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1.  Post-translational modifications of voltage-gated sodium channels in chronic pain syndromes 
In the peripheral sensory nervous system the neuronal expression of voltage-gated sodium channels (Navs) is very important for the transmission of nociceptive information since they give rise to the upstroke of the action potential (AP). Navs are composed of nine different isoforms with distinct biophysical properties. Studying the mutations associated with the increase or absence of pain sensitivity in humans, as well as other expression studies, have highlighted Nav1.7, Nav1.8, and Nav1.9 as being the most important contributors to the control of nociceptive neuronal electrogenesis. Modulating their expression and/or function can impact the shape of the AP and consequently modify nociceptive transmission, a process that is observed in persistent pain conditions. Post-translational modification (PTM) of Navs is a well-known process that modifies their expression and function. In chronic pain syndromes, the release of inflammatory molecules into the direct environment of dorsal root ganglia (DRG) sensory neurons leads to an abnormal activation of enzymes that induce Navs PTM. The addition of small molecules, i.e., peptides, phosphoryl groups, ubiquitin moieties and/or carbohydrates, can modify the function of Navs in two different ways: via direct physical interference with Nav gating, or via the control of Nav trafficking. Both mechanisms have a profound impact on neuronal excitability. In this review we will discuss the role of Protein Kinase A, B, and C, Mitogen Activated Protein Kinases and Ca++/Calmodulin-dependent Kinase II in peripheral chronic pain syndromes. We will also discuss more recent findings that the ubiquitination of Nav1.7 by Nedd4-2 and the effect of methylglyoxal on Nav1.8 are also implicated in the development of experimental neuropathic pain. We will address the potential roles of other PTMs in chronic pain and highlight the need for further investigation of PTMs of Navs in order to develop new pharmacological tools to alleviate pain.
PMCID: PMC4633509  PMID: 26594175
voltage-gated sodium channels; post-translational modification; chronic pain; hyperexcitability; nociceptive neurons
2.  Functional up-regulation of Nav1.8 sodium channel in Aβ afferent fibers subjected to chronic peripheral inflammation 
Functional alterations in the properties of Aβ afferent fibers may account for the increased pain sensitivity observed under peripheral chronic inflammation. Among the voltage-gated sodium channels involved in the pathophysiology of pain, Nav1.8 has been shown to participate in the peripheral sensitization of nociceptors. However, to date, there is no evidence for a role of Nav1.8 in controlling Aβ-fiber excitability following persistent inflammation.
Distribution and expression of Nav1.8 in dorsal root ganglia and sciatic nerves were qualitatively or quantitatively assessed by immunohistochemical staining and by real time-polymerase chain reaction at different time points following complete Freund’s adjuvant (CFA) administration. Using a whole-cell patch-clamp configuration, we further determined both total INa and TTX-R Nav1.8 currents in large-soma dorsal root ganglia (DRG) neurons isolated from sham or CFA-treated rats. Finally, we analyzed the effects of ambroxol, a Nav1.8-preferring blocker on the electrophysiological properties of Nav1.8 currents and on the mechanical sensitivity and inflammation of the hind paw in CFA-treated rats.
Our findings revealed that Nav1.8 is up-regulated in NF200-positive large sensory neurons and is subsequently anterogradely transported from the DRG cell bodies along the axons toward the periphery after CFA-induced inflammation. We also demonstrated that both total INa and Nav1.8 peak current densities are enhanced in inflamed large myelinated Aβ-fiber neurons. Persistent inflammation leading to nociception also induced time-dependent changes in Aβ-fiber neuron excitability by shifting the voltage-dependent activation of Nav1.8 in the hyperpolarizing direction, thus decreasing the current threshold for triggering action potentials. Finally, we found that ambroxol significantly reduces the potentiation of Nav1.8 currents in Aβ-fiber neurons observed following intraplantar CFA injection and concomitantly blocks CFA-induced mechanical allodynia, suggesting that Nav1.8 regulation in Aβ-fibers contributes to inflammatory pain.
Collectively, these findings support a key role for Nav1.8 in controlling the excitability of Aβ-fibers and its potential contribution to the development of mechanical allodynia under persistent inflammation.
PMCID: PMC4007624  PMID: 24606981
Aβ-fibers; Allodynia; Complete Freund’s adjuvant; Electrophysiology; Sodium channel blocker
3.  Functional properties and toxin pharmacology of a dorsal root ganglion sodium channel viewed through its voltage sensors 
The voltage-activated sodium (Nav) channel Nav1.9 is expressed in dorsal root ganglion (DRG) neurons where it is believed to play an important role in nociception. Progress in revealing the functional properties and pharmacological sensitivities of this non-canonical Nav channel has been slow because attempts to express this channel in a heterologous expression system have been unsuccessful. Here, we use a protein engineering approach to dissect the contributions of the four Nav1.9 voltage sensors to channel function and pharmacology. We define individual S3b–S4 paddle motifs within each voltage sensor, and show that they can sense changes in membrane voltage and drive voltage sensor activation when transplanted into voltage-activated potassium channels. We also find that the paddle motifs in Nav1.9 are targeted by animal toxins, and that these toxins alter Nav1.9-mediated currents in DRG neurons. Our results demonstrate that slowly activating and inactivating Nav1.9 channels have functional and pharmacological properties in common with canonical Nav channels, but also show distinctive pharmacological sensitivities that can potentially be exploited for developing novel treatments for pain.
