Isolation and Structure-Activity of μ-Conotoxin TIIIA, A Potent Inhibitor of Tetrodotoxin-Sensitive Voltage-Gated Sodium Channels

Lewis, Richard J.; Schroeder, Christina I.; Ekberg, Jenny; Nielsen, Katherine J.; Loughnan, Marion L.; Thomas, Linda; Adams, Denise A.; Drinkwater, R. D.; Adams, David J.; Alewood, Paul F.

doi:10.1124/mol.106.028225

Cited by 60 publications

(85 citation statements)

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“…Although Nav1.2 was not the most abundantly expressed Nav isoform in SH-SY5Y cells as determined by RT-PCR, the Nav1.2/1.4-selective blocker TIIIA [8] reduced veratridine-induced responses by 42.6 ± 6.8 % (Fig 4 E; (Fig 4 F).…”

Section: Nav Subtypes Contributing To the Veratridine-induced Responsementioning

confidence: 90%

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Characterisation of Nav types endogenously expressed in human SH-SY5Y neuroblastoma cells

Vetter

Mozar

Durek

et al. 2012

Biochemical Pharmacology

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Abbreviations: Nav, voltage-gated sodium channel; TTX, tetrodotoxin; ProTxII, β/ω-theraphotoxin-Tp2a; Ca 2+ , calcium ion; DMEM, Dulbecco's Modified Eagle's Medium; FBS, foetal bovine serum; PBS, phosphate buffered saline; PSS, physiological salt solution; HBS, HEPES-buffered saline; AFU, arbitrary fluorescence unit; SEM, standard error of the mean; Fluo-4 AM, Fluo-4 acetoxymethylester; RPMI, Roswell Park Memorial Institute; BSA, bovine serum albumin; CCD, charge-coupled device; DAPI, 4',6-diamidino-2-phenylindole 2 AbstractThe human neuroblastoma cell line SH-SY5Y is a potentially useful model for the identification and characterisation of Nav modulators, but little is known about the pharmacology of their endogenously expressed Navs. The aim of this study was to determine the expression of endogenous Nav α and β subunits in SH-SY5Y cells using PCR and immunohistochemical approaches, and pharmacologically characterise the Nav isoforms endogenously expressed in this cell line using electrophysiological and fluorescence approaches. SH-SY5Y human neuroblastoma cells were found to endogenously express several Nav isoforms including Nav1.2 and Nav1.7. Activation of endogenously expressed Navs with veratridine or the scorpion toxin OD1 caused membrane depolarization and subsequent Ca 2+ influx through voltage-gated L-and N-type calcium channels, allowing Nav activation to be detected with membrane potential and fluorescent Ca 2 dyes. -Conotoxin TIIIA and ProTxII identified Nav1.2 and Nav1.7 as the major contributors of this response. The Nav1.7-selective scorpion toxin OD1 in combination with veratridine produced a Nav1.7-selective response, confirming that endogenously expressed human Nav1.7 in SH-SY5Y cells is functional and can be synergistically activated, providing a new assay format for ligand screening.Key Words: SH-SY5Y; Ca 2+ ; Nav1.7; ProTxII; OD1 3 IntroductionVoltage-gated sodium channels (Nav) are complex transmembrane proteins comprised of a poreforming α subunit and accessory β subunits that play an essential role in the initiation and propagation of action potentials in excitable cells. To date, apart from the related Nax which appears to function as a sodium sensor [1,2], nine isoforms termed Nav1.1 -Nav1.9 have been functionally defined as sodium-selective ion channels [3]. Their distinct tissue distribution and amenability to modulation by toxins and drugs has led to significant interest in Nav as therapeutic targets in a number of poorly treated conditions ranging from epilepsy to cardiac arrhythmias and pain [4]. In recent years Nav1.7 has emerged as an attractive drug target, with expression restricted to a subset of nociceptive neurons that is expected to limit on-target side effects of pharmacological modulators of Nav1.7 [5]. In addition, loss-of-function mutations in humans have highlighted the possibility that Nav1.7 inhibition could produce complete loss of pain sensations without dose-limiting side effects [6]. However, it remains unclear if such an effect can be translated to the clinic...

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Section: Nav Subtypes Contributing To the Veratridine-induced Responsementioning

confidence: 90%

“…Synthetic CVID, TIIIA, GIIIA were obtained through total synthesis as described previously [7][8][9]. Scorpion toxin OD1 was prepared by a combination of solid-phase peptide synthesis and chemical ligation [10].…”

Section: Methodsmentioning

confidence: 99%

Characterisation of Nav types endogenously expressed in human SH-SY5Y neuroblastoma cells

Vetter

Mozar

Durek

et al. 2012

Biochemical Pharmacology

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“…More recently, μ-conotoxins active at neuronal subtypes in addition to Na v 1.4 have been identified, including PIIIA and TIIIA which inhibit Na v 1.2 (Shon et al 1998a;Lewis et al 2007), and KIIIA which inhibits Na v 1.2, 1.6, 1.7 (Zhang et al 2007). However, despite subtype selective sodium channel inhibitors having considerable therapeutic potential, little progress has been made towards the development of μ-conotoxin inhibitors for specific therapeutically relevant VGSCs, including Na v 1.3, 1.6, 1.7 and 1.8.…”

Section: M-conotoxinsmentioning

confidence: 99%

“…Residues are numbered according to their individual primary sequence (see Table 3). Reproduced from Lewis et al (2007) with permission Table 4 Selected μO-and δ-conotoxin modulators of VGSCs…”

Section: O-conotoxinsmentioning

confidence: 99%

Developmental Biology of Neoplastic Growth

Macieira‐Coelho¹

2005

Progress in Molecular and Subcellular Biology

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“…GIIIA also exhibits different effects in different species, with the human Na V 1.4 isoform being resistant to GIIIA-mediated inhibition 130 despite the high potency of the toxin in the rat Na V 1.4 isoform 130 . In order to examine the effect of Na V 1.6, GIIIA is administered together with TIIIA, another conotoxin-derived peptide that inhibits Na V 1.1, 1.2, 1.3, and Na V 1.4 212 , but not Na V 1.6. The differential inhibitory activity of the two peptides allows for assessment of the effect of Na V 1.6 inhibition independent of Na V 1.1, Na V 1.2, and Na V 1.4 inhibition in animal models 2,133,200 .…”

Section: Introductionmentioning

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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Isolation and Structure-Activity of μ-Conotoxin TIIIA, A Potent Inhibitor of Tetrodotoxin-Sensitive Voltage-Gated Sodium Channels

Cited by 60 publications

References 40 publications

Characterisation of Nav types endogenously expressed in human SH-SY5Y neuroblastoma cells

Characterisation of Nav types endogenously expressed in human SH-SY5Y neuroblastoma cells

Developmental Biology of Neoplastic Growth

A pharmacological and transcriptomic approach to exploring novel pain targets

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