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1.  µ-Conotoxin KIIIA Derivatives with Divergent Affinities versus Efficacies in Blocking Voltage-gated Sodium Channels 
Biochemistry  2010;49(23):4804-4812.
The possibility of independently manipulating the affinity and efficacy of pore-blocking ligands of sodium channels is of interest for the development of new drugs for the treatment of pain. The analgesic µ-conotoxin KIIIA, a 16-residue peptide with three disulfide bridges, is a pore-blocker of voltage-gated sodium channels, including the neuronal subtype NaV1.2 (Kd of 5 nM). At saturating concentrations, µ-KIIIA incompletely blocks the sodium current of NaV1.2, leaving a 5% residual current (rINa). Lys7 is an important residue: the mutation K7A decreases both the efficacy (i.e., increases rINa to 23%) and the affinity of the peptide (Kd, 115 nM). In this report, various replacements of residue 7 were examined to determine whether affinity and efficacy were inexorably linked. Because of their facile chemical synthesis, KIIIA analogs were used that had as a core structure the disulfide-depleted KIIIA[C1A,C2U,C9A,C5U] (where U is selenocysteine) or ddKIIIA. The analogs ddKIIIA and ddKIIIA[K7X], where X represents one of nine different amino acids, were tested on voltage-clamped Xenopus oocytes expressing rat NaV1.2 or NaV1.4. Their affinities ranged from 0.01 to 36 µM and rINa's from 2 to 42%, and these two variables appeared uncorrelated. Instead, rINa varied inversely with side chain size, and remarkably charge and hydrophobicity appeared inconsequential. The ability to manipulate a µ-conopeptide's affinity and efficacy, as well as its capacity to interfere with subsequent tetrodotoxin-binding, greatly expands its scope as a reagent to probe sodium channel structure and function, and may also lead to the development of µ-conotoxins as safe analgesics.
doi:10.1021/bi100207k
PMCID: PMC2907105  PMID: 20459109
2.  Synergistic and Antagonistic Interactions between Tetrodotoxin and μ-Conotoxin in Blocking Voltage-gated Sodium Channels 
Channels (Austin, Tex.)  2009;3(1):32-38.
Tetrodotoxin (TTX) is the quintessential ligand of voltage-gated sodium channels (NaVs). Like TTX, μ-conotoxin peptides are pore blockers, and both toxins have helped to define the properties of neurotoxin receptor Site 1 of NaVs. Here, we report unexpected results showing that the recently discovered μ-conotoxin KIIIA and TTX can simultaneously bind to Site 1 and act in concert. Results with saturating concentrations of peptide applied to voltage-clamped Xenopus oocytes expressing brain NaV1.2, and single-channel recordings from brain channels in lipid bilayers, show that KIIIA or its analog, KIIIA[K7A], block partially, with a residual current that can be completely blocked by TTX. In addition, the kinetics of block by TTX and peptide are each affected by the prior presence of the other toxin. For example, bound peptide slows subsequent binding of TTX (an antagonistic interaction) and slows TTX dissociation when both toxins are bound (a synergistic effect on block). The overall functional consequence resulting from the combined action of the toxins depends on the quantitative balance between these opposing actions. The results lead us to postulate that in the bi-liganded NaV complex, TTX is bound between the peptide and the selectivity filter. These observations refine our view of Site 1 and open new possibilities in NaV pharmacology.
PMCID: PMC2878737  PMID: 19221510
conotoxin; contratoxin; NaV1.2; oocyte; sodium channel; site 1; syntoxin; tetrodotoxin; voltage clamp
3.  Disulfide-Depleted Selenoconopeptides: a Minimalist Strategy to Oxidative Folding of Cysteine-Rich Peptides 
ACS medicinal chemistry letters  2010;1(4):140-144.
Despite the therapeutic promise of disulfide-rich, peptidic natural products, their discovery and structure/function studies have been hampered by inefficient oxidative folding methods for their synthesis. Here we report that converting the three disulfide-bridged μ-conopeptide KIIIA into a disulfide-depleted selenoconopeptide (by removal of a noncritical disulfide bridge and substitution of a disulfide- with a diselenide-bridge) dramatically simplified its oxidative folding while preserving the peptide’s ability to block voltage-gated sodium channels. The simplicity of synthesizing disulfide-depleted selenopeptide analogs containing a single disulfide bridge allowed rapid positional scanning at Lys7 of μ-KIIIA, resulting in the identification of K7L as a mutation that improved the peptide’s selectivity in blocking a neuronal (Nav1.2) over a muscle (Nav1.4) subtype of sodium channel. The disulfide-depleted selenopeptide strategy offers regioselective folding compatible with high throughput chemical synthesis and on-resin oxidation methods, and thus shows great promise to accelerate the use of disulfide-rich peptides as research tools and drugs.
