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European Journal of 582 (2008) 12–16 www.elsevier.com/locate/ejphar

Inhibition of insect channels by -V, a from Chinese tarantula Ornithoctonus huwena ⁎ 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 channels. Whole- patch-clamp configuration indicated that huwentoxin-V specifically inhibited high-voltage-activated calcium channels in cockroach dorsal unpaired median (IC50 ≈219 nM) while having no evident effect on voltage-gated potassium and sodium channels. Omega- GVIA is a well-known neuronal N-type 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 , 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 ), 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 cells. In channels also have been found to exist in dorsal unpaired median 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% 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 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 is composed of 35 residues and its six 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 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 , 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 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 . 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 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, 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. The maximal change and reverse-phase high-performance liquid chromato- inhibition (~81.1±2.3%, n=5) could be achieved within 2 min graphy (RP-HPLC) as described earlier (Zhang et al., 2003). after treated by 10 μM huwentoxin-V (Fig. 2E). When the The purity of huwentoxin-V was over 98%, as judged by RP- dose–response data was fit to a Hill equation, the yielded IC50 HPLC and mass spectrometry analysis (data not shown). value and Hill coefficient values were 219±4 nM and 0.9, Sequence alignment indicated that huwentoxin-V shared 65%, respectively. (Fig. 2F). The Hill coefficient was close to 1, 62% and 57% sequence identities with other known tarantula suggesting that huwentoxin-V should interact with HVA cal- toxins such as ProTx-I, HmTX-I and Ccotoxin-III, respectively cium channels in a 1:1 manner on cockroach dorsal unpaired (Fig. 1). The six cysteine residues in huwentoxin-V also well median neurons. conserved at similar positions among these toxins. Omega-conotoxin GVIA is a well-characterized vertebrate neuronal N-type calcium channel blocker from the venom of sea 3.2. Effects of huwentoxin-V on voltage-gated calcium channels snail Conus geographus (Olivera et al., 1984; Olivera and Mcintosh, 1985). Wicher and Penzlin (1994). demonstrated that It is well known that there are two main categories of the toxin also could partially block calcium currents in voltage-gated calcium channels in rat dorsal root ganglion cockroach dorsal unpaired median neurons. In accordance neurons and cockroach dorsal unpaired median neurons: LVA with these findings, in our study, we found that 10 μM omega- and HVA calcium channels. LVA calcium channels can be conotoxin GVIA could completely block N-type calcium activated by a 100-ms depolarization of −20 mV from a holding channels on adult rat dorsal root ganglion neurons (data not potential (Vh) of −90 mV, while only HVA currents are elicited shown). The toxin at the same dose suppressed the HVA by a depolarization of 20 mV from a Vh of −50 mV (Linfis calcium currents on cockroach dorsal unpaired median neurons et al., 1989; Nowicky et al., 1985; Wheeler et al., 1994; Wicher only by 28.8±3.3% and no further blockade was detected at and Penzlin, 1997). higher concentrations. Diltiazem is another well-known calcium As shown in Fig. 2,1 μM huwentoxin-Vexhibited no evident channel blocker that not only completely inhibits vertebrate L- effects on LVA currents from both rat dorsal root ganglion and type channel but also partially depresses insect HVA calcium cockroach dorsal unpaired median neurons (Fig. 2A, C, n=4). channels (Wicher and Penzlin, 1997). In this study, 31.1±4.2% Only weak inhibition (15.2±3.1%, n=4) was detected on HVA calcium currents could be blocked on adult cockroach dorsal unpaired median neurons by 10 µM diltiazem and 100 µM diltiazem did not cause further blockade. Interestingly, 10 µM diltiazem (or 10 µM omega-conotoxin GVIA) could reduce 27–32% of the remaining HVA currents after pretreat- ment with omega-conotoxin GVIA (or with diltiazem). Even at a concentration of 50 μM, omega-conotoxin GVIA did not weaken the blocking potency of diltiazem (Fig. 3A, C, n=4). Similar to omega-conotoxin GVIA and diltiazem, huwentoxin- V also partially blocked insect HVA calcium channels. After treated by 100 µM huwentoxin-V, 19.1±2.1% of calcium currents remained available. The residual currents pretreated with 10 μM omega-conotoxin GVIA and 10 μM diltiazem were further inhibited by 80.9±2.1% when exposed to 10 μM huwentoxin-V (Fig. 3A, C, n=4). However, the remaining HVA currents pretreated with 10 µM huwentoxin-V were resistant to both omega-conotoxin GVIA and diltiazem (Fig. 3B). These findings indicated that huwentoxin-V might block Fig. 2. Effects of huwentoxin-Von voltage-gated calcium channels. A, LVAcalcium omega-conotoxin GVIA-sensitive, diltiazem-sensitive and par- μ channels were unaffected by 1 M huwentoxin-V on rat dorsal root ganglion tial currents resistant to both omega-conotoxin GVIA and neurons. B, 1 μM huwentoxin-V had weak effect on HVA calcium channels in rat dorsal root ganglion (DRG) neurons. C, 1 μM huwentoxin-V had no significant diltiazem in cockroach dorsal unpaired median cells. effect on LVA calcium channels in cockroach dorsal unpaired median (DUM) Steady-state activation of the insect calcium channels was neurons. D, 100 nM and 1 μM huwentoxin-V evidently reduced the control HVA studied by 100-ms depolarizing steps to various potentials from currents amplitude in dorsal unpaired median neurons by 29.3±3.6% and 65±3.9% a holding potential of −90 mV. Fig. 4B shows the conductance– μ (n=5).E,10 M huwentoxin-V eliminated the HVA calcium current on cockroach voltage (G–V) curves of HVA calcium channels from American dorsal unpaired median neurons and rat dorsal root ganglion neurons in a time- dependent manner. F, Dose-dependent blockage of HVA calcium channels by cockroach dorsal unpaired median neurons. Under control huwentoxin-Von cockroach dorsal unpaired median (DUM) neurons and rat dorsal conditions, half-maximal activation occurred at −13.9±0.3 mV root ganglion (DRG) neurons. (n=5). After 200 nM huwentoxin-V treatments for 3 min, the M. Deng et al. / European Journal of Pharmacology 582 (2008) 12–16 15