PMCID: PMC3135324  PMID: 21670206
4.  Phyla- and Subtype-Selectivity of CgNa, a Na+ Channel Toxin from the Venom of the Giant Caribbean Sea Anemone Condylactis Gigantea 
Because of their prominent role in electro-excitability, voltage-gated sodium (NaV) channels have become the foremost important target of animal toxins. These toxins have developed the ability to discriminate between closely related NaV subtypes, making them powerful tools to study NaV channel function and structure. CgNa is a 47-amino acid residue type I toxin isolated from the venom of the Giant Caribbean Sea Anemone Condylactis gigantea. Previous studies showed that this toxin slows the fast inactivation of tetrodotoxin-sensitive NaV currents in rat dorsal root ganglion neurons. To illuminate the underlying NaV subtype-selectivity pattern, we have assayed the effects of CgNa on a broad range of mammalian isoforms (NaV1.2–NaV1.8) expressed in Xenopus oocytes. This study demonstrates that CgNa selectively slows the fast inactivation of rNaV1.3/β1, mNaV1.6/β1 and, to a lesser extent, hNaV1.5/β1, while the other mammalian isoforms remain unaffected. Importantly, CgNa was also examined on the insect sodium channel DmNaV1/tipE, revealing a clear phyla-selectivity in the efficacious actions of the toxin. CgNa strongly inhibits the inactivation of the insect NaV channel, resulting in a dramatic increase in peak current amplitude and complete removal of fast and steady-state inactivation. Together with the previously determined solution structure, the subtype-selective effects revealed in this study make of CgNa an interesting pharmacological probe to investigate the functional role of specific NaV channel subtypes. Moreover, further structural studies could provide important information on the molecular mechanism of NaV channel inactivation.
PMCID: PMC3153007  PMID: 21833172
sea anemone; toxin; inactivation; sodium channel; subtype; selectivity
5.  Correlation of the electrophysiological profiles and sodium channel transcripts of individual rat dorsal root ganglia neurons 
Voltage gated sodium channels (Nav channels) play an important role in nociceptive transmission. They are intimately tied to the genesis and transmission of neuronal firing. Five different isoforms (Nav1.3, Nav1.6, Nav1.7, Nav1.8, and Nav1.9) have been linked to nociceptive responses. A change in the biophysical properties of these channels or in their expression levels occurs in different pathological pain states. However, the precise involvement of the isoforms in the genesis and transmission of nociceptive responses is unknown. The aim of the present study was to investigate the synergy between the different populations of Nav channels that give individual neurons a unique electrophysical profile. We used the patch-clamp technique in the whole-cell configuration to record Nav currents and action potentials from acutely dissociated small diameter DRG neurons (<30 μm) from adult rats. We also performed single cell qPCR on the same neurons. Our results revealed that there is a strong correlation between Nav currents and mRNA transcripts in individual neurons. A cluster analysis showed that subgroups formed by Nav channel transcripts by mRNA quantification have different biophysical properties. In addition, the firing frequency of the neurons was not affected by the relative populations of Nav channel. The synergy between populations of Nav channel in individual small diameter DRG neurons gives each neuron a unique electrophysiological profile. The Nav channel remodeling that occurs in different pathological pain states may be responsible for the sensitization of the neurons.
PMCID: PMC4168718  PMID: 25285069
voltage-gated sodium channel; neuronal excitability; pain; biophysical properties; dorsal root ganglia neurons
6.  Effects of ranolazine on wild-type and mutant hNav1.7 channels and on DRG neuron excitability 
Molecular Pain  2010;6:35.
A direct role of sodium channels in pain has recently been confirmed by establishing a monogenic link between SCN9A, the gene which encodes sodium channel Nav1.7, and pain disorders in humans, with gain-of-function mutations causing severe pain syndromes, and loss-of-function mutations causing congenital indifference to pain. Expression of sodium channel Nav1.8 in DRG neurons has also been shown to be essential for the manifestation of mutant Nav1.7-induced neuronal hyperexcitability. These findings have confirmed key roles of Nav1.7 and Nav1.8 in pain and identify these channels as novel targets for pain therapeutic development. Ranolazine preferentially blocks cardiac late sodium currents at concentrations that do not significantly reduce peak sodium current. Ranolazine also blocks wild-type Nav1.7 and Nav1.8 channels in a use-dependent manner. However, ranolazine's effects on gain-of-function mutations of Nav1.7 and on DRG neuron excitability have not been investigated. We used voltage- and current-clamp recordings to evaluate the hypothesis that ranolazine may be effective in regulating Nav1.7-induced DRG neuron hyperexcitability.
We show that ranolazine produces comparable block of peak and ramp currents of wild-type Nav1.7 and mutant Nav1.7 channels linked to Inherited Erythromelalgia and Paroxysmal Extreme Pain Disorder. We also show that ranolazine, at a clinically-relevant concentration, blocks high-frequency firing of DRG neurons expressing wild-type but not mutant channels.
Our data suggest that ranalozine can attenuate hyperexcitability of DRG neurons over-expressing wild-type Nav1.7 channels, as occurs in acquired neuropathic and inflammatory pain, and thus merits further study as an alternative to existing non-selective sodium channel blockers.
PMCID: PMC2898769  PMID: 20529343
7.  Scorpion toxin BmK I directly activates Nav1.8 in primary sensory neurons to induce neuronal hyperexcitability in rats 
Protein & Cell  2015;6(6):443-452.
