Novel ω-Conotoxins from Conus catus Discriminate among Neuronal Calcium Channel Subtypes

Lewis, Richard J.; Nielsen, Katherine J.; Craik, David J.; Loughnan, Marion L.; Adams, Denise A.; Sharpe, Iain A.; Luchian, Tudor; Adams, David J.; Bond, Trudy; Thomas, Linda; Jones, Ashley; Matheson, J; Drinkwater, R. D.; Andrews, P. R.; Alewood, Paul F.

doi:10.1074/jbc.m002252200

Cited by 211 publications

(230 citation statements)

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“…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%

“…To assess which voltage-gated calcium channels contribute to the depolarization-induced Ca 2+ influx after addition of veratridine, we assessed the effects of nifedipine to block L-type voltagegated calcium channels (VGCC) [19], ω-conotoxin CVID to block N-type VGCC [7], and ω-agatoxin TK to block P/Q-type VGCC [20], on the veratridine-induced responses (Fig 4 C and D). Pre-treatment with nifedipine concentration-dependently inhibited veratridine-induced responses with an IC50 of 10.7 nM (pIC50 7.97 ± 0.2) (Fig 4 C).…”

Section: L-type and N-type Calcium Channels Contribute To The Veratrimentioning

confidence: 99%

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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: Methodsmentioning

confidence: 99%

Section: L-type and N-type Calcium Channels Contribute To The Veratrimentioning

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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“…The resulting pellet was resuspended in TEM buffer containing 10% glycerol. Rat brain homogenates were prepared as described previously (17). Protein concentration was determined using the BCA protein assay kit (Pierce, Rockford, IL) following the manufacturer's protocol.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of the Norepinephrine Transporter by the Venom Peptide χ-MrIA

Sharpe

Palant²,

Schroeder

et al. 2003

Journal of Biological Chemistry

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-Conopeptide MrIA (-MrIA) is a 13-residue peptide contained in the venom of the predatory marine snail Conus marmoreus that has been found to inhibit the norepinephrine transporter (NET). We investigated whether -MrIA targeted the other members of the monoamine transporter family and found no effect of the peptide (100 M) on the activity of the dopamine transporter and the serotonin transporter, indicating a high specificity of action. The binding of the NET inhibitors, [ Because of its poor lipid solubility and degree of ionization at physiological pH, norepinephrine crosses cell membranes poorly by diffusion (1) and so relies on the operation of the norepinephrine transporter (NET) 1 for uptake into cells. Clearance by this integral membrane protein constitutes the major mechanism for the termination of action of this neurotransmitter at noradrenergic synapses (2), and disturbances in the functioning of the NET are associated with pathological states including depression (3), congestive heart failure (4), and orthostatic intolerance, and tachycardia (5). Known inhibitors of the NET include antidepressants (e.g. desipramine and nisoxetine), the appetite suppressant mazindol, and the abused drug cocaine (for review, see Ref. 6). The NET, together with the dopamine transporter (DAT) and the serotonin transporter (SERT), forms a family of Na ϩ -and Cl

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“…Isolation and Sequencing-Specimens of Conus tulipa were collected from the Great Barrier Reef, Australia. Venom ducts were dissected, and crude venom prepared as previously described (15). Initial fractionation of the venom was carried out by RP-HPLC, and fractions were stored at 4°C until further use.…”

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confidence: 99%

Conopressin-T from Conus tulipa Reveals an Antagonist Switch in Vasopressin-like Peptides

Dutertre

Croker²,

Daly

et al. 2008

Journal of Biological Chemistry

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We report the discovery of conopressin-T, a novel bioactive peptide isolated from Conus tulipa venom. Conopressin-T belongs to the vasopressin-like peptide family and displays high sequence homology to the mammalian hormone oxytocin (OT) and to vasotocin, the endogenous vasopressin analogue found in teleost fish, the cone snail's prey. Conopressin-T was found to act as a selective antagonist at the human V 1a receptor. All peptides in this family contain two conserved amino acids within the exocyclic tripeptide (Pro 7 and Gly 9 ), which are replaced with Leu 7 and Val 9 in conopressin-T. Whereas conopressin-T binds only to OT and V 1a receptors, an L7P analogue had increased affinity for the V 1a receptor and weak V 2 receptor binding. Surprisingly, replacing Gly 9 with Val 9 in OT and vasopressin revealed that this position can function as an agonist/antagonist switch at the V 1a receptor. NMR structures of both conopressin-T and L7P analogue revealed a marked difference in the orientation of the exocyclic tripeptide that may serve as templates for the design of novel ligands with enhanced affinity for the V 1a receptor.The vasopressin (AVP) 3 and oxytocin (OT) peptides were originally discovered and identified as neurohypophysial hormones in mammals (1). In humans, AVP acts via three vasopressin receptors (vascular V 1a R, pituitary V 1b R, and renal V 2 R), whereas OT acts via one OT receptor (OTR). All targets are members of the G protein-coupled receptor family (2). Peripherally, they regulate water balance, the control of blood pressure, and contraction of uterine smooth muscle and mammary myoepithelium (3). Centrally, these peptides affect levels of aggression, depression, and young parent bonding (4 -6). Endogenous analogues of OT and AVP have been reported in nonmammalian vertebrates, annelids, molluscs, and insects, suggesting an old lineage for these peptides (7). Surprisingly, two variants were also found in the venom of predatory cone snails. The original discovery of these two AVP analogues, named conopressins, was based on the characteristic "scratching" effect observed upon intracerebral injection into mice (8). Although the sequences of conopressins are similar to vasopressin itself, they have an additional positive charge in position 4, which is only found in two other endogenous vasopressin analogues, cephalotocin (Octopus vulgaris) and annetocin (Eisenia foetida). Conopressin-S was isolated from Conus striatus, whereas conopressin-G was first isolated from Conus geographus venom but later found in Conus imperialis venom as well as in tissue extracts of the nonvenomous snails Lymnea stagnalis and Aplysia californica and the leech Erpobdella octoculata (9 -11).Molluscs of the genus Conus produce bioactive peptides in a combinatorial fashion. As demonstrated for the snake toxins (12), most conotoxins or conopeptides are believed to be derived from an endogenous structural template (13). Because conopressin-G is widely distributed, it may represent the endogenous hormone in Gastropods and Annelid...

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Novel ω-Conotoxins from Conus catus Discriminate among Neuronal Calcium Channel Subtypes

Cited by 211 publications

References 56 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

Inhibition of the Norepinephrine Transporter by the Venom Peptide χ-MrIA

Conopressin-T from Conus tulipa Reveals an Antagonist Switch in Vasopressin-like Peptides

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