PMCID: PMC2911238  PMID: 20676359
conotoxins; diselenide bridges; selenocysteines; oxidative folding; disulfide-rich peptides
4.  The Tetrodotoxin Receptor of Voltage-Gated Sodium Channels—Perspectives from Interactions with μ-Conotoxins 
Marine Drugs  2010;8(7):2153-2161.
Neurotoxin receptor site 1, in the outer vestibule of the conducting pore of voltage-gated sodium channels (VGSCs), was first functionally defined by its ability to bind the guanidinium-containing agents, tetrodotoxin (TTX) and saxitoxin (STX). Subsequent studies showed that peptide μ-conotoxins competed for binding at site 1. All of these natural inhibitors block single sodium channels in an all-or-none manner on binding. With the discovery of an increasing variety of μ-conotoxins, and the synthesis of numerous derivatives, observed interactions between the channel and these different ligands have become more complex. Certain μ-conotoxin derivatives block single-channel currents partially, rather than completely, thus enabling the demonstration of interactions between the bound toxin and the channel’s voltage sensor. Most recently, the relatively small μ-conotoxin KIIIA (16 amino acids) and its variants have been shown to bind simultaneously with TTX and exhibit both synergistic and antagonistic interactions with TTX. These interactions raise new pharmacological possibilities and place new constraints on the possible structures of the bound complexes of VGSCs with these toxins.
doi:10.3390/md8072153
PMCID: PMC2920548  PMID: 20714429
guanidinium toxins; conopeptides; pore block
5.  Conotoxins Targeting Neuronal Voltage-Gated Sodium Channel Subtypes: Potential Analgesics? 
Toxins  2012;4(11):1236-1260.
Voltage-gated sodium channels (VGSC) are the primary mediators of electrical signal amplification and propagation in excitable cells. VGSC subtypes are diverse, with different biophysical and pharmacological properties, and varied tissue distribution. Altered VGSC expression and/or increased VGSC activity in sensory neurons is characteristic of inflammatory and neuropathic pain states. Therefore, VGSC modulators could be used in prospective analgesic compounds. VGSCs have specific binding sites for four conotoxin families: μ-, μO-, δ- and ί-conotoxins. Various studies have identified that the binding site of these peptide toxins is restricted to well-defined areas or domains. To date, only the μ- and μO-family exhibit analgesic properties in animal pain models. This review will focus on conotoxins from the μ- and μO-families that act on neuronal VGSCs. Examples of how these conotoxins target various pharmacologically important neuronal ion channels, as well as potential problems with the development of drugs from conotoxins, will be discussed.
doi:10.3390/toxins4111236
PMCID: PMC3509706  PMID: 23202314
voltage-gated sodium channel; Nav1.3; Nav1.7; Nav1.8; Nav1.9; μ-conotoxin; μO-conotoxin; nociception; analgesic; pain
6.  An in vivo tethered toxin approach for the cell-autonomous inactivation of voltage-gated sodium channel currents in nociceptors 
The Journal of Physiology  2010;588(10):1695-1707.
Understanding information flow in sensory pathways requires cell-selective approaches to manipulate the activity of defined neurones. Primary afferent nociceptors, which detect painful stimuli, are enriched in specific voltage-gated sodium channel (VGSC) subtypes. Toxins derived from venomous animals can be used to dissect the contributions of particular ion currents to cell physiology. Here we have used a transgenic approach to target a membrane-tethered isoform of the conotoxin MrVIa (t-MrVIa) only to nociceptive neurones in mice. T-MrVIa transgenic mice show a 44 ± 7% reduction of tetrodotoxin-resistant (TTX-R) VGSC current densities. This inhibition is permanent, reversible and does not result in functional upregulation of TTX-sensitive (TTX-S) VGSCs, voltage-gated calcium channels (VGCCs) or transient receptor potential (TRP) channels present in nociceptive neurones. As a consequence of the reduction of TTX-R VGSC currents, t-MrVIa transgenic mice display decreased inflammatory mechanical hypersensitivity, cold pain insensitivity and reduced firing of cutaneous C-fibres sensitive to noxious cold temperatures. These data validate the use of genetically encoded t-toxins as a powerful tool to manipulate VGSCs in specific cell types within the mammalian nervous system. This novel genetic methodology can be used for circuit mapping and has the key advantage that it enables the dissection of the contribution of specific ionic currents to neuronal function and to behaviour.
doi:10.1113/jphysiol.2010.187112
PMCID: PMC2887988  PMID: 20308253
7.  An in vivo tethered toxin approach for the cell-autonomous inactivation of voltage-gated sodium channel currents in nociceptors 
The Journal of Physiology  2010;588(Pt 10):1695-1707.
