Structure and Function of Hainantoxin-III, a Selective Antagonist of Neuronal Tetrodotoxin-sensitive Voltage-gated Sodium Channels Isolated from the Chinese Bird Spider Ornithoctonus hainana

Z, Liu; Cai, Tianfu; Zhu, Qi; Deng, Mei; Li, Jiayan; Zhou, Xi; Zhang, Fang; Li, Dan; Li, Jing; Liu, Yu; Hu, Weijun; Liang, Songping

doi:10.1074/jbc.m112.426627

Cited by 57 publications

(66 citation statements)

References 56 publications

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“…However, domain swapping, site-directed mutagenesis, and competitive binding experiments have localized the O-conotoxin binding site to the voltage-sensing regions located on Domain II, providing evidence that O-conotoxins act as gating modifiers that inhibit Na ϩ current by trapping the Domain II voltage sensor in the closed position to prevent opening of the channel (21,22). This mechanism of action is also reported for several spider peptides that inhibit Na V channels by binding to the extracellular loops of the same domain (55)(56)(57). Accordingly, MfVIA inhibited peak Na ϩ current, causing small hyperpolarizing shifts in the voltage dependence of activation and inactivation at Na V 1.8.…”

Section: Discussionmentioning

confidence: 76%

Development of a μO-Conotoxin Analogue with Improved Lipid Membrane Interactions and Potency for the Analgesic Sodium Channel NaV1.8

Deuis

Dekan

Inserra

et al. 2016

Journal of Biological Chemistry

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The O-conotoxins MrVIA, MrVIB, and MfVIA inhibit the voltage-gated sodium channel Na V 1.8, a well described target for the treatment of pain; however, little is known about the residues or structural elements that define this activity. In this study, we determined the three-dimensional structure of MfVIA, examined its membrane binding properties, performed alaninescanning mutagenesis, and identified residues important for its activity at human Na V 1.8. A second round of mutations resulted in (E5K,E8K)MfVIA, a double mutant with greater positive surface charge and greater affinity for lipid membranes compared with MfVIA. This analogue had increased potency at Na V 1.8 and was analgesic in the mouse formalin assay.Voltage-gated sodium channels (Na V s) 5 are responsible for the initiation and propagation of action potentials (1). Nine subtypes have been described to date (Na V 1.1-1.9), each with a distinct expression profile and subsequent functional role (1). For example, Na V 1.4 is predominantly expressed in skeletal muscle where it is important in the generation and propagation of action potentials that initiate muscle contraction (2), whereas Na V 1.5 is predominately expressed in cardiac tissue where it is crucial in setting the electrical conduction properties of the heart (3). Several subtypes (Na V 1.1 and Na V 1.6 -1.9) are expressed in peripheral sensory neurons, and it is due to this expression profile that some of these subtypes have gained attention as potential therapeutic targets for pain (4). Na V 1.8 is of particular interest as an analgesic target because it is expressed almost exclusively in peripheral sensory neurons and is present in the majority of nociceptive neurons (5, 6) where it is a major contributor to the sodium current underlying action potentials (7-9). Accordingly, Na V 1.8-null mice have reduced responses to nociceptive mechanical and thermal stimuli as well as deficits in the development of inflammatory pain, visceral pain, and to a lesser extent neuropathic pain (10 -14). Therefore, pharmacological inhibition of Na V 1.8 is considered a promising therapeutic strategy for the treatment of pain.The recently discovered O-conotoxin MfVIA (15) and the related MrVIA and MrVIB (16) (see Fig. 1A) are some of a few venom-derived peptides that inhibit Na V 1.8 and the only peptides known to selectively inhibit Na V 1.8 over the other neuronal Na V subtypes expressed in peripheral sensory neurons (15,17,18). However, despite this unique selectivity, little is known about structure-activity relationships of the O-conotoxins. This gap in knowledge arises from difficulties in the synthesis of sufficient quantities to permit detailed studies (19), which is in part due to the hydrophobic nature of these peptides. Characterization of the structural elements contributing to the high affinity inhibition of Na V 1.8 by the O-conotoxins is of particular interest given that the O-conotoxins also potently inhibit the skeletal muscle isoform Na V 1.4, and reduced activity at Na V 1.4 while maintain...