Fig. 5. Effects of huwentoxin-V on the voltage-gated potassium channels and sodium channels expressed in cockroach dorsal unpaired median neurons. A, potassium current traces were evoked by a 300-ms depolarization to 30 mV from a holding potential of −90 mV. 1 μM huwentoxin-V failed to affect on the

dorsal unpaired median neuron IK. B, sodium current traces were evoked by a 50-ms depolarization to −10 mV from a holding potential of −80 mV. 1 μM huwentoxin-V did not affect the amplitude of the sodium current on the dorsal unpaired median neuron.

Fig. 3. Effects of huwentoxin-V on the omega-conotoxin GVIA-sensitive, 3.3. Effects of huwentoxin-V on voltage-gated potassium diltiazem-sensitive currents and omega-conotoxin GVIA and diltiazem-resistant channels and sodium channels currents in cockroach dorsal unpaired median neurons. A, in the presence of 10 μM omega-conotoxin GVIA and 10 μM diltiazem, 10 μM huwentoxin-V has additional effect on the HVA calcium currents. B, 10 μM omega-conotoxin Multiple potassium and sodium channels express in both GVIA and 10 μM diltiazem have no effect on the huwentoxin-V-resistant vertebrate dorsal root ganglion neurons and insect dorsal unpaired currents. C, representative time courses of development from block by omega- median neurons and underlie spontaneous electrical activity. conotoxin GVIA, diltiazem and huwentoxin-V on HVA calcium channels in Blocking these channels can significantly affect neuronal cockroach dorsal unpaired median neurons. excitability (Matthew et al., 2001; Grolleau and Lapied, 1995). Tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels inhibition of currents could be observed at all test pulses, but co-express in adult rat dorsal root ganglion neurons (Xiao et al., half-maximal activation voltage was unchanged: −13.8±0.5 mV. 2005), whereas only tetrodotoxin-sensitive type is distributed in It implied that huwentoxin-V could only suppress peak currents cockroach dorsal unpaired median neurons (Lapied et al., 2001). without affecting the kinetics of HVA calcium channels on cock- Spider are demonstrated to contain many components roach dorsal unpaired median neurons. interacting with potassium and sodium channels. In order to determine the selectivity of huwentoxin-V toward ion channels, we further tested the potency of huwentoxin-Von potassium and sodium channels. As shown in Fig. 5, 1 μM huwentoxin-V failed to change the ion currents elicited from voltage-gated potassium channels (e.g. A-like or delayed rectifier types) and sodium channels on cockroach dorsal unpaired median neurons. No effect was detected on either potassium channels or sodium channels from vertebrate sensory neurons (data not shown).

