A Novel Peptide from Funnel Web Spider Venom, ω-Aga-TK, Selectively Blocks P-Type Calcium Channels

Teramoto, Takashi; Kuwada, Manabu; Niidome, Takuro; Sawada, Kouhei; Nishizawa, Yuji; Katayama, Kouichi

doi:10.1006/bbrc.1993.2225

Cited by 61 publications

(40 citation statements)

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“…Using this approach, both pore blockers and gating 20 modifiers of human sodium channels could be detected using fluorescence, providing a flexible, low cost alternative for the primary identification of novel Nav modulators.…”

Section: Discussionmentioning

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: Discussionmentioning

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 less specific, P/Q/N-type Ca 2ϩ -channel blocker -conotoxin MVIIC (1 M) (McDonough et al 1996) blocked 22.7 Ϯ 9.9% (n ϭ 7) of the total Ca 2ϩ current. Because the potent, reversible P/Q-type blocker -Aga-TK (50 nM) (Teramoto et al 1993) exerted no effect on I Ca amplitude in six cells tested (8.7 Ϯ 4.5%, P Ͼ 0.05), we concluded that both the -conotoxins (GVIA and MVIIC) acted on N-type channels. In support of this notion, GVIA treatment evoked an average block of the I Ca that was not significantly different from the MVIIC-evoked inhibition (P Ͼ 0.05).…”

Section: Pharmacological Identification Of the Voltage-gated Ca 2ϩ Cumentioning

confidence: 99%

Ionic Mechanisms Mediating Oscillatory Membrane Potentials in Wide-Field Retinal Amacrine Cells

Vı́gh

Solessio

Morgans³

et al. 2003

Journal of Neurophysiology

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Particular types of amacrine cells of the vertebrate retina show oscillatory membrane potentials (OMPs) in response to light stimulation. Historically it has been thought the oscillations arose as a result of circuit properties. In a previous study we found that in some amacrine cells, the ability to oscillate was an intrinsic property of the cell. Here we characterized the ionic mechanisms responsible for the oscillations in wide-field amacrine cells (WFACs) in an effort to better understand the functional properties of the cell. The OMPs were found to be calcium (Ca2+) dependent; blocking voltage-gated Ca2+ channels eliminated the oscillations, whereas elevating extracellular Ca2+ enhanced them. Strong intracellular Ca2+ buffering (10 mM EGTA or bis-( o-aminophenoxy)- N,N,N′ ,N′-tetraacetic acid) eliminated any attenuation in the OMPs as well as a Ca2+-dependent inactivation of the voltage-gated Ca2+ channels. Pharmacological and immunohistochemical characterization revealed that WFACs express L- and N-type voltage-sensitive Ca2+ channels. Block of the L-type channels eliminated the OMPs, but ω-conotoxin GVIA did not, suggesting a different function for the N-type channels. The L-type channels in WFACs are functionally coupled to a set of calcium-dependent potassium ( K(Ca)) channels to mediate OMPs. The initiation of OMPs depended on penitrem-A-sensitive (BK) K(Ca) channels, whereas their duration is under apamin-sensitive (SK) K(Ca) channel control. The Ca2+ current is essential to evoke the OMPs and triggering the K(Ca) currents, which here act as resonant currents, enhances the resonance as an amplifying current, influences the filtering characteristics of the cell membrane, and attenuates the OMPs via CDI of the L-type Ca2+ channel.

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“…Cells dissected from anterior pituitary were pre-treated with E 2 for 48 h, and the efficacy of GnRH-induced LH release was measured in the presence or absence of specific blockers of the chosen channel proteins. u-Agatoxin TK was used to selectively block Ca v 2.1 (P/Q type) channels (Teramoto et al 1993), r-Kurtoxin to (nZ3; *P!0 . 05; **P!0 .…”

Section: Functional Validation Of Era-regulated Genes In the Primary mentioning

confidence: 99%

Identification of estradiol/ERα-regulated genes in the mouse pituitary

Kim¹,

Gieske²,

Trudgen³

et al. 2011

Journal of Endocrinology

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Estrogen acts to prime the pituitary prior to the GnRHinduced LH surge by undiscovered mechanisms. This study aimed to identify the key components that mediate estrogen action in priming the pituitary. RNA extracted from the pituitaries of metestrous (low estrogen) and proestrus (high estrogen) stage mice, as well as from ovariectomized wildtype and estrogen receptor a (ERa) knockout mice treated with 17b-estradiol (E 2 ) or vehicle, was used for gene expression microarray. Microarray data were then aggregated, built into a functional electronic database, and used for further characterization of E 2 /ERa-regulated genes. These data were used to compile a list of genes representing diverse biological pathways that are regulated by E 2 via an ERa-mediated pathway in the pituitary. This approach substantiates ERa regulation of membrane potential regulators and intracellular vesicle transporters, among others, but not the basic components of secretory machinery. Subsequent characterization of six selected genes (Cacna1a, Cacna1g, Cited1, Abep1, Opn3, and Kcne2) confirmed not only ERa dependency for their pituitary expression but also the significance of their expression in regulating GnRH-induced LH secretion. In conclusion, findings from this study suggest that estrogen primes the pituitary via ERa by equipping pituitary cells with critical cellular components that potentiate LH release on subsequent GnRH stimulations.

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A Novel Peptide from Funnel Web Spider Venom, ω-Aga-TK, Selectively Blocks P-Type Calcium Channels

Cited by 61 publications

References 0 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

Ionic Mechanisms Mediating Oscillatory Membrane Potentials in Wide-Field Retinal Amacrine Cells

Identification of estradiol/ERα-regulated genes in the mouse pituitary

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