C 2006) Cellular and Molecular Neurobiology, Vol. 27, No. 1, January 2007 ( DOI: 10.1007/s10571-006-9123-z
Leftward Shift in the Voltage-Dependence for Ca2+ Currents Activation Induced by a New Toxin from Phoneutria reidyi (Aranae, Ctenidae) Venom L. B. Vieira,1,8 A. M. C. Pimenta,2,3,8,9 M. Richardson,4 M. P. Bemquerer,2 H. J. Reis,1 J. S. Cruz,5 M. V. Gomez,1 M. M. Santoro,3 R. Ferreira-de-Oliveira,4 S. G. Figueiredo,6 T. P. Snutch,7 and M. N. Cordeiro4 Received April 3, 2006; accepted September 27, 2006; Published online: December 7, 2006 SUMMARY Various neurotoxins have been described from the venom of the Brazilian spider Phoneutria nigriventer, but little is known about the venoms of the other species of this genus. In the present work, we describe the purification and some structural and pharmacological features of a new toxin (PRTx3-7) from Phoneutria reidyi that causes flaccid paralysis in mice. The observed molecular mass (4627.26 Da) was in accordance with the calculated mass for the amidated form of the amino acid sequence (4627.08 Da). The presence of an α-amidated C-terminus was confirmed by MS/MS analysis of the C-terminal peptide, isolated after enzymatic digestion of the native protein with Glu-C endoproteinase. The purified protein was injected (intracerebro-ventricular) into mice at dose levels of 5 µg/mouse causing immediate agitation and clockwise gyration, followed by the gradual development of general flaccid paralysis. PRTx3-7 at 1 µM inhibited by 20% the KCl-induced increase on [Ca2+ ]i in rat brain synaptosomes. The HEK cells permanently expressing L, N, P/Q and R HVA Ca2+ channels were also used to better characterize the pharmacological features of PRTx3-7. To our surprise, PRTx3-7 shifted the voltage-dependence for activation towards hyperpolarized membrane potentials for L ( −4 mV), P/Q ( −8 mV) and R ( −5 mV) type Ca2+ currents. In addition, the new toxin also affected the steady state of inactivation of L-, N- and P/Q-type Ca2+ currents. KEY WORDS: Phoneutria reidyi; spider venom; neurotoxin; amidated C-terminus; Ca2+ channel toxin.
1 Laboratorio ´
de Neurofarmacologia, Departamento de Farmacologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil. 2 Laboratorio ´ ˆ ´ de Venenos e Toxinas Animais, Instituto de Ciencias Biologicas, Universidade Federal de ˆ Minas Gerais, Av. Antonio Carlos, 6627, Campus Pampulha, Caixa Postal 486, CEP 31270-901, Belo Horizonte, MG, Brazil. 3 Nucleo ´ ´ de Biomoleculas, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil. 4 Centro de Pesquisa Professor Carlos Ribeiro Diniz, Fundac¸ao ˜ Ezequiel Dias, Belo Horizonte, MG, Brazil. 5 Laboratorio ´ ´ de Membranas Excitaveis, Departamento de Bioqu´ımica e Imunologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil. 6 Departamento de Ciencias ˆ ´ ´ Biologicas, Universidade Federal de Esp´ırito Santo, Vitoria, ES, Brazil. 7 Biotechnology Laboratory, University of British Columbia, Vancouver, BC, Canada. 8 L. B. Vieira and A. M. C. Pimenta contributed equally to this work. 9 To whom correspondence should be addressed; e-mail:
[email protected]. 129 C 2006 Springer Science+Business Media, LLC 0272-4340/07/0100-0129/1