Voltage-gated sodium channels (VGSCs) in primary sensory neurons play a key role in transmitting pain signals to the central nervous system. BmK I, a site-3 sodium channel-specific toxin from scorpion Buthus martensi Karsch, induces pain behaviors in rats. However, the subtypes of VGSCs targeted by BmK I were not entirely clear. We therefore investigated the effects of BmK I on the current amplitude, gating and kinetic properties of Nav1.8, which is associated with neuronal hyperexcitability in DRG neurons. It was found that BmK I dose-dependently increased Nav1.8 current in small-sized (<25 μm) acutely dissociated DRG neurons, which correlated with its inhibition on both fast and slow inactivation. Moreover, voltage-dependent activation and steady-state inactivation curves of Nav1.8 were shifted in a hyperpolarized direction. Thus, BmK I reduced the threshold of neuronal excitability and increased action potential firing in DRG neurons. In conclusion, our data clearly demonstrated that BmK I modulated Nav1.8 remarkably, suggesting BmK I as a valuable probe for studying Nav1.8. And Nav1.8 is an important target related to BmK I-evoked pain.
PMCID: PMC4444811  PMID: 25903152
voltage-gated sodium channel; Nav1.8; primary sensory neurons; BmK I
8.  Scorpion toxin BmK I directly activates Nav1.8 in primary sensory neurons to induce neuronal hyperexcitability in rats 
Protein & Cell  2015;6(6):443-452.
Voltage-gated sodium channels (VGSCs) in primary sensory neurons play a key role in transmitting pain signals to the central nervous system. BmK I, a site-3 sodium channel-specific toxin from scorpion Buthus martensi Karsch, induces pain behaviors in rats. However, the subtypes of VGSCs targeted by BmK I were not entirely clear. We therefore investigated the effects of BmK I on the current amplitude, gating and kinetic properties of Nav1.8, which is associated with neuronal hyperexcitability in DRG neurons. It was found that BmK I dose-dependently increased Nav1.8 current in small-sized (<25 μm) acutely dissociated DRG neurons, which correlated with its inhibition on both fast and slow inactivation. Moreover, voltage-dependent activation and steady-state inactivation curves of Nav1.8 were shifted in a hyperpolarized direction. Thus, BmK I reduced the threshold of neuronal excitability and increased action potential firing in DRG neurons. In conclusion, our data clearly demonstrated that BmK I modulated Nav1.8 remarkably, suggesting BmK I as a valuable probe for studying Nav1.8. And Nav1.8 is an important target related to BmK I-evoked pain.
PMCID: PMC4444811  PMID: 25903152
voltage-gated sodium channel; Nav1.8; primary sensory neurons; BmK I
9.  The hitchhiker’s guide to the voltage-gated sodium channel galaxy 
Eukaryotic voltage-gated sodium (Nav) channels contribute to the rising phase of action potentials and served as an early muse for biophysicists laying the foundation for our current understanding of electrical signaling. Given their central role in electrical excitability, it is not surprising that (a) inherited mutations in genes encoding for Nav channels and their accessory subunits have been linked to excitability disorders in brain, muscle, and heart; and (b) Nav channels are targeted by various drugs and naturally occurring toxins. Although the overall architecture and behavior of these channels are likely to be similar to the more well-studied voltage-gated potassium channels, eukaryotic Nav channels lack structural and functional symmetry, a notable difference that has implications for gating and selectivity. Activation of voltage-sensing modules of the first three domains in Nav channels is sufficient to open the channel pore, whereas movement of the domain IV voltage sensor is correlated with inactivation. Also, structure–function studies of eukaryotic Nav channels show that a set of amino acids in the selectivity filter, referred to as DEKA locus, is essential for Na+ selectivity. Structures of prokaryotic Nav channels have also shed new light on mechanisms of drug block. These structures exhibit lateral fenestrations that are large enough to allow drugs or lipophilic molecules to gain access into the inner vestibule, suggesting that this might be the passage for drug entry into a closed channel. In this Review, we will synthesize our current understanding of Nav channel gating mechanisms, ion selectivity and permeation, and modulation by therapeutics and toxins in light of the new structures of the prokaryotic Nav channels that, for the time being, serve as structural models of their eukaryotic counterparts.
PMCID: PMC4692491  PMID: 26712848
10.  Single-cell analysis of sodium channel expression in dorsal root ganglion neurons 
Sensory neurons of the dorsal root ganglia (DRG) express multiple voltage-gated sodium (Na) channels that substantially differ in gating kinetics and pharmacology. Small-diameter (<25 µm) neurons isolated from the rat DRG express a combination of fast tetrodotoxin-sensitive (TTX-S) and slow TTX-resistant (TTX-R) Na currents while large-diameter neurons (>30 µm) predominately express fast TTX-S Na current. Na channel expression was further investigated using single-cell RT-PCR to measure the transcripts present in individually harvested DRG neurons. Consistent with cellular electrophysiology, the small neurons expressed transcripts encoding for both TTX-S (Nav1.1, Nav1.2, Nav1.6, Nav1.7) and TTX-R (Nav1.8, Nav1.9) Na channels. Nav1.7, Nav1.8 and Nav1.9 were the predominant Na channels expressed in the small neurons. The large neurons highly expressed TTX-S isoforms (Nav1.1, Nav1.6, Nav1.7) while TTX-R channels were present at comparatively low levels. A unique subpopulation of the large neurons was identified that expressed TTX-R Na current and high levels of Nav1.8 transcript. DRG neurons also displayed substantial differences in the expression of neurofilaments (NF200, peripherin) and Necl-1, a neuronal adhesion molecule involved in myelination. The preferential expression of NF200 and Necl-1 suggests that large-diameter neurons give rise to thick myelinated axons. Small-diameter neurons expressed peripherin, but reduced levels of NF200 and Necl-1, a pattern more consistent with thin unmyelinated axons. Single-cell analysis of Na channel transcripts indicates that TTX-S and TTX-R Na channels are differentially expressed in large myelinated (Nav1.1, Nav1.6, Nav1.7) and small unmyelinated (Nav1.7, Nav1.8, Nav1.9) sensory neurons.