Understanding information flow in sensory pathways requires cell-selective approaches to manipulate the activity of defined neurones. Primary afferent nociceptors, which detect painful stimuli, are enriched in specific voltage-gated sodium channel (VGSC) subtypes. Toxins derived from venomous animals can be used to dissect the contributions of particular ion currents to cell physiology. Here we have used a transgenic approach to target a membrane-tethered isoform of the conotoxin MrVIa (t-MrVIa) only to nociceptive neurones in mice. T-MrVIa transgenic mice show a 44 ± 7% reduction of tetrodotoxin-resistant (TTX-R) VGSC current densities. This inhibition is permanent, reversible and does not result in functional upregulation of TTX-sensitive (TTX-S) VGSCs, voltage-gated calcium channels (VGCCs) or transient receptor potential (TRP) channels present in nociceptive neurones. As a consequence of the reduction of TTX-R VGSC currents, t-MrVIa transgenic mice display decreased inflammatory mechanical hypersensitivity, cold pain insensitivity and reduced firing of cutaneous C-fibres sensitive to noxious cold temperatures. These data validate the use of genetically encoded t-toxins as a powerful tool to manipulate VGSCs in specific cell types within the mammalian nervous system. This novel genetic methodology can be used for circuit mapping and has the key advantage that it enables the dissection of the contribution of specific ionic currents to neuronal function and to behaviour.
doi:10.1113/jphysiol.2010.187112
PMCID: PMC2887988  PMID: 20308253
8.  Synthesis and evaluation of a 125I-labeled iminodihydroquinoline-derived tracer for imaging of voltage-gated sodium channels☆ 
Graphical abstract
In vivo imaging of voltage-gated sodium channels (VGSCs) can potentially provide insights into the activation of neuronal pathways and aid the diagnosis of a number of neurological diseases. The iminodihydroquinoline WIN17317-3 is one of the most potent sodium channel blockers reported to date and binds with high affinity to VGSCs throughout the rat brain. We have synthesized a 125I-labeled analogue of WIN17317-3 and evaluated the potential of the tracer for imaging of VGSCs with SPECT. Automated patch clamp studies with CHO cells expressing the Nav1.2 isoform and displacement studies with [3H]BTX yielded comparable results for the non-radioactive iodinated iminodihydroquinoline and WIN17317-3. However, the 125I-labeled tracer was rapidly metabolized in vivo, and suffered from low brain uptake and high accumulation of radioactivity in the intestines. The results suggest that iminodihydroquinolines are poorly suited for tracer development.
doi:10.1016/j.bmcl.2013.07.014
PMCID: PMC3764405  PMID: 23910595
SPECT; Voltage-gated sodium channel; WIN17317-3; Iodine-125; Imaging
9.  Voltage-gated sodium channels and metastatic disease 
Channels (Austin, Tex.)  2012;6(5):352-361.
Voltage-gated Na+ channels (VGSCs) are macromolecular protein complexes containing a pore-forming α subunit and smaller non-pore-forming β subunits. VGSCs are expressed in metastatic cells from a number of cancers. In these cells, Na+ current carried by α subunits enhances migration, invasion and metastasis in vivo. In contrast, the β subunits mediate cellular adhesion and process extension. The prevailing hypothesis is that VGSCs are up-regulated in cancer, in general favoring an invasive/metastatic phenotype, although the mechanisms are still not fully clear. Expression of the Nav1.5 α subunit associates with poor prognosis in clinical breast cancer specimens, suggesting that VGSCs may have utility as prognostic markers for cancer progression. Furthermore, repurposing existing VGSC-blocking therapeutic drugs may provide a new strategy to improve outcomes in patients suffering from metastatic disease, which is the major cause of cancer-related deaths, and for which there is currently no cure.
doi:10.4161/chan.21910
PMCID: PMC3508774  PMID: 22992466
Anticonvulsant; Cancer; Invasion; Metastasis; Migration; Phenytoin; Voltage-gated Na+ channel
10.  Voltage-gated sodium channels and metastatic disease 
Channels  2012;6(5):352-361.
Voltage-gated Na+ channels (VGSCs) are macromolecular protein complexes containing a pore-forming α subunit and smaller non-pore-forming β subunits. VGSCs are expressed in metastatic cells from a number of cancers. In these cells, Na+ current carried by α subunits enhances migration, invasion and metastasis in vivo. In contrast, the β subunits mediate cellular adhesion and process extension. The prevailing hypothesis is that VGSCs are upregulated in cancer, in general favoring an invasive/metastatic phenotype, although the mechanisms are still not fully clear. Expression of the Nav1.5 α subunit associates with poor prognosis in clinical breast cancer specimens, suggesting that VGSCs may have utility as prognostic markers for cancer progression. Furthermore, repurposing existing VGSC-blocking therapeutic drugs may provide a new strategy to improve outcomes in patients suffering from metastatic disease, which is the major cause of cancer-related deaths, and for which there is currently no cure.
doi:10.4161/chan.21910
PMCID: PMC3508774  PMID: 22992466
anticonvulsant; cancer; invasion; metastasis; migration; phenytoin; voltage-gated Na+ channel
11.  Structural Basis for the Modulation of the Neuronal Voltage-Gated Sodium Channel NaV1.6 by Calmodulin 
Scientific Reports  2013;3:2435.
