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Inhibition of Insect Calcium Channels by Huwentoxin-V, a Neurotoxin From Available online at www.sciencedirect.com European Journal of Pharmacology 582 (2008) 12–16 www.elsevier.com/locate/ejphar Inhibition of insect calcium channels by huwentoxin-V, a neurotoxin from Chinese tarantula Ornithoctonus huwena venom ⁎ Meichun Deng, Xuan Luo, Er Meng, Yucheng Xiao, Songping Liang College of Life Sciences, Hunan Normal University, Changsha 410081, China Received 9 May 2007; received in revised form 2 December 2007; accepted 16 December 2007 Available online 27 December 2007 Abstract The effects of huwentoxin-V, an insect neurotoxic peptide from Chinese tarantula Ornithoctonus huwena venom, were studied on neuronal voltage-gated ion channels. Whole-cell patch-clamp configuration indicated that huwentoxin-V specifically inhibited high-voltage-activated calcium channels in adult cockroach dorsal unpaired median neurons (IC50 ≈219 nM) while having no evident effect on voltage-gated potassium and sodium channels. Omega-conotoxin GVIA is a well-known neuronal N-type calcium channel blocker from the venom of the sea snail Conus geographus and it also can partially block calcium currents in cockroach dorsal unpaired median neurons. In our study, huwentoxin-V inhibited omega-conotoxin GVIA-sensitive, diltiazem-sensitive and partial omega-conotoxin GVIA and diltiazem-resistant calcium currents elicited from insect neurons. Based on the sensitivity of calcium currents to these toxins, insect neuronal HVA calcium channels might be classified into four types: Type I, omega-conotoxin GVIA-sensitive and huwentoxin-V-sensitive; type II, diltiazem-sensitive and huwentoxin-V-sensitive; type III, huwentoxin-V-sensitive but omega-conotoxin GVIA and diltiazem-resistant; type IV, omega-conotoxin GVIA and diltiazem-resistant and huwentoxin-V-resistant. Its specificity, incomplete inhibition and insect-toxic effects make it an interesting tool for investigating insect voltage- gated calcium channels and development of new insecticidal compounds. © 2008 Published by Elsevier B.V. Keywords: Huwentoxin-V; Insect; Calcium channel; Whole-cell; Patch-clamp 1. Introduction particular, long-lasting HVA currents (L-type-like currents) in insect neurons are blocked by phenylalkylamines (as in Ca2+entry into the cells through voltage-gated Ca2+channels vertebrates), but not by dihydropyridines (unlike in vertebrates) mediates many physiological processes including neurotrans- (Wicher and Penzlin, 1997). Voltage-gated calcium currents in mitter release, neurosecretion, neuronal excitation, survival of the somata of insect dorsal unpaired median neurons are found neurons, and regulation of gene expression. Currently, there are to be necessary for maintaining repetitive activity because the at least five types of voltage-gated calcium channels that have repetitive activity disappeared in Ca2+-free bath solution been found and functionally characterized from vertebrates. (Wicher and Penzlin, 1994). Using a variety of criteria including According to their distinct properties and kinetics, they can be ionic selectivity, voltage and ionic sensitivity, pharmacological divided into two groups: low-voltage-activated (LVA) calcium profile and sensitivity to physiological ligands, voltage-gated channels (T-type) and high-voltage-activated (HVA) channels calcium channels have been classified into several distinct (L-, N-, P/Q- and R-type channels) (Nowicky et al., 1985; Linfis classes including most notably low-voltage-activated and high- et al., 1989; Zhang et al., 1993; Wheeler et al., 1994). voltage-activated calcium channels. Moreover, the subtypes Calcium channels from insect neurons are demonstrated to maintained LVA calcium channels and M-LVA calcium have properties different from those in vertebrate cells. In channels also have been found to exist in dorsal unpaired median neuron somata (Grolleau and Lapied, 1996; Grolleau ⁎ Corresponding author. Tel.: +86 731 8872556; fax: +86 731 8861304. and Lapied, 2000). Toxins exhibiting high selectivity to calcium E-mail address: [email protected] (S. Liang). channel isoforms have proved to be crucial for the identification 0014-2999/$ - see front matter © 2008 Published by Elsevier B.V. doi:10.1016/j.ejphar.2007.12.014 M. Deng et al. / European Journal of Pharmacology 582 (2008) 12–16 13 of calcium channels (King, 2007). There are many peptide cockroaches (P. Americana) were killed in 75% alcohol and toxins have been found to act on vertebrate calcium channels, washed in double-distilled water. After wiping off the enteron, such as huwentoxin-X, omega-conotoxin CVID, omega-con- the fifth abdominal ganglia were excised, digested and otoxin MVIIA, and so on (Liu et al., 2006). However, only a incubated for 3 min at room temperature in saline solution (in few peptide toxins have been reported to be able to target mM): 200 NaCl, 3 KCl, 5 CaCl2, 4 MgCl2, 50 sucrose and 10 calcium channels in cockroach dorsal unpaired median neurons, HEPES at pH 7.4, with 0.5 mg/ml trypsin (type III) and 0.5 mg/ml such as ω-AgaTx IVA, isolated from the venom of spider collagenase (type IA). The enzymatic digestion was stopped in Agelenopsis aperta, and ω-conotoxin MVIIC, isolated from the 35-mm-culture dishes with culture medium containing 200 NaCl, venom of snail Conus magus (Wicher and Penzlin, 1997). 