PMCID: PMC3005531  PMID: 20816971
Sodium channel; dorsal root ganglia; single-cell RT-PCR; Necl-1; NF200; peripherin
11.  Systematic Study of Binding of µ-Conotoxins to the Sodium Channel NaV1.4 
Toxins  2014;6(12):3454-3470.
Voltage-gated sodium channels (NaV) are fundamental components of the nervous system. Their dysfunction is implicated in a number of neurological disorders, such as chronic pain, making them potential targets for the treatment of such disorders. The prominence of the NaV channels in the nervous system has been exploited by venomous animals for preying purposes, which have developed toxins that can block the NaV channels, thereby disabling their function. Because of their potency, such toxins could provide drug leads for the treatment of neurological disorders associated with NaV channels. However, most toxins lack selectivity for a given target NaV channel, and improving their selectivity profile among the NaV1 isoforms is essential for their development as drug leads. Computational methods will be very useful in the solution of such design problems, provided accurate models of the protein-ligand complex can be constructed. Using docking and molecular dynamics simulations, we have recently constructed a model for the NaV1.4-µ-conotoxin-GIIIA complex and validated it with the ample mutational data available for this complex. Here, we use the validated NaV1.4 model in a systematic study of binding other µ-conotoxins (PIIIA, KIIIA and BuIIIB) to NaV1.4. The binding mode obtained for each complex is shown to be consistent with the available mutation data and binding constants. We compare the binding modes of PIIIA, KIIIA and BuIIIB to that of GIIIA and point out the similarities and differences among them. The detailed information about NaV1.4-µ-conotoxin interactions provided here will be useful in the design of new NaV channel blocking peptides.
PMCID: PMC4280544  PMID: 25529306
sodium channels; conotoxins; homology modeling; docking; molecular dynamics; potential of mean force; binding free energy
12.  Localization of Sodium Channel Subtypes in Mouse Ventricular Myocytes Using Quantitative Immunocytochemistry 
Journal of molecular and cellular cardiology  2013;64:10.1016/j.yjmcc.2013.08.004.
Voltage-gated sodium channels are responsible for the rising phase of the action potential in cardiac muscle. Previously, both TTX-sensitive neuronal sodium channels (NaV1.1, NaV1.2, NaV1.3, NaV1.4 and NaV1.6) and the TTX-resistant cardiac sodium channel (NaV1.5) have been detected in cardiac myocytes, but relative levels of protein expression of the isoforms were not determined. Using a quantitative approach, we analyzed z-series of confocal microscopy images from individual mouse myocytes stained with either anti-NaV1.1, anti-NaV1.2, anti-NaV1.3, anti-NaV1.4, anti-NaV1.5, or anti-NaV1.6 antibodies and calculated the relative intensity of staining for these sodium channel isoforms. Our results indicate that the TTX-sensitive channels represented approximately 23% of the total channels, whereas the TTX-resistant NaV1.5 channel represented 77% of the total channel staining in mouse ventricular myocytes. These ratios are consistent with previous electrophysiological studies in mouse ventricular myocytes. NaV1.5 was located at the cell surface, with high density at the intercalated disc, but was absent from the transverse (t)-tubular system, suggesting that these channels support surface conduction and inter-myocyte transmission. Low-level cell surface staining of NaV1.4 and NaV1.6 channels suggest a minor role in surface excitation and conduction. Conversely, NaV1.1 and NaV1.3 channels are localized to the t-tubules and are likely to support t-tubular transmission of the action potential to the myocyte interior. This quantitative immunocytochemical approach for assessing sodium channel density and localization provides a more precise view of the relative importance and possible roles of these individual sodium channel protein isoforms in mouse ventricular myocytes and may be applicable to other species and cardiac tissue types.
PMCID: PMC3851329  PMID: 23982034
13.  Pain without Nociceptors? Nav1.7-Independent Pain Mechanisms 
Cell Reports  2014;6(2):301-312.
Nav1.7, a peripheral neuron voltage-gated sodium channel, is essential for pain and olfaction in mice and humans. We examined the role of Nav1.7 as well as Nav1.3, Nav1.8, and Nav1.9 in different mouse models of chronic pain. Constriction-injury-dependent neuropathic pain is abolished when Nav1.7 is deleted in sensory neurons, unlike nerve-transection-related pain, which requires the deletion of Nav1.7 in sensory and sympathetic neurons for pain relief. Sympathetic sprouting that develops in parallel with nerve-transection pain depends on the presence of Nav1.7 in sympathetic neurons. Mechanical and cold allodynia required distinct sets of neurons and different repertoires of sodium channels depending on the nerve injury model. Surprisingly, pain induced by the chemotherapeutic agent oxaliplatin and cancer-induced bone pain do not require the presence of Nav1.7 sodium channels or Nav1.8-positive nociceptors. Thus, similar pain phenotypes arise through distinct cellular and molecular mechanisms. Therefore, rational analgesic drug therapy requires patient stratification in terms of mechanisms and not just phenotype.