The neuronal-voltage gated sodium channel (VGSC), NaV1.6, plays an important role in propagating action potentials along myelinated axons. Calmodulin (CaM) is known to modulate the inactivation kinetics of NaV1.6 by interacting with its IQ motif. Here we report the crystal structure of apo-CaM:NaV1.6IQ motif, along with functional studies. The IQ motif of NaV1.6 adopts an α-helical conformation in its interaction with the C-lobe of CaM. CaM uses different residues to interact with NaV1.6IQ motif depending on the presence or absence of Ca2+. Three residues from NaV1.6, Arg1902, Tyr1904 and Arg1905 were identified as the key common interacting residues in both the presence and absence of Ca2+. Substitution of Arg1902 and Tyr1904 with alanine showed a reduced rate of NaV1.6 inactivation in electrophysiological experiments in vivo. Compared with other CaM:NaV complexes, our results reveal a different mode of interaction for CaM:NaV1.6 and provides structural insight into the isoform-specific modulation of VGSCs.
doi:10.1038/srep02435
PMCID: PMC3743062  PMID: 23942337
12.  β-Spectrin Is Colocalized with Both Voltage-gated Sodium Channels and AnkyrinG at the Adult Rat Neuromuscular Junction  
The Journal of Cell Biology  1998;140(3):675-684.
Voltage-gated sodium channels (VGSCs) are concentrated in the depths of the postsynaptic folds at mammalian neuromuscular junctions (NMJs) where they facilitate action potential generation during neuromuscular transmission. At the nodes of Ranvier and the axon hillocks of central neurons, VGSCs are associated with the cytoskeletal proteins, β-spectrin and ankyrin, which may help to maintain the high local density of VGSCs. Here we show in skeletal muscle, using immunofluorescence, that β-spectrin is precisely colocalized with both VGSCs and ankyrinG, the nodal isoform of ankyrin. In en face views of rat NMJs, acetylcholine receptors (AChRs), and utrophin immunolabeling are organized in distinctive linear arrays corresponding to the crests of the postsynaptic folds. In contrast, β-spectrin, VGSCs, and ankyrinG have a punctate distribution that extends laterally beyond the AChRs, consistent with a localization in the depths of the folds. Double antibody labeling shows that β-spectrin is precisely colocalized with both VGSCs and ankyrinG at the NMJ. Furthermore, quantification of immunofluorescence in labeled transverse sections reveals that β-spectrin is also concentrated in perijunctional regions, in parallel with an increase in labeling of VGSCs and ankyrinG, but not of dystrophin. These observations suggest that interactions with β-spectrin and ankyrinG help to maintain the concentration of VGSCs at the NMJ and that a common mechanism exists throughout the nervous system for clustering VGSCs at a high density.
PMCID: PMC2140176  PMID: 9456326
13.  Voltage-gated Na+ channel SCN5A is a key regulator of a gene transcriptional network that controls colon cancer invasion 
Cancer research  2010;70(17):6957-6967.
Voltage-gated Na+ channels (VGSCs) have been implicated in the metastatic potential of human breast, prostate and lung cancer cells. Specifically, the SCN5A gene encoding the VGSC isotype Nav1.5 has been defined as a key driver of human cancer cell invasion. In this study, we examined the expression and function of VGSCs in a panel of colon cancer cell lines by electrophysiological recordings. Na+ channel activity and invasive potential were inhibited pharmacologically by tetrodotoxin or genetically by siRNAs specifically targeting SCN5A. Clinical relevance was established by immunohistochemistry of patient biopsies, where there was strong Nav1.5 protein staining in colon cancer specimens but little to no staining in matched-paired normal colon tissues. We explored the mechanism of VGSC-mediated invasive potential on the basis of reported links between VGSC activity and gene expression in excitable cells. Probabilistic modeling of loss-of-function screens and microarray data established an unequivocal role of VGSC SCN5A as a high level regulator of a colon cancer invasion network, involving genes that encompass Wnt signaling, cell migration, ectoderm development, response to biotic stimulus, steroid metabolic process and cell cycle control. siRNA-mediated knockdown of predicted downstream network components caused a loss of invasive behavior, demonstrating network connectivity and its function in driving colon cancer invasion.
doi:10.1158/0008-5472.CAN-10-1169
PMCID: PMC2936697  PMID: 20651255
voltage-gated Na+ channels; invasion; colon cancer; gene network
14.  Lactam Constraints Provide Insights into the Receptor-Bound Conformation of Secretin and Stabilize a Receptor Antagonist 
Biochemistry  2011;50(38):8181-8192.
