistent with an interpretation that TTX relieves a tonic block of the TTXR VGSC by lanthanum. We conclude that TTX binds to the TTXR VGSC at low concentrations, without blocking it. This appears to be the first demonstration of a clear distinction between binding affinity and blocking potency of a channel-blocking agent.
| S-EPMC2527855 | biostudies-other
Project description:Background: Endometrial cancer is the most common gynecologic malignancy in women in the developed countries. Despite recent progress in functional characterization of voltage-gated sodium channel (Nav) in multiple cancers, very little was known about the expression of Nav in human endometrial cancer. The present study sought to determine the role of Nav and molecular nature of this channel in the endometrial cancer. Methods: PCR approach was introduced to determine expression level of Nav subunits in endometrial cancer specimens. Pharmacological agents were used to investigate Nav function in endometrial cancer cells. Flow cytometry were used to test cancer apoptosis, and invasion assays were applied to test tumor metastasis. Results: Transcriptional levels of the all Nav ? and ? subunits were determined by real time-PCR in endometrial cancer with pair tissues of carcinoma and adjacent nonneoplastic tissue, Nav1.7 was the most highly expressed Nav subtype in endometrial cancer tissues. Nav1.7 level was closely associated with tumor size, local lymph node metastasis, and 5-year and 10-year survival ratio. Inhibition of this channel by Nav1.7 blocker PF-05089771, promoted cancer apoptosis and attenuated cancer cell invasion. Conclusion: These results establish a relationship between voltage-gated sodium channel protein and endometrial cancer, and suggest that Nav1.7 is a potential prognostic biomarker and could serve as a novel therapeutic target for endometrial cancer.
| S-EPMC6775510 | biostudies-literature
Project description:BackgroundVoltage-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.MethodsThe 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.ResultsFrom a holding potential of -70 mV, isoflurane (0.53 +/- 0.06 mM, 1.8 minimum alveolar concentration at 24 degrees 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 it 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 minimum alveolar concentration. 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 versus 0.78 +/- 0.01 for control.ConclusionIsoflurane 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.
| S-EPMC2756082 | biostudies-literature
Project description:Here we report the pharmacologic blockade of voltage-gated sodium ion channels (NaVs) by a synthetic saxitoxin derivative affixed to a photocleavable protecting group. We demonstrate that a functionalized saxitoxin (STX-eac) enables exquisite spatiotemporal control of NaVs to interrupt action potentials in dissociated neurons and nerve fiber bundles. The photo-uncaged inhibitor (STX-ea) is a nanomolar potent, reversible binder of NaVs. We use STX-eac to reveal differential susceptibility of myelinated and unmyelinated axons in the corpus callosum to NaV-dependent alterations in action potential propagation, with unmyelinated axons preferentially showing reduced action potential fidelity under conditions of partial NaV block. These results validate STX-eac as a high precision tool for robust photocontrol of neuronal excitability and action potential generation.
| S-EPMC8263607 | biostudies-literature
Project description:Veratridine is a lipid-soluble neurotoxin derived from plants in the family Liliaceae. It has been broadly investigated for its action as a sodium channel agonist. However, the effects of veratridine on subtypes of sodium channels, especially Nav1.7, remain to be studied. Here, we investigated the effects of veratridine on human Nav1.7 ectopically expressed in HEK293A cells and recorded Nav1.7 currents from the cells using whole-cell patch clamp technique. We found that veratridine exerted a dose-dependent inhibitory effect on the peak current of Nav1.7, with the half-maximal inhibition concentration (IC50) of 18.39?µM. Meanwhile, veratridine also elicited tail current (linearly) and sustained current [half-maximal concentration (EC50): 9.53?µM], also in a dose-dependent manner. Veratridine (75?µM) shifted the half-maximal activation voltage of the Nav1.7 activation curve in the hyperpolarized direction, from -21.64?±?0.75?mV to -28.14?±?0.66?mV, and shifted the half-inactivation voltage of the steady-state inactivation curve from -59.39?±?0.39?mV to -73.78?±?0.5?mV. An increased frequency of stimulation decreased the peak and tail currents of Nav1.7 for each pulse along with pulse number, and increased the accumulated tail current at the end of train stimulation. These findings reveal the different modulatory effects of veratridine on the Nav1.7 peak current and tail current.