Graphical Abstract
•Phenotypically identical pain models have different underlying molecular mechanisms•Nav1.7 expression is required for sympathetic sprouting after neuronal damage•Oxaliplatin and cancer-induced bone pain are both Nav1.7-independent•Deleting Nav1.7 in adult mice reverses nerve damage-induced neuropathic pain
Wood and colleagues describe two pain syndromes that occur in the absence of Nav1.7, a sodium channel considered to be essential for pain perception and olfaction in humans. They provide evidence that pain phenotypes such as cold and mechanical allodynia can arise through distinct cell and molecular mechanisms after nerve injury in mouse peripheral sensory neurons. The existence of redundant mechanistically distinct peripheral pain mechanisms may help to explain recent difficulties with the development of new analgesic drugs.
PMCID: PMC3969273  PMID: 24440715
14.  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
15.  Molecular Surface of JZTX-V (β-Theraphotoxin-Cj2a) Interacting with Voltage-Gated Sodium Channel Subtype NaV1.4 
Toxins  2014;6(7):2177-2193.
Voltage-gated sodium channels (VGSCs; NaV1.1–NaV1.9) have been proven to be critical in controlling the function of excitable cells, and human genetic evidence shows that aberrant function of these channels causes channelopathies, including epilepsy, arrhythmia, paralytic myotonia, and pain. The effects of peptide toxins, especially those isolated from spider venom, have shed light on the structure–function relationship of these channels. However, most of these toxins have not been analyzed in detail. In particular, the bioactive faces of these toxins have not been determined. Jingzhaotoxin (JZTX)-V (also known as β-theraphotoxin-Cj2a) is a 29-amino acid peptide toxin isolated from the venom of the spider Chilobrachys jingzhao. JZTX-V adopts an inhibitory cysteine knot (ICK) motif and has an inhibitory effect on voltage-gated sodium and potassium channels. Previous experiments have shown that JZTX-V has an inhibitory effect on TTX-S and TTX-R sodium currents on rat DRG cells with IC50 values of 27.6 and 30.2 nM, respectively, and is able to shift the activation and inactivation curves to the depolarizing and the hyperpolarizing direction, respectively. Here, we show that JZTX-V has a much stronger inhibitory effect on NaV1.4, the isoform of voltage-gated sodium channels predominantly expressed in skeletal muscle cells, with an IC50 value of 5.12 nM, compared with IC50 values of 61.7–2700 nM for other heterologously expressed NaV1 subtypes. Furthermore, we investigated the bioactive surface of JZTX-V by alanine-scanning the effect of toxin on NaV1.4 and demonstrate that the bioactive face of JZTX-V is composed of three hydrophobic (W5, M6, and W7) and two cationic (R20 and K22) residues. Our results establish that, consistent with previous assumptions, JZTX-V is a Janus-faced toxin which may be a useful tool for the further investigation of the structure and function of sodium channels.
PMCID: PMC4113750  PMID: 25055801
spider toxin; voltage gated sodium channels; JZTX-V; NaV1.4
16.  Characterization of Endogenous Sodium Channels in the ND7-23 Neuroblastoma Cell Line: Implications for Use as a Heterologous Ion Channel Expression System Suitable for Automated Patch Clamp Screening 
The rodent neuroblastoma cell line, ND7-23, is used to express voltage-dependent sodium (Nav) and other neuronal ion channels resistant to heterologous expression in Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells. Their advantage is that they provide endogenous factors and signaling pathways to promote ion channel peptide folding, expression, and function at the cell surface and are also amenable to automated patch clamping. However, ND7-23 cells exhibit endogenous tetrodotoxin (TTX)-sensitive Nav currents, and molecular profiling has revealed the presence of Nav1.2, Nav1.3, Nav1.6, and Nav1.7 transcripts, but no study has determined which subtypes contribute to functional channels at the cell surface. We profiled the repertoire of functional Nav channels endogenously expressed in ND7-23 cells using the QPatch automated patch clamp platform and selective toxins and small molecules. The potency and subtype selectivity of the ligands (Icagen compound 68 from patent US-20060025415-A1-20060202, 4,9 anhydro TTX, and Protoxin-II) were established in human Nav1.3, Nav1.6, and Nav1.7 channel cell lines before application of selective concentrations to ND7-23 cells. Our data confirm previous studies that >97% of macroscopic Nav current in ND7-23 cells is carried by TTX-sensitive channels (300 nM TTX) and that Nav1.7 is the predominant channel contributing to this response (65% of peak inward current), followed by Nav1.6 (∼20%) and negligible Nav1.3 currents (∼2%). In addition, our data are the first to assess the Nav1.6 potency (50% inhibitory concentration [IC50] of 33 nM) and selectivity (50-fold over Nav1.7) of 4,9 anhydro TTX in human Nav channels expressed in mammalian cells, confirming previous studies of rodent Nav channels expressed in oocytes and HEK cells.
PMCID: PMC4800267  PMID: 26991361
17.  Assessment of TTX-s and TTX-r Action Potential Conduction along Neurites of NGF and GDNF Cultured Porcine DRG Somata 
PLoS ONE  2015;10(9):e0139107.