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INTRODUCTION Spider venoms are an important part of biodiversity. They may be regarded as a “treasure chest” of potential future discoveries, because they are a valuable source of a range of novel chemicals with different biological activities. In recent years, there has been an explosion of interest in the small (1.5–9.5 kDa) polypeptides/proteins in venoms that exhibit varying toxicities towards the nervous systems of mammals and/or insects. This is because their mode of action in the prey targets neuronal ion channels (Ca2+ , Na+ , K+ or Cl− ), various ionotropic (NMDA, AMPA or kainate) and metabotropic glutamate receptors, pre-synaptic membrane proteins involved in neurotransmitter release, and/or post-synaptic cholinergic receptors (Rash and Hodgson, 2002 (2002). As a result, they have potential use: (i) as laboratory tools in gaining a better understanding of the structure and function of neuronal ion channels; (ii) in medicine as lead compounds for the development of new drug therapies and (iii) in agriculture for the design of new, highly selective and environmentally friendly insecticides (Corzo and Escoubas, 2003; Rash and Hodgson, 2002; Escoubas et al., 2000; Pimenta and De Lima, 2005). The extremely aggressive Brazilian spider Phoneutria nigriventer, responsible for many accidents involving envenomation of humans, is a wandering spider, which does not utilize a web, and therefore produces particularly neurotoxic venom to rapidly subdue its prey. Various neurotoxins active on Na+ (Cordeiro et al., 1992; Matavel et al., 2002), K+ (Cordeiro et al., 1993; Carneiro et al., 2003) and Ca2+ (Diniz et al., 1990, 1993; Santos et al., 2002, Gomez et al., 2002) ion channels, and other highly insecticidal proteins (Figueiredo et al., 1995, 2001; Mafra et al., 1999; De Lima et al., 2002; Oliveira et al., 2003) have been described from the venom of this spider, which is commonly found in Central and South-East Brazil, but very little is known about the venoms of the other species of this genus. Recently, our group has published the comparison of the partial proteomes of venoms from different species of the genus Phoneutria (Richardson et al., 2006). Using different separation methodologies, including 2D-gel and multi-dimensional chromatographic procedures, we were able to find about 400 peptides and proteins of which, nearly 120 have their partial/complete primary structure determined. We now describe the purification, primary structure and identify the molecular target of a new toxin from the venom of Phoneutria reidyi, a spider from the northern Brazilian states Amazonas, Para´ and Roraima, which causes flaccid paralysis in mice at dose levels of 5 µg/mouse. This toxin, named PRTx3-7, has its C-terminal residue amidated and was found to be homologous to PNTx3-2, a Ca2+ -type toxin isolated from Phoneutria nigriventer venom. It has been also reported that some toxins from the venom of P. nigriventer, like PRTx3-7, cause flaccid paralysis when injected intracerebroventricular (i.c.v.) into mice (Cordeiro et al., 1993). PNTx3-2 is known to partially block L-type Ca2+ channels in GH3 cells (Kalapothakis et al., 1998; Gomez et al., 2002), whereas PNTx3-1 is a potent and selective blocker of A-type K+ channels (Kushmerick et al., 1999) and PNTx3-3 inhibits all known HVA Ca2+ channels, and ˜ et al., 2000). most effectively the P/Q and R-type currents (Leao Electrophysiogical data in HEK cells expressing high-voltage-activated (HVA) Ca2+ channels suggest that PRTx3-7 inhibits these channels, change their
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voltage-dependent properties and has more affinity for N-type Ca2+ channels. The investigation of these natural mutants, along with the related biochemical and biological data will contribute to the global understanding of the mechanisms involving spider toxins and their related targets.