The natural ligands for family B G protein-coupled receptors are moderate length linear peptides having diffuse pharmacophores. The amino-terminal regions of these ligands are critical for biological activity, with their amino-terminal truncation leading to production of orthosteric antagonists. The carboxyl-terminal regions of these peptides are thought to occupy a ligand-binding cleft within the disulfide-bonded amino-terminal domains of these receptors, with the peptides in amphipathic helical conformations. In the current work, we have characterized the binding and activity of a series of 11 truncated and lactam-constrained secretin(5-27) analogues at the prototypic member of this family, the secretin receptor. One peptide in this series with lactam connecting residues 16 and 20 (c[E16,K20][Y10]sec(5-27)) improved the binding affinity of its unconstrained parental peptide 22-fold, while retaining absence of endogenous biological activity and competitive antagonist characteristics. Homology modeling with molecular mechanics and molecular dynamics simulations established that this constrained peptide occupies the ligand-binding cleft in orientation similar to natural full-length secretin, and provided insights into why this peptide was more effective than other truncated conformationally-constrained peptides in the series. This lactam bridge is believed to stabilize an extended α-helical conformation of this peptide while in solution and to not interfere with critical residue-residue approximations while docked to the receptor.
doi:10.1021/bi2008036
PMCID: PMC3177990  PMID: 21851058
Secretin; secretin receptor; family B G protein-coupled receptor; antagonist; lactam bridge; ligand binding
15.  Tetrodotoxin (TTX) as a Therapeutic Agent for Pain 
Marine Drugs  2012;10(2):281-305.
Tetrodotoxin (TTX) is a potent neurotoxin that blocks voltage-gated sodium channels (VGSCs). VGSCs play a critical role in neuronal function under both physiological and pathological conditions. TTX has been extensively used to functionally characterize VGSCs, which can be classified as TTX-sensitive or TTX-resistant channels according to their sensitivity to this toxin. Alterations in the expression and/or function of some specific TTX-sensitive VGSCs have been implicated in a number of chronic pain conditions. The administration of TTX at doses below those that interfere with the generation and conduction of action potentials in normal (non-injured) nerves has been used in humans and experimental animals under different pain conditions. These data indicate a role for TTX as a potential therapeutic agent for pain. This review focuses on the preclinical and clinical evidence supporting a potential analgesic role for TTX. In addition, the contribution of specific TTX-sensitive VGSCs to pain is reviewed.
doi:10.3390/md10020281
PMCID: PMC3296997  PMID: 22412801
tetrodotoxin; TTX; TTX-sensitive voltage-gated sodium channels; pain; neuropathic pain
16.  Mechanism of μ-Conotoxin PIIIA Binding to the Voltage-Gated Na+ Channel NaV1.4 
PLoS ONE  2014;9(3):e93267.
Several subtypes of voltage-gated Na+ (NaV) channels are important targets for pain management. μ-Conotoxins isolated from venoms of cone snails are potent and specific blockers of different NaV channel isoforms. The inhibitory effect of μ-conotoxins on NaV channels has been examined extensively, but the mechanism of toxin specificity has not been understood in detail. Here the known structure of μ-conotoxin PIIIA and a model of the skeletal muscle channel NaV1.4 are used to elucidate elements that contribute to the structural basis of μ-conotoxin binding and specificity. The model of NaV1.4 is constructed based on the crystal structure of the bacterial NaV channel, NaVAb. Six different binding modes, in which the side chain of each of the basic residues carried by the toxin protrudes into the selectivity filter of NaV1.4, are examined in atomic detail using molecular dynamics simulations with explicit solvent. The dissociation constants (Kd) computed for two selected binding modes in which Lys9 or Arg14 from the toxin protrudes into the filter of the channel are within 2 fold; both values in close proximity to those determined from dose response data for the block of NaV currents. To explore the mechanism of PIIIA specificity, a double mutant of NaV1.4 mimicking NaV channels resistant to μ-conotoxins and tetrodotoxin is constructed and the binding of PIIIA to this mutant channel examined. The double mutation causes the affinity of PIIIA to reduce by two orders of magnitude.