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Section: Discussionmentioning

confidence: 76%

Development of a μO-Conotoxin Analogue with Improved Lipid Membrane Interactions and Potency for the Analgesic Sodium Channel NaV1.8

Deuis

Dekan

Inserra

et al. 2016

Journal of Biological Chemistry

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“…Although a few of the jingzhaotoxins (Chilobrachys jingzhao) so far discovered have been shown to inhibit Na V current by acting as gating modifiers [244,[257][258][259], only one -β/κ-TRTX-Cj1a (jingzhaotoxin-III) -has so far demonstrated direct binding to the site 4 DIIS3-S4 loop [245]. Two other spider venom peptides that have been shown to bind the DIIS3-S4 linker through mutational analysis include µ-TRTX-Hh2a (huwentoxin-IV isolated from Ornithoctonus huwena) [243] and µ-TRTX-Hhn2a (hainantoxin-III isolated from Ornithoctonus hainana) [255]. Both of these toxins also inhibit Na V current by interacting with the DIIS3-S4 linker and trapping the voltage sensor in the closed conformation.…”

Section: Site 3 and Sitementioning

confidence: 99%

“…Multiple other spider venom peptides have since been discovered with critical binding domains located on the site 4 DIIS3-S4 loop [239] (Figure 4), as determined by either mutagenesis of key residues on this region of the Na V channel [243,245,255] or competitive assay with β-scorpion toxins [244]. However, many of these spider venom peptides exhibit a distinct functional profile to the β-scorpion toxins, whereby the peptide traps the DIIS4 voltage sensor in the closed state resulting in an inhibition of peak current [245,255,256].…”

Section: Site 3 and Sitementioning

confidence: 99%

“…However, many of these spider venom peptides exhibit a distinct functional profile to the β-scorpion toxins, whereby the peptide traps the DIIS4 voltage sensor in the closed state resulting in an inhibition of peak current [245,255,256]. Although a few of the jingzhaotoxins (Chilobrachys jingzhao) so far discovered have been shown to inhibit Na V current by acting as gating modifiers [244,[257][258][259], only one -β/κ-TRTX-Cj1a (jingzhaotoxin-III) -has so far demonstrated direct binding to the site 4 DIIS3-S4 loop [245].…”

Section: Site 3 and Sitementioning

confidence: 99%

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Discovery and characterization of NaV modulatory venom peptides

Wingerd¹

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Voltage-gated sodium channels (Na V ) are integral membrane proteins that are responsible for the increase in sodium permeability that initiates and propagates the rising phase of action potentials, carrying electrical signals along nerve fibers and through excitable cells. Na V channels play a diverse role in neurophysiology and neurotransmission, as well as serving as molecular targets for several groups of neurotoxins that bind to different receptor sites and alter voltage-dependent activation, inactivation and conductance. There are nine Na V channel isoforms so far discovered, each of which display distinct functional profiles and tissue-specific expression patterns. The modulation of specific isoforms for therapeutic purposes has become an important research objective for the treatment of conductance diseases exhibiting phenotypes of chronic pain, epilepsy, myotonia, seizure, and cardiac arrhythmia. However, because of the high sequence similarity and structural homology between Na V channel isoforms, many current therapeutics that target Na V channels -the vast majority of which are small molecules -lack specificity between isoforms, or even other voltage-gated ion channels. The current push for greater selectivity while maintaining a relevant degree of potency has led the focus away from small molecules and towards the discovery and development of peptidic ligands for therapeutic use. Venom derived peptides have proven to be naturally potent and selective bioactive molecules, exhibiting inherent secondary structures that add stability through the formation of disulfide bonds. Organisms such as cone snails and spiders have evolved venom for the purpose of prey capture and defense, therefore many of the components exhibit paralytic qualities and specifically target Na V channels. Much of the discovery process has focused on screening crude venoms for a particular function and then isolating the molecule responsible for that function using assay guided purification.The first section of this thesis describes the development of a cell-based, high-throughput functional assay for the detection of Na V modulating venom peptides in crude venom. This assay was successfully implemented and resulted in the isolation and sequencing of MVIA from Conus magus. Initial results and sequence similarity placed MVIA into the δ-conotoxin family, a poorly described class of peptides that inhibits fast inactivation. However, multiple solid-phase synthesis and bacterial recombinant expression methods were unsuccessful in producing enough of the extremely hydrophobic δ-MVIA for further characterization. As no more C. magus crude venom was available, this project was left until optimized methods of production for highly hydrophobic peptides could be developed.ii An optimized method of bacterial recombinant expression was successful in producing large yields of another venom peptide, β/δ-TRTX-Pre1a, isolated from the spider Psalmopoeus reduncus. The same recombinant expression method was also used to produce uniformly label...