4. Discussion

In the present study, we characterized the actions of huwentoxin-V, isolated from the venom of Chinese tarantula O. huwena, on ionic channels. It was found that the toxin showed no effect on voltage-gated potassium channels, sodium channels and LVA calcium channels from both cockroach dorsal unpaired median neurons and rat dorsal root ganglion neurons. Fig. 4. Effects of huwentoxin-Von the conductance–voltage (G–V) relationship of voltage-gated calcium channels in dorsal unpaired median neurons. A, family However, huwentoxin-V obviously depressed the amplitude of of currents was elicited by 100-ms depolarizing steps to various potentials from HVA calcium currents on cockroach dorsal unpaired median a holding potential of −90 mV. Test potentials ranged from −60 mV to 60 mVat neurons and it exhibited much higher affinity toward insect HVA increments of +10 mV. The G–V curve (B) of calcium currents showed the calcium channels than that of vertebrate isoforms. From the relationship between current traces before (above) and after (below) adding conductance–voltage (G–V) curves of insect HVA calcium 200 nM huwentoxin-V in A. Data were fit with a Boltzmann equation g/g = max currents (see Fig. 4), it has been found that HWTX-V can only [1+exp({V−Va}/k)]−1 where g/gmax is the normalized peak conductance occurring during a depolarization to the potential V, Va is the half-activation suppress peak currents without affecting the kinetics of HVA potential, and k is the slope factor for activation. calcium channels, which distinguishes HWTX-V from ω-AgaTx 16 M. Deng et al. / European Journal of Pharmacology 582 (2008) 12–16

IVA, a peptide isolated from the venom of the funnel-web spider Grolleau, F., Lapied, B., 1995. Separation and identification of multiple Agelenopsis aperta, inhibits voltage-gated calcium channels by potassium currents regulating the pacemaker activity of insect neurosecre- tory cells (DUM neurons). J. Neurophysiol. 73, 160–171. altering the voltage-dependency of gating (Mintz et al., 1992). Grolleau, F., Lapied, B., 1996. Two distincts low-voltage-activated Ca2+ currents Importantly, huwentoxin-V was found to block omega- contribute to the pacemaker mechanism in cockroach dorsal unpaired median conotoxin GVIA-sensitive, diltiazem-sensitive currents and even neurons. J. Neurophysiol. 76, 963–976. partial omega-conotoxin GVIA and diltiazem-resistant currents on Grolleau, F., Lapied, B., 2000. Dorsal unpaired median neurones in the insect cockroach dorsal unpaired median neurons. Therefore, based on the central : towards a better understanding of the ionic mechanisms underlying spontaneous electrical activity. J. Exp. Biol. 203 (11), 1633–1648. sensitivity of calcium currents to omega-conotoxin GVIA, King, G.F., 2007. Modulation of insect Cav channels by peptidic spider toxins. diltiazem and huwentoxin-V, cockroach dorsal unpaired median Toxicon. 49, 513–530. HVA calcium channels might be divided into four categories: Type Lapied, B., Grolleau, F., Sattelle, D.B., 2001. Indoxacarb, an oxadiazine , I, omega-conotoxin GVIA-sensitive and huwentoxin-V-sensitive; blocks insect neuronal sodium channels. British. J. Pharmacol. 132, 587–595. type II, diltiazem-sensitive and huwentoxin-V-sensitive; type III, Liang, S.P., 2004. 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