| S-EPMC6289357 | biostudies-literature
Project description:Mutations in voltage-gated sodium channels (Navs) can cause alterations in pain sensation, such as chronic pain diseases like inherited erythromelalgia. The mutation causing inherited erythromelalgia, Nav1.7 p.I848T, is known to induce a hyperpolarized shift in the voltage dependence of activation in Nav1.7. So far, however, the mechanism to explain this increase in voltage sensitivity remains unknown. In the present study, we show that phosphorylation of the newly introduced Thr residue explains the functional change. We expressed wildtype human Nav1.7, the I848T mutant, or other mutations in HEK293T cells and performed whole-cell patch-clamp electrophysiology. As the insertion of a Thr residue potentially creates a novel phosphorylation site for Ser/Thr kinases and because Nav1.7 had been shown in Xenopus oocytes to be affected by protein kinases C and A, we used different nonselective and selective kinase inhibitors and activators to test the effect of phosphorylation on Nav1.7 in a human system. We identify protein kinase C, but not protein kinase A, to be responsible for the phosphorylation of T848 and thereby for the shift in voltage sensitivity. Introducing a negatively charged amino acid instead of the putative phosphorylation site mimics the effect on voltage gating to a lesser extent. 3D modeling using the published cryo-EM structure of human Nav1.7 showed that introduction of this negatively charged site seems to alter the interaction of this residue with the surrounding amino acids and thus to influence channel function. These results could provide new opportunities for the development of novel treatment options for patients with chronic pain.
| S-EPMC7948457 | biostudies-literature
Project description:IntroductionChronic pain is a massive clinical problem. We discuss the potential of subtype selective sodium channel blockers that may provide analgesia with limited side effects.Areas coveredSodium channel subtypes have been linked to human pain syndromes through genetic studies. Gain of function mutations in Nav1.7, 1.8 and 1.9 can cause pain, whilst loss of function Nav1.7 mutations lead to loss of pain in otherwise normal people. Intriguingly, both human and mouse Nav1.7 null mutants have increased opioid drive, because naloxone, an opioid antagonist, can reverse the analgesia associated with the loss of Nav1.7 expression.Expert opinionWe believe there is a great future for sodium channel antagonists, particularly Nav1.7 antagonists in treating most pain syndromes. This review deals with recent attempts to develop specific sodium channel blockers, the mechanisms that underpin the Nav1.7 null pain-free phenotype and new routes to analgesia using, for example, gene therapy or combination therapy with subtype specific sodium channel blockers and opioids. The use of selective Nav1.7 antagonists together with either enkephalinase inhibitors or low dose opioids has the potential for side effect-free analgesia, as well as an important opioid sparing function that may be clinically very significant.