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“…However, its efficacy at Na V 1.6 was not examined 230 . Similarly, hainantoxin-IV was found to inhibit TTX-s rat Na V isoforms 231 and was shown to inhibit Na V 1.1, 1.2, and 1.7, while having no effect over Na V 1.4 and 1.5 232 .…”

Section: Pme1a Exhibits Agonistic Activity Towards Trpv1 and Induces mentioning

confidence: 99%

A pharmacological and transcriptomic approach to exploring novel pain targets

Yin¹

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Ever since the discovery that mutations in the voltage-gated sodium channel 1.7 protein are responsible for human congenital insensitivity to pain, the voltage-gated sodium channel (Na V ) family of ion channels has been the subject of intense research with the hope of discovering novel analgesics. We now know that Na V 1.7 deletion in select neuronal populations yield different phenotypes, with the deletion of Na V 1.7 in all sensory neurons being successful at abolishing mechanical and heat-induced pain. However, it is rapidly becoming apparent that a number of pain syndromes are not modulated by Na V 1.7 at all, such as oxaliplatin-mediated neuropathy. It is particularly interesting to note that the loss of Na V 1.7 function is also associated with the selective inhibition of pain mediated by specific stimuli, such as that observed in burn-induced pain where Na V 1.7 gene knockout abolished thermal allodynia but did not affect mechanical allodynia. Accordingly, significant interests exist in delineating the contribution of other Na V isoforms in modality-specific pain pathways. Two other isoforms, Na V 1.6 and Na V 1.8, are now specifically implicated in some Na V 1.7-independent conditions such as oxaliplatin-induced cold allodynia. Such selective contributions of specific ion channel isoforms to pain highlight the need to discover other putative protein targets involved in mediating nociception.The aim of my work is therefore to discover selective molecular inhibitors of Na V 1.6 and Na V 1.8, to find useful cell models for peripheral nociceptors, to investigate the roles of Na V 1.6 and Na V 1.8 in an animal model of burn-induced pain, and to screen for putative new targets for analgesia in burn-related pain.Chapter 2 of this thesis describes activity-guided discovery of novel Na V 1.6 and Na V 1.8-modulting peptides from crude spider venoms. Crude venom from Poecilotheria metallica successfully reduced Na V 1.8-mediated voltage changes in HEK293-expressing cells. A new Na V 1.8-inhibitory peptide was sequenced from the venom and named Pme1a. However, Pme1a exhibited TRPV1 agonist activity and induced nocifensive behaviours, such as paw licking, after injection into the hind paws of mice, indicating the peptide was not a selective Na V 1.8 modulator and was unsuitable as a tool for Na V 1.8 investigation.iii With the unsuccessful exploration of spider venoms for new specific inhibitors, I then investigated in vitro cell models as tools to research specific nociceptor subtypes that express Na V 1.6 or Na V 1.8. In Chapter 3, I provide the first complete transcriptome of three common in vitro neuronal cell lines (SH-SY5Y, F-11, and ND7/23) to examine whether they were appropriate for investigating the functions of Na V 1.6 and Na V 1.8, and to identify if the cell lines resemble in vivo dorsal root ganglion (DRG) neuronal subclasses. The three cell lines examined all expressed proteins belonging to similar pathways and of similar proportions to native DRG neurons. However, they did not express any ce...

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Structure and Function of Hainantoxin-III, a Selective Antagonist of Neuronal Tetrodotoxin-sensitive Voltage-gated Sodium Channels Isolated from the Chinese Bird Spider Ornithoctonus hainana

Cited by 57 publications

References 56 publications

Development of a μO-Conotoxin Analogue with Improved Lipid Membrane Interactions and Potency for the Analgesic Sodium Channel NaV1.8

Development of a μO-Conotoxin Analogue with Improved Lipid Membrane Interactions and Potency for the Analgesic Sodium Channel NaV1.8

Discovery and characterization of NaV modulatory venom peptides

A pharmacological and transcriptomic approach to exploring novel pain targets

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