doi:10.1371/journal.pone.0093267
PMCID: PMC3968119  PMID: 24676211
17.  Therapeutic potential for phenytoin: targeting Nav1.5 sodium channels to reduce migration and invasion in metastatic breast cancer 
Voltage-gated Na+ channels (VGSCs) are heteromeric membrane protein complexes containing pore-forming α subunits and smaller, non-pore-forming β subunits. VGSCs are classically expressed in excitable cells, including neurons and muscle cells, where they mediate action potential firing, neurite outgrowth, pathfinding, and migration. VGSCs are also expressed in metastatic cells from a number of cancers. The Nav1.5 α subunit (encoded by SCN5A) is expressed in breast cancer (BCa) cell lines, where it enhances migration and invasion. We studied the expression of SCN5A in BCa array data, and tested the effect of the VGSC-blocking anticonvulsant phenytoin (5,5-diphenylhydantoin) on Na+ current, migration, and invasion in BCa cells. SCN5A was up-regulated in BCa samples in several datasets, and was more highly expressed in samples from patients who had a recurrence, metastasis, or died within 5 years. SCN5A was also overexpressed as an outlier in a subset of samples, and associated with increased odds of developing metastasis. Phenytoin inhibited transient and persistent Na+ current recorded from strongly metastatic MDA-MB-231 cells, and this effect was more potent at depolarized holding voltages. It may thus be an effective VGSC-blocking drug in cancer cells, which typically have depolarized membrane potentials. At a concentration within the therapeutic range used to treat epilepsy, phenytoin significantly inhibited the migration and invasion of MDA-MB-231 cells, but had no effect on weakly metastatic MCF-7 cells, which do not express Na+ currents. We conclude that phenytoin suppresses Na+ current in VGSC-expressing metastatic BCa cells, thus inhibiting VGSC-dependent migration and invasion. Together, our data support the hypothesis that SCN5A is up-regulated in BCa, favoring an invasive/metastatic phenotype. We therefore propose that repurposing existing VGSC-blocking therapeutic drugs should be further investigated as a potential new strategy to improve patient outcomes in metastatic BCa.
doi:10.1007/s10549-012-2102-9
PMCID: PMC3401508  PMID: 22678159
Electrophysiology; Invasion; Metastasis; Migration; Phenytoin; Voltage-gated Na+ channel
18.  Therapeutic potential for phenytoin: targeting Nav1.5 sodium channels to reduce migration and invasion in metastatic breast cancer 
Voltage-gated Na+ channels (VGSCs) are heteromeric membrane protein complexes containing pore-forming α subunits and smaller, non-pore-forming β subunits. VGSCs are classically expressed in excitable cells, including neurons and muscle cells, where they mediate action potential firing, neurite outgrowth, pathfinding, and migration. VGSCs are also expressed in metastatic cells from a number of cancers. The Nav1.5 α subunit (encoded by SCN5A) is expressed in breast cancer (BCa) cell lines, where it enhances migration and invasion. We studied the expression of SCN5A in BCa array data, and tested the effect of the VGSC-blocking anticonvulsant phenytoin (5,5-diphenylhydantoin) on Na+ current, migration, and invasion in BCa cells. SCN5A was up-regulated in BCa samples in several datasets, and was more highly expressed in samples from patients who had a recurrence, metastasis, or died within 5 years. SCN5A was also overexpressed as an outlier in a subset of samples, and associated with increased odds of developing metastasis. Phenytoin inhibited transient and persistent Na+ current recorded from strongly metastatic MDA-MB-231 cells, and this effect was more potent at depolarized holding voltages. It may thus be an effective VGSC-blocking drug in cancer cells, which typically have depolarized membrane potentials. At a concentration within the therapeutic range used to treat epilepsy, phenytoin significantly inhibited the migration and invasion of MDA-MB-231 cells, but had no effect on weakly metastatic MCF-7 cells, which do not express Na+ currents. We conclude that phenytoin suppresses Na+ current in VGSC-expressing metastatic BCa cells, thus inhibiting VGSC-dependent migration and invasion. Together, our data support the hypothesis that SCN5A is up-regulated in BCa, favoring an invasive/metastatic phenotype. We therefore propose that repurposing existing VGSC-blocking therapeutic drugs should be further investigated as a potential new strategy to improve patient outcomes in metastatic BCa.
doi:10.1007/s10549-012-2102-9
PMCID: PMC3401508  PMID: 22678159
Electrophysiology; Invasion; Metastasis; Migration; Phenytoin; Voltage-gated Na+ channel
19.  Comparison of Design Strategies for Promotion of β-Peptide 14-Helix Stability in Water 
Many short β-peptides adopt well-defined conformations in organic solvents, but specialized stabilizing elements are required for folding to occur in aqueous solution. Several different strategies have been developed to stabilize the 14-helical secondary structure in water, and here we provide a direct comparison of three such strategies. We have synthesized and characterized β-peptide heptamers in which either a salt bridge between side chains or a covalent link between side chains or a few cyclically constrained residues have been incorporated to promote 14-helicity. The incorporation of a salt bridge does not generate significant 14-helicity in water, according to CD and 2D NMR data. In contrast, incorporation of either a lactam bridge between side chains or cyclic residues results in stable 14-helices in water. The β-peptides featuring trans-2-aminocyclohexane-carboxylic acid (ACHC) residues show the highest 14-helical backbone stability, with hardly any sensitivity to pH or ionic strength. The β-peptides featuring a side chain-to-side chain cyclization show lower 14-helical backbone stability and higher sensitivity to pH and ionic strength, but an increased order between the side chains because of the cyclization.
doi:10.1002/cbic.200800355
PMCID: PMC3551619  PMID: 18756554
β-peptides; 14-helix; cyclic peptides; foldamers
20.  Screening for Voltage-Gated Sodium Channel Interacting Peptides 
Scientific Reports  2014;4:4569.