MATERIALS AND METHODS Toxin Purification Venom collected from spiders captured near the Usina Tucurui in the state of ˜ Paulo, Brazil). Para in Northern Brazil was provided by the Butantan Institute (Sao Aliquots (25 mg) of lyophilised venom were dissolved in 2 mL of 0.1% (v/v) trifluoroacetic acid (TFA) in water and insoluble materials removed by centrifugation at 10,000 × g for 10 min. Each aliquot of venom was initially fractionated by reverse phase HPLC on a Vydac C4 (214TP1022) preparative column (2.2 cm × 25 cm), equilibrated with 0.1% TFA in water, and eluted at a flow rate of 5 mL/min with the following solutions: 0–20 min, 0.1% TFA in water; 20–30 min, a gradient of 0–20% acetonitrile in 0.1% TFA in water; 30–110 min, gradient of 20–40% acetonitrile containing 0.1% TFA in water; 110–130 min, 40–50% acetonitrile containing 0.1% TFA in water; 130–140 min, 50–70% acetonitrile with 0.1% TFA in water. The material in the 17th peak (detected by A214 nm ) that eluted after 68 min (at a concentration of 29% acetonitrile) was collected and lyophilised. It was then dissolved in 2 mL of 10 mM sodium phosphate buffer pH 6.1, before being subjected to cation exchange FPLC on a column of ResourceTM S 1 mL (Pharmacia Biotech), equilibrated in the same buffer and eluted at a flow rate of 1 mL/min with a linear gradient of 0–0.5 M NaCl. The material in the third major peak (A280 nm ) that eluted at 0.32 M NaCl was desalted by a final step of reverse phase FPLC on a column of C2/C18 PepRPCTM 15 µm (HR10/10, Pharmacia), using a linear gradient of acetonitrile in 0.1% aqueous TFA. The homogeneity of the isolated protein was examined by high-resolution propionic acid/urea PAGE (Chettibi and Lawrence, 1989). The toxicity of the material was assayed by intracerebro-ventricular injection in mice as described previously (Rezende et al., 1991). Mass Spectrometry Analyses The molecular mass of the protein was determined by electrospray mass spectrometry (Q-TOF Micro® , Micromass, UK) in positive ion mode as previously described (Pimenta et al., 2005). The protein (20 µg) was solubilised in 100 µL of 50% acetonitrile in aqueous 0.1% TFA and applied to mass spectrometer by a syringe pump system at a flow rate of 5–10 µL/min. Alternatively, a nanospray interface was used to increase sensitivity (flow rate of 0.5–1.0 µL/min). The capillary voltages were 3–3.5 kV and cone voltages were 40–60 V. The spectrum used was the result from 20 scans (2.4 s) combined. Original data (m/z) were treated (base line subtraction, smoothing and centring) and transformed into a mass (Da) spectrum.
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Sequencing by tandem mass spectrometry (MS/MS) was performed using collisioninduced dissociation (CID) technique, carried out using argon and collision energies in the range 30–45 eV. Data were analysed by MassLynx® 3.5 software. Edman Degradation Sequencing The protein was S-reduced and alkylated using 4-vinylpyridine as described (Wilson and Yuan, 1988). Samples of the native and alkylated protein (3–4 nmol) were sequenced by Edman degradation using an automated Shimadzu PPSQ-21A protein sequencer coupled to reversed phase separation of PTH-amino acids on a WAKOSIL-PTH (4.6 mm × 250 mm) column. Enzymatic Hydrolysis of PRTx3-7 A sample (20 nmol) of the native protein was digested with 5 µg of the Glu-C endoproteinase from S. aureus V8 strain (Sigma) in 0.1 M Na phosphate buffer, pH 7.8, for 24 h at 37◦ C. The resulting peptides were separated by reverse phase HPLC on an analytical column (0.46 cm × 25 cm) of Vydac C18 small pore (201SP54), using a gradient of 0–50% acetonitrile in 0.1% (v/v) aqueous TFA (with 1.0 mL/min flow and detection at 214 nm) and the C-terminal peptide was submitted to MS/MS sequencing. Solutions and Drugs Stock solutions of PRTx3-7 were prepared in distilled water and stored at −20◦ C. Toxin was thawed shortly before the experiments