Nine isoforms of voltage-gated sodium channels (NaV) have been characterized and in excitable tissues they are responsible for the initiation and conduction of action potentials. For primary afferent neurons residing in dorsal root ganglia (DRG), individual neurons may express multiple NaV isoforms extending the neuron’s functional capabilities. Since expression of NaV isoforms can be differentially regulated by neurotrophic factors we have examined the functional consequences of exposure to either nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF) on action potential conduction in outgrowing cultured porcine neurites of DRG neurons. Calcium signals were recorded using the exogenous intensity based calcium indicator Fluo-8®, AM. In 94 neurons, calcium signals were conducted along neurites in response to electrical stimulation of the soma. At an image acquisition rate of 25 Hz it was possible to discern calcium transients in response to individual electrical stimuli. The peak amplitude of electrically-evoked calcium signals was limited by the ability of the neuron to follow the stimulus frequency. The stimulus frequency required to evoke a half-maximal calcium response was approximately 3 Hz at room temperature. In 13 of 14 (93%) NGF-responsive neurites, TTX-r NaV isoforms alone were sufficient to support propagated signals. In contrast, calcium signals mediated by TTX-r NaVs were evident in only 4 of 11 (36%) neurites from somata cultured in GDNF. This establishes a basis for assessing action potential signaling using calcium imaging techniques in individual cultured neurites and suggests that, in the pig, afferent nociceptor classes relying on the functional properties of TTX-r NaV isoforms, such as cold-nociceptors, most probably derive from NGF-responsive DRG neurons.
PMCID: PMC4583387  PMID: 26407014
18.  Roles of Voltage-Gated Tetrodotoxin-Sensitive Sodium Channels NaV1.3 and NaV1.7 in Diabetes and Painful Diabetic Neuropathy 
Diabetes mellitus (DM) is a common chronic medical problem worldwide; one of its complications is painful peripheral neuropathy, which can substantially erode quality of life and increase the cost of management. Despite its clinical importance, the pathogenesis of painful diabetic neuropathy (PDN) is complex and incompletely understood. Voltage-gated sodium channels (VGSCs) link many physiological processes to electrical activity by controlling action potentials in all types of excitable cells. Two isoforms of VGSCs, NaV1.3 and NaV1.7, which are encoded by the sodium voltage-gated channel alpha subunit 3 and 9 (Scn3A and Scn9A) genes, respectively, have been identified in both peripheral nociceptive neurons of dorsal root ganglion (DRG) and pancreatic islet cells. Recent advances in our understanding of tetrodotoxin-sensitive (TTX-S) sodium channels NaV1.3 and NaV1.7 lead to the rational doubt about the cause–effect relation between diabetes and painful neuropathy. In this review, we summarize the roles of NaV1.3 and NaV1.7 in islet cells and DRG neurons, discuss the link between DM and painful neuropathy, and present a model, which may provide a starting point for further studies aimed at identifying the mechanisms underlying diabetes and painful neuropathy.
PMCID: PMC5037757  PMID: 27608006
diabetes mellitus; painful diabetic neuropathy; NaV1.3; NaV1.7; dorsal root ganglion neurons; pancreatic islet cells
19.  Quantitative Proteomics Reveals Protein–Protein Interactions with Fibroblast Growth Factor 12 as a Component of the Voltage-Gated Sodium Channel 1.2 (Nav1.2) Macromolecular Complex in Mammalian Brain*  
Voltage-gated sodium channels (Nav1.1–Nav1.9) are responsible for the initiation and propagation of action potentials in neurons, controlling firing patterns, synaptic transmission and plasticity of the brain circuit. Yet, it is the protein–protein interactions of the macromolecular complex that exert diverse modulatory actions on the channel, dictating its ultimate functional outcome. Despite the fundamental role of Nav channels in the brain, information on its proteome is still lacking. Here we used affinity purification from crude membrane extracts of whole brain followed by quantitative high-resolution mass spectrometry to resolve the identity of Nav1.2 protein interactors. Of the identified putative protein interactors, fibroblast growth factor 12 (FGF12), a member of the nonsecreted intracellular FGF family, exhibited 30-fold enrichment in Nav1.2 purifications compared with other identified proteins. Using confocal microscopy, we visualized native FGF12 in the brain tissue and confirmed that FGF12 forms a complex with Nav1.2 channels at the axonal initial segment, the subcellular specialized domain of neurons required for action potential initiation. Co-immunoprecipitation studies in a heterologous expression system validate Nav1.2 and FGF12 as interactors, whereas patch-clamp electrophysiology reveals that FGF12 acts synergistically with CaMKII, a known kinase regulator of Nav channels, to modulate Nav1.2-encoded currents. In the presence of CaMKII inhibitors we found that FGF12 produces a bidirectional shift in the voltage-dependence of activation (more depolarized) and the steady-state inactivation (more hyperpolarized) of Nav1.2, increasing the channel availability. Although providing the first characterization of the Nav1.2 CNS proteome, we identify FGF12 as a new functionally relevant interactor. Our studies will provide invaluable information to parse out the molecular determinant underlying neuronal excitability and plasticity, and extending the relevance of iFGFs signaling in the normal and diseased brain.
PMCID: PMC4424400  PMID: 25724910
20.  Voltage-dependent sodium (NaV) channels in group IV sensory afferents 
Molecular Pain  2016;12:1744806916660721.