The voltage-gated sodium channel (VGSC) interacting peptide is of special interest for both basic research and pharmaceutical purposes. In this study, we established a yeast-two-hybrid based strategy to detect the interaction(s) between neurotoxic peptide and the extracellular region of VGSC. Using a previously reported neurotoxin JZTX-III as a model molecule, we demonstrated that the interactions between JZTX-III and the extracellular regions of its target hNav1.5 are detectable and the detected interactions are directly related to its activity. We further applied this strategy to the screening of VGSC interacting peptides. Using the extracellular region of hNav1.5 as the bait, we identified a novel sodium channel inhibitor SSCM-1 from a random peptide library. This peptide selectively inhibits hNav1.5 currents in the whole-cell patch clamp assays. This strategy might be used for the large scale screening for target-specific interacting peptides of VGSCs or other ion channels.
doi:10.1038/srep04569
PMCID: PMC3972499
21.  A novel adhesion molecule in human breast cancer cells: Voltage-gated Na+ channel β1 subunit 
Voltage-gated Na+ channels (VGSCs), predominantly the ‘neonatal’ splice form of Nav1.5 (nNav1.5), are upregulated in metastatic breast cancer (BCa) and potentiate metastatic cell behaviours. VGSCs comprise one pore-forming α subunit and one or more β subunits. The latter modulate VGSC expression and gating, and can function as cell adhesion molecules of the immunoglobulin superfamily. The aims of this study were (1) to determine which β subunits were expressed in weakly metastatic MCF-7 and strongly metastatic MDA-MB-231 human BCa cells, and (2) to investigate the possible role of β subunits in adhesion and migration. In both cell lines, the β subunit mRNA expression profile was SCN1B (encoding β1) ≫ SCN4B (encoding β4) > SCN2B (encoding β2); SCN3B (encoding β3) was not detected. MCF-7 cells had much higher levels of all β subunit mRNAs than MDA-MB-231 cells, and β1 mRNA was the most abundant. Similarly, β1 protein was strongly expressed in MCF-7 and barely detectable in MDA-MB-231 cells. In MCF-7 cells transfected with siRNA targeting β1, adhesion was reduced by 35 %, while migration was increased by 121 %. The increase in migration was reversed by tetrodotoxin (TTX). In addition, levels of nNav1.5 mRNA and protein were increased following β1 down-regulation. Stable expression of β1 in MDA-MB-231 cells increased functional VGSC activity, process length and adhesion, and reduced lateral motility and proliferation. We conclude that β1 is a novel cell adhesion molecule in BCa cells and can control VGSC (nNav1.5) expression and, concomitantly, cellular migration.
doi:10.1016/j.biocel.2008.11.001
PMCID: PMC2678854  PMID: 19041953
Adhesion; breast cancer; metastasis; migration; voltage-gated Na+ channel
22.  Design, Synthesis, and Biological Evaluation of New Cyclic Melanotropin Peptide Analogues Selective for the Human Melanocortin-4 Receptor 
Journal of medicinal chemistry  2006;49(23):6888-6896.
Intensive efforts have been made to develop potent and selective ligands for certain human melanocortin receptors as possible treatments for obesity and sexual dysfunction due to the role of these receptors in feeding behavior, energy homeostasis, sexual function, etc. A number of novel α-MSH analogues were designed and synthesized primarily on the basis of our previous MTII NMR structure. In these peptide analogues, a disulfide or lactam bridge between residues at positions 5 and 8 was used as a conformational constraint to enhance the β-turn spanning His6 and d-Phe7, while the pharmacophore group in Arg8 was mimicked via Nα-alkylation of residues 8 or 9 with the guanidinylbutyl group. Biological assays for binding affinities and adenylate cyclase activities for the hMC1R, hMC3R, hMC4R, and hMC5R showed that three analogues have good binding affinity for the hMC4R (0.7–4.1 nM), but have no binding affinity up to 10 μM at the other three melanocortin receptors. Interestingly, the three hMC4R selective analogues display only 50% binding efficiency, suggesting there is allosteric modulation of the melanocortin-4 receptor. These analogues were found to act as antagonists of the hMC4R. This result represents a discovery of very selective peptide-based antagonists for the hMC4R. The high selectivity may be due to the strong conformational constraint via ring contraction as compared to MTII, and the rigid conformation preferred by these new ligands allows them to recognize only the hMC4R, but not to activate the second messenger. The MTII NMR structure-based design thus not only examined the structural model of melanocortin ligands, but also yielded new biologically unique α-MSH analogues.
doi:10.1021/jm060768f
PMCID: PMC1764620  PMID: 17154518
23.  Effect of amitriptyline on tetrodotoxin-resistant Nav1.9 currents in nociceptive trigeminal neurons 
Molecular Pain  2013;9:31.