| S-EPMC4950419 | biostudies-literature
Project description:ND7/23 cells are gaining traction as a host model to express peripheral sodium channels such as NaV1.8 and NaV1.9 that have been difficult to express in widely utilized heterologous cells, like CHO and HEK293. Use of ND7/23 as a model cell to characterize the properties of sodium channels requires clear understanding of the endogenous ion channels. To define the nature of the background sodium currents in ND7/23 cells, we aimed to comprehensively profile the voltage-gated sodium channel subunits by endpoint and quantitative reverse transcription-PCR and by whole-cell patch clamp electrophysiology. We found that untransfected ND7/23 cells express endogenous peak sodium currents that average -2.12nA (n = 15) and with kinetics typical of fast sodium currents having activation and inactivation completed within few milliseconds. Furthermore, sodium currents were reduced to virtually nil upon exposure to 100nM tetrodotoxin, indicating that ND7/23 cells have essentially null background for tetrodotoxin-resistant (TTX-R) currents. qRT-PCR profiling indicated a major expression of TTX-sensitive (TTX-S) NaV1.6 and NaV1.7 at similar levels and very low expression of TTX-R NaV1.9 transcripts. There was no expression of TTX-R NaV1.8 in ND7/23 cells. There was low expression of NaV1.1, NaV1.2, NaV1.3 and no expression of cardiac or skeletal muscle sodium channels. As for the sodium channel auxiliary subunits, ?1 and ?3 subunits were expressed, but not the ?2 and ?4 subunits that covalently associate with the ?-subunits. In addition, our results also showed that only the mouse forms of NaV1.6, NaV1.7 and NaV1.9 sodium channels were expressed in ND7/23 cells that was originally generated as a hybridoma of rat embryonic DRG and mouse neuroblastoma cell-line. By molecular profiling of auxiliary ?- and principal ?-subunits of the voltage gated sodium channel complex, our results define the background sodium channels expressed in ND7/23 cells, and confirm their utility for detailed functional studies of emerging pain channelopathies ascribed to mutations of the TTX-R sodium channels of sensory neurons.
| S-EPMC6697327 | biostudies-literature
Project description:Tetrodotoxin (TTX) is a potent neurotoxin associated with human poisonings through the consumption of pufferfish. More recently, TTX has been identified in bivalve molluscs from diverse geographical environments, including Europe, and is therefore recognised as an emerging threat to food safety. A recent scientific opinion of the EFSA Panel on Contaminants in the Food Chain recognised the need for further data on the acute oral toxicity of TTX and suggested that, since saxitoxin (STX) and TTX had similar modes of action, it was possible that their toxicities were additive so could perhaps be combined to yield one health-based guideline value. The present study determined the toxicity of TTX by various routes of administration. The testing of three different mixtures of STX and TTX and comparing the experimentally determined values to those predicted on the basis of additive toxicity demonstrated that the toxicities of STX and TTX are additive. This illustrates that it is appropriate to treat TTX as a member of the paralytic shellfish group of toxins. Since the toxicity of TTX was found to be the same as STX by feeding, a molar toxicity equivalence factor of 1.0 for TTX can be applied.
| S-EPMC6266834 | biostudies-literature
Project description:The antidiabetic drug metformin has been shown to reduce pain hypersensitivity in preclinical models of chronic pain and in neuropathic pain in humans. Multiple intracellular pathways have been described as metformin targets. Among them, metformin is an activator of the adenosine 5'-monophosphate protein kinase that can in turn modulate the activity of the E3 ubiquitin ligase NEDD4-2 and thus post-translational expression of voltage-gated sodium channels (NaVs). In this study, we found that the bulk of the effect of metformin on Na1.7 is dependent on NEDD4-2. In HEK cells, the expression of NaV1.7 at the membrane fraction, obtained by a biotinylation approach, is only reduced by metformin when cotransfected with NEDD4-2. Similarly, in voltage-clamp recordings, metformin significantly reduced NaV1.7 current density when cotransfected with NEDD4-2. In mouse dorsal root ganglion (DRG) neurons, without changing the biophysical properties of NaV1.7, metformin significantly decreased NaV1.7 current densities, but not in Nedd4L knock-out mice (SNS-Nedd4L-/-). In addition, metformin induced a significant reduction in NEDD4-2 phosphorylation at the serine-328 residue in DRG neurons, an inhibitory phosphorylation site of NEDD4-2. In current-clamp recordings, metformin reduced the number of action potentials elicited by DRG neurons from Nedd4Lfl/fl , with a partial decrease also present in SNS-Nedd4L-/- mice, suggesting that metformin can also change neuronal excitability in an NEDD4-2-independent manner. We suggest that NEDD4-2 is a critical player for the effect of metformin on the excitability of nociceptive neurons; this action may contribute to the relief of neuropathic pain.
| S-EPMC8906783 | biostudies-literature