Patients with intermittent claudication suffer from both muscle pain and an exacerbated exercise pressor reflex. Excitability of the group III and group IV afferent fibers mediating these functions is controlled in part by voltage-dependent sodium (NaV) channels. We previously found tetrodotoxin-resistant NaV1.8 channels to be the primary type in muscle afferent somata. However, action potentials in group III and IV afferent axons are blocked by TTX, supporting a minimal role of NaV1.8 channels. To address these apparent differences in NaV channel expression between axon and soma, we used immunohistochemistry to identify the NaV channels expressed in group IV axons within the gastrocnemius muscle and the dorsal root ganglia sections. Positive labeling by an antibody against the neurofilament protein peripherin was used to identify group IV neurons and axons. We show that >67% of group IV fibers express NaV1.8, NaV1.6, or NaV1.7. Interestingly, expression of NaV1.8 channels in group IV somata was significantly higher than in the fibers, whereas there were no significant differences for either NaV1.6 or NaV1.7. When combined with previous work, our results suggest that NaV1.8 channels are expressed in most group IV axons, but that, under normal conditions, NaV1.6 and/or NaV1.7 play a more important role in action potential generation to signal muscle pain and the exercise pressor reflex.
PMCID: PMC4956173  PMID: 27385723
Voltage-dependent sodium channels; dorsal root ganglia; exercise pressor reflex; muscle afferents
21.  Endogenous opioids contribute to insensitivity to pain in humans and mice lacking sodium channel Nav1.7 
Nature Communications  2015;6:8967.
Loss-of-function mutations in the SCN9A gene encoding voltage-gated sodium channel Nav1.7 cause congenital insensitivity to pain in humans and mice. Surprisingly, many potent selective antagonists of Nav1.7 are weak analgesics. We investigated whether Nav1.7, as well as contributing to electrical signalling, may have additional functions. Here we report that Nav1.7 deletion has profound effects on gene expression, leading to an upregulation of enkephalin precursor Penk mRNA and met-enkephalin protein in sensory neurons. In contrast, Nav1.8-null mutant sensory neurons show no upregulated Penk mRNA expression. Application of the opioid antagonist naloxone potentiates noxious peripheral input into the spinal cord and dramatically reduces analgesia in both female and male Nav1.7-null mutant mice, as well as in a human Nav1.7-null mutant. These data suggest that Nav1.7 channel blockers alone may not replicate the analgesic phenotype of null mutant humans and mice, but may be potentiated with exogenous opioids.
Nav1.7 channels are known to regulate pain perception in humans and mice. Here, the authors provide evidence that Nav1.7 deletion leads to transcriptional upregulation of opioid peptides in sensory neurons, and that treatment with the opioid blocker naloxone helps reverse analgesia in mice and human Nav1.7 nulls.
PMCID: PMC4686868  PMID: 26634308
22.  Nav1.7 is the predominant sodium channel in rodent olfactory sensory neurons 
Molecular Pain  2011;7:32.
Voltage-gated sodium channel Nav1.7 is preferentially expressed in dorsal root ganglion (DRG) and sympathetic neurons within the peripheral nervous system. Homozygous or compound heterozygous loss-of-function mutations in SCN9A, the gene which encodes Nav1.7, cause congenital insensitivity to pain (CIP) accompanied by anosmia. Global knock-out of Nav1.7 in mice is neonatal lethal reportedly from starvation, suggesting anosmia. These findings led us to hypothesize that Nav1.7 is the main sodium channel in the peripheral olfactory sensory neurons (OSN, also known as olfactory receptor neurons).
We used multiplex PCR-restriction enzyme polymorphism, in situ hybridization and immunohistochemistry to determine the identity of sodium channels in rodent OSNs.
We show here that Nav1.7 is the predominant sodium channel transcript, with low abundance of other sodium channel transcripts, in olfactory epithelium from rat and mouse. Our in situ hybridization data show that Nav1.7 transcripts are present in rat OSNs. Immunostaining of Nav1.7 and Nav1.6 channels in rat shows a complementary accumulation pattern with Nav1.7 in peripheral presynaptic OSN axons, and Nav1.6 primarily in postsynaptic cells and their dendrites in the glomeruli of the olfactory bulb within the central nervous system.
Our data show that Nav1.7 is the dominant sodium channel in rat and mouse OSN, and may explain anosmia in Nav1.7 null mouse and patients with Nav1.7-related CIP.
PMCID: PMC3101130  PMID: 21569247
23.  Pharmacological fractionation of tetrodotoxin-sensitive sodium currents in rat dorsal root ganglion neurons by μ-conotoxins 
British Journal of Pharmacology  2013;169(1):102-114.
Background and Purpose
Adult rat dorsal root ganglion (DRG) neurons normally express transcripts for five isoforms of the α-subunit of voltage-gated sodium channels: NaV1.1, 1.6, 1.7, 1.8 and 1.9. Tetrodotoxin (TTX) readily blocks all but NaV1.8 and 1.9, and pharmacological agents that discriminate among the TTX-sensitive NaV1-isoforms are scarce. Recently, we used the activity profile of a panel of μ-conotoxins in blocking cloned rodent NaV1-isoforms expressed in Xenopus laevis oocytes to conclude that action potentials of A- and C-fibres in rat sciatic nerve were, respectively, mediated primarily by NaV1.6 and NaV1.7.