Background
Amitriptyline (AMI) is tricyclic antidepressant that has been widely used to manage various chronic pains such as migraines. Its efficacy is attributed to its blockade of voltage-gated sodium channels (VGSCs). However, the effects of AMI on the tetrodotoxin-resistant (TTX-r) sodium channel Nav1.9 currents have been unclear to present.
Results
Using a whole-cell patch clamp technique, this study showed that AMI efficiently inhibited Nav1.9 currents in a concentration-dependent manner and had an IC50 of 15.16 μM in acute isolated trigeminal ganglion (TG) neurons of the rats. 10 μM AMI significantly shifted the steady-state inactivation of Nav1.9 channels in the hyperpolarizing direction without affecting voltage-dependent activation. Surprisingly, neither 10 nor 50 μM AMI caused a use-dependent blockade of Nav1.9 currents elicited by 60 pulses at 1 Hz.
Conclusion
These data suggest that AMI is a state-selective blocker of Nav1.9 channels in rat nociceptive trigeminal neurons, which likely contributes to the efficacy of AMI in treating various pains, including migraines.
doi:10.1186/1744-8069-9-31
PMCID: PMC3691845  PMID: 24228717
Amitriptyline; Nav1.9; Patch clamp; Trigeminal ganglion; Pain
24.  Human voltage-gated sodium channel mutations that cause inherited neuronal and muscle channelopathies increase resurgent sodium currents 
Inherited mutations in voltage-gated sodium channels (VGSCs; or Nav) cause many disorders of excitability, including epilepsy, chronic pain, myotonia, and cardiac arrhythmias. Understanding the functional consequences of the disease-causing mutations is likely to provide invaluable insight into the roles that VGSCs play in normal and abnormal excitability. Here, we sought to test the hypothesis that disease-causing mutations lead to increased resurgent currents, unusual sodium currents that have not previously been implicated in disorders of excitability. We demonstrated that a paroxysmal extreme pain disorder (PEPD) mutation in the human peripheral neuronal sodium channel Nav1.7, a paramyotonia congenita (PMC) mutation in the human skeletal muscle sodium channel Nav1.4, and a long-QT3/SIDS mutation in the human cardiac sodium channel Nav1.5 all substantially increased the amplitude of resurgent sodium currents in an optimized adult rat–derived dorsal root ganglion neuronal expression system. Computer simulations indicated that resurgent currents associated with the Nav1.7 mutation could induce high-frequency action potential firing in nociceptive neurons and that resurgent currents associated with the Nav1.5 mutation could broaden the action potential in cardiac myocytes. These effects are consistent with the pathophysiology associated with the respective channelopathies. Our results indicate that resurgent currents are associated with multiple channelopathies and are likely to be important contributors to neuronal and muscle disorders of excitability.
doi:10.1172/JCI40801
PMCID: PMC2799199  PMID: 20038812
25.  Association between Tetrodotoxin Resistant Channels and Lipid Rafts Regulates Sensory Neuron Excitability 
PLoS ONE  2012;7(8):e40079.
Voltage-gated sodium channels (VGSCs) play a key role in the initiation and propagation of action potentials in neurons. NaV1.8 is a tetrodotoxin (TTX) resistant VGSC expressed in nociceptors, peripheral small-diameter neurons able to detect noxious stimuli. NaV1.8 underlies the vast majority of sodium currents during action potentials. Many studies have highlighted a key role for NaV1.8 in inflammatory and chronic pain models. Lipid rafts are microdomains of the plasma membrane highly enriched in cholesterol and sphingolipids. Lipid rafts tune the spatial and temporal organisation of proteins and lipids on the plasma membrane. They are thought to act as platforms on the membrane where proteins and lipids can be trafficked, compartmentalised and functionally clustered. In the present study we investigated NaV1.8 sub-cellular localisation and explored the idea that it is associated with lipid rafts in nociceptors. We found that NaV1.8 is distributed in clusters along the axons of DRG neurons in vitro and ex vivo. We also demonstrated, by biochemical and imaging studies, that NaV1.8 is associated with lipid rafts along the sciatic nerve ex vivo and in DRG neurons in vitro. Moreover, treatments with methyl-β-cyclodextrin (MβCD) and 7-ketocholesterol (7KC) led to the dissociation between rafts and NaV1.8. By calcium imaging we demonstrated that the lack of association between rafts and NaV1.8 correlated with impaired neuronal excitability, highlighted by a reduction in the number of neurons able to conduct mechanically- and chemically-evoked depolarisations. These findings reveal the sub-cellular localisation of NaV1.8 in nociceptors and highlight the importance of the association between NaV1.8 and lipid rafts in the control of nociceptor excitability.
doi:10.1371/journal.pone.0040079
PMCID: PMC3411591  PMID: 22870192

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