3KCl,5CaCl2, 4 MgCl2, 50 sucrose and 10 HEPES, 5% fetal Huwentoxin-V is an insect neurotoxic peptide isolated from bovine serum, penicillin and streptomycin at pH 6.8. The cells the venom of Chinese tarantula Ornithoctonus huwena. The were incubated in CO2 incubator (5% CO2,28°C)for2–3h toxin is composed of 35 amino acid residues and its six cysteine before the patch-clamp experiment. residues are all involved in forming three disulfide bridges (I–IV, Rat dorsal root ganglion neurons were acutely dissociated II–V, III–VI) (Fig. 1)(Liang, 2004). As revealed in our previous and maintained in a short-term primary culture according to the work, this toxin can paralyze locusts and cockroaches and can procedures adapted from Xiao et al. (2005). Briefly, 30-day cause a dose-dependent reversible paralysis after intra-peritoneal adult Sprague–Dawley rats of either sex were killed by CO2 injection (Zhang et al., 2003). In this study, we describe the effect anesthesia followed by decapitation, the dorsal root ganglia of huwentoxin-V on voltage-gated ion channels (including were removed quickly from the spinal cord, and then they were calcium, potassium and sodium channels) expressed in both transferred into Dulbecco's modified Eagle's medium (DMEM) cockroach dorsal unpaired median neurons and rat dorsal root containing trypsin (0.5 g/l, type III), collagenase (1.0 g/l, type ganglion neurons. IA) to incubate at 34 °C for 30 min. Trypsin inhibitor (1.5 g/l, type II-S) was used to terminate enzyme treatment. The isolated 2. Materials and methods dorsal root ganglion cells were suspended in essential DMEM medium supplemented with 10% newborn calf serum and 2.1. Animals and materials NaHCO3 (3.6 g/l). The cells were plated in 35-mm-culture dishes (Corning, Sigma) and incubated in CO2 incubator (5% Adult Sprague–Dawley rats and American cockroaches were CO2, 37 °C). Cells were incubated for 3–24 h before the patch- from our laboratory stock. Omega-conotoxin GVIA, tetrodo- clamp experiment. toxin, trypsin (type III), collagenase (type IA), Dulbecco's modified Eagle's medium, ATP-Mg, and EGTA were purchased 2.3. Electrophysiological recordings from Sigma Chemical Co., USA. All reagents were of analytical grade. Calcium currents were recorded using an EPC-9 patch-clamp amplifier (HEKA, Germany) under whole-cell patch-clamp 2.2. Cell preparation configuration at room temperature (22–25 °C). The resistances of recording pipettes were 2.0–3.0 MΩ when filled with Cockroach dorsal unpaired median neurons were acutely internal solution containing (in mM): 140 CsCl, 2 MgC12,2 dissociated and maintained in a short-term primary culture as ATP-Mg, 10 HEPES and 1.5 EGTA at pH 7.2. The culture described by Wicher and Penzlin (1997).Briefly,adult media was replaced with an external solution containing (in mM): 150 Choline Chloride, 2 MgC12, 10 BaC12, 10 HEPES, 25 TEA and 1 tetrodotoxin at pH 7.4. Ba2+was used as the charge carrier. After the establishment of the whole-cell con- figuration, the currents were monitored for 20 min. The cells showing noticeable run-down currents during this period were discarded. Series resistance was kept near 5 MΩ and com- pensated by 65–70%. Linear capacitance and leakage currents were digitally subtracted using P/4 protocol. Experimental data were acquired and analyzed by the program Pulse-Pulsefit8.0 (HEKA, Germany) and Sigmaplot (Sigma, USA). To construct conductance–voltage curves, the Fig. 1. Sequence alignment of huwentoxin-V with some other toxins from the spiders. Identical amino acids are shown in gray, the sequence length, the conductance (G) as a function of test potential (Vtest) was percentage identity (I %) and bioactivity are shown to the right of the sequences. calculated as The disulfide linkage (I–IV, II–V, III–VI) indicated below the sequences. m huwentoxin-V, from Ornithoctonus huwena (Zhang et al., 2003); ProTx-I, from G ¼ I=ðÞVtest À VR Thrixopelma pruriens and (Middleton et al., 2002); HmTX-I, from Heteroscodra maculate (Escoubas et al., 2002); Ccotoxin-III, from Ceratogyrus cornuatus (Bosmans et al., 2005); SGTX-I, from Scodra grisepes (Marvin et al., 1999); Where I is the peak current generated by the step SNX-482, from Hysterocrates gigas (Newcomb et al., 1998); HaTx1/HaTx2, depolarization to Vtest, and VR is reversal potential determined from Grammostola spatulata (Swartz and MacKinnon, 1995). from the I–V relationship. 14 M. Deng et al. / European Journal of Pharmacology 582 (2008) 12–16 3. Results mammalian neuronal HVA calcium currents (Fig. 2B, n=5). However, application of 0.1 or 1 μM huwentoxin-V rapidly 3.1. Toxin purification and sequence similarity inhibited HVA calcium currents on cockroach dorsal unpaired median neurons (Fig. 2D, n=5). The inhibition was in a time- Huwentoxin-V was purified by a combination of ion-ex- dependent and concentration-dependent manner.
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