Experimental Approach
We used three μ-conotoxins, μ-TIIIA, μ-PIIIA and μ-SmIIIA, applied individually and in combinations, to pharmacologically differentiate the TTX-sensitive INa of voltage-clamped neurons acutely dissociated from adult rat DRG. We examined only small and large neurons whose respective INa were >50% and >80% TTX-sensitive.
Key Results
In both small and large neurons, the ability of the toxins to block TTX-sensitive INa was μ-TIIIA < μ-PIIIA < μ-SmIIIA, with the latter blocking ≳90%. Comparison of the toxin-susceptibility profiles of the neuronal INa with recently acquired profiles of rat NaV1-isoforms, co-expressed with various NaVβ-subunits in X. laevis oocytes, were consistent: NaV1.1, 1.6 and 1.7 could account for all of the TTX-sensitive INa, with NaV1.1 < NaV1.6 < NaV1.7 for small neurons and NaV1.7 < NaV1.1 < NaV1.6 for large neurons.
Conclusions and Implications
Combinations of μ-conotoxins can be used to determine the probable NaV1-isoforms underlying the INa in DRG neurons. Preliminary experiments with sympathetic neurons suggest that this approach is extendable to other neurons.
PMCID: PMC3632242  PMID: 23351163
μ-conotoxin PIIIA; μ-conotoxin SmIIIA; μ-conotoxin TIIIA; dorsal root ganglion; superior cervical ganglion; tetrodotoxin; voltage-gated sodium channel; whole-cell patch clamp
24.  Comparison of Gating Properties and Use-Dependent Block of Nav1.5 and Nav1.7 Channels by Anti-Arrhythmics Mexiletine and Lidocaine 
PLoS ONE  2015;10(6):e0128653.
Mexiletine and lidocaine are widely used class IB anti-arrhythmic drugs that are considered to act by blocking voltage-gated open sodium currents for treatment of ventricular arrhythmias and relief of pain. To gain mechanistic insights into action of anti-arrhythmics, we characterized biophysical properties of Nav1.5 and Nav1.7 channels stably expressed in HEK293 cells and compared their use-dependent block in response to mexiletine and lidocaine using whole-cell patch clamp recordings. While the voltage-dependent activation of Nav1.5 or Nav1.7 was not affected by mexiletine and lidocaine, the steady-state fast and slow inactivation of Nav1.5 and Nav1.7 were significantly shifted to hyperpolarized direction by either mexiletine or lidocaine in dose-dependent manner. Both mexiletine and lidocaine enhanced the slow component of closed-state inactivation, with mexiletine exerting stronger inhibition on either Nav1.5 or Nav1.7. The recovery from inactivation of Nav1.5 or Nav1.7 was significantly prolonged by mexiletine compared to lidocaine. Furthermore, mexiletine displayed a pronounced and prominent use-dependent inhibition of Nav1.5 than lidocaine, but not Nav1.7 channels. Taken together, our findings demonstrate differential responses to blockade by mexiletine and lidocaine that preferentially affect the gating of Nav1.5, as compared to Nav1.7; and mexiletine exhibits stronger use-dependent block of Nav1.5. The differential gating properties of Nav1.5 and Nav1.7 in response to mexiletine and lidocaine may help explain the drug effectiveness and advance in new designs of safe and specific sodium channel blockers for treatment of cardiac arrhythmia or pain.
PMCID: PMC4465899  PMID: 26068619
25.  Nav1.9 Channel Contributes to Mechanical and Heat Pain Hypersensitivity Induced by Subacute and Chronic Inflammation 
PLoS ONE  2011;6(8):e23083.
Inflammation is known to be responsible for the sensitization of peripheral sensory neurons, leading to spontaneous pain and invalidating pain hypersensitivity. Given its role in regulating neuronal excitability, the voltage-gated Nav1.9 channel is a potential target for the treatment of pathological pain, but its implication in inflammatory pain is yet not fully described. In the present study, we examined the role of the Nav1.9 channel in acute, subacute and chronic inflammatory pain using Nav1.9-null mice and Nav1.9 knock-down rats. In mice we found that, although the Nav1.9 channel does not contribute to basal pain thresholds, it plays an important role in heat pain hypersensitivity induced by subacute paw inflammation (intraplantar carrageenan) and chronic ankle inflammation (complete Freund's adjuvant-induced monoarthritis). We showed for the first time that Nav1.9 also contributes to mechanical hypersensitivity in both models, as assessed using von Frey and dynamic weight bearing tests. Consistently, antisense-based Nav1.9 gene silencing in rats reduced carrageenan-induced heat and mechanical pain hypersensitivity. While no changes in Nav1.9 mRNA levels were detected in dorsal root ganglia (DRGs) during subacute and chronic inflammation, a significant increase in Nav1.9 immunoreactivity was observed in ipsilateral DRGs 24 hours following carrageenan injection. This was correlated with an increase in Nav1.9 immunolabeling in nerve fibers surrounding the inflamed area. No change in Nav1.9 current density could be detected in the soma of retrolabeled DRG neurons innervating inflamed tissues, suggesting that newly produced channels may be non-functional at this level and rather contribute to the observed increase in axonal transport. Our results provide evidence that Nav1.9 plays a crucial role in the generation of heat and mechanical pain hypersensitivity, both in subacute and chronic inflammatory pain models, and bring new elements for the understanding of its regulation in those models.
PMCID: PMC3155549  PMID: 21857998

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