and diluted into the bath solution at the appropriate concentration. BSA (0.025%, Jackson Immuno Res., West Grove, PA, USA) was included in external solutions to avoid non-specific binding. For experiments with permanently expressed HVA Ca2+ channels, the perfusion system consisted of a custom-made multiple solution perfusion manifolds with four input and four output capillary tubes (custom microfil, 28 gauge, 250 µm inner diameter and 350 µm outer diameter; World Precision Instruments, Sarasota, FL) sheathed in a glass pipette. High chemical-resistant Tygon Chemfluor FEP (Norton Performance Plastics, Akron, OH) and Silastic (Fisher Scientific, Nepean, ON) tubing was used to connect the perfusion manifold to the syringe valve. Gravity-driven perfusion occurred at a rate of approximately 400 µL/min. The outputs of the manifold were placed within close proximity of the cell, resulting in the cell being bathed in new solutions with minimal delay (within 1 s). Preparation of Synaptosomes Adult Wistar rats of both sexes (180–200 g) were decapitated and had their cortices removed and homogenized 1:10 (w/v) in 0.32 M sucrose solution containing dithiothreitol (0.25 mM) and EDTA (1 mM). Homogenates were then submitted to low-speed centrifugation (1000 × g for 10 min) and synaptosomes were purified from the supernatant by discontinuous PercollTM -density-gradient
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centrifugation (Dunkley et al., 1988; essentially as described by Romano-Silva et al., 1993). The isolated nerve terminals were re-suspended in Krebs–Ringer–Hepes solution (124 mM NaCl, 4 mM KCl, 1.2 mM MgSO4 , 10 mM glucose, 25 mM Hepes, pH 7.4) with no added CaCl2 , to a concentration of approximately 10 mg/mL, divided into aliquots of 200 µL and kept on ice until loaded with fura-2AMTM . Measurements of Intracellular Calcium Concentration [Ca2+ ]i Fura-2AMTM (stock solution 1 mM in DMSO) was added to the synaptosomal suspensions to give a final concentration of 5 µM and the mixture incubated for 30 min (35◦ C) and then diluted with medium to a concentration of 1.2 mg/mL followed by a further 30 min incubation period. Fura-2-loaded isolated nerve terminals were washed, re-suspended with medium (1 mg/mL) and immediately used for ratiometric quantification of intracellular free calcium (Grynkiewicz et al., 1985) in a PTITM spectrofluorimeter. Fluorescence emission ratio was calculated for emission at 500 nm using excitation at 330/370 (FR = I330/I370, where I330 and I370 are the emission intensities for excitation at corresponding wavelengths). Calibration of fura-2 signals and estimation of synaptosomal background fluorescence were performed as described elsewhere (Romano-Silva et al., 1993). CaCl2 was added to the synaptosomal suspension at the beginning of each fluorimetric assay (for 1.0 mM final concentration). Synaptosomes were stirred throughout the experiment in a cuvette maintained at 35◦ C. Toxin was added to synaptosomal suspension 60 s prior to membrane depolarisation with 33 mM KCl. The [Ca2+ ]i increase induced by membrane depolarisation is strictly dependent on the presence of extracellular Ca2+ . Cell Culture Human embryonic kidney cells (HEK 293) expressing a simian virus 40Tantigen (tsA-201), were grown in standard DMEM (10% fetal bovine serum and 50 U/mL penicillin–50 µg/mL streptomycin). HEK cells were split at 80% confluence using trypsin–EDTA, plated on 35-mm culture dishes at 5–10% confluence and maintained at 37◦ C in a humidified atmosphere of 95% O2 /5% CO2 . The cells were allowed to recover for 12 h before transfection. Transient Transfection with L-, P/Q- and R-type Ca Channels HEK cells were grown to approximately 80% confluency, enzymatically dissociated using 0.25% trypsin in DMEM containing 1 mM EDTA (trypsin/EDTA), plated on 35-mm culture dishes at 5–10% confluency, and allowed to recover for 12 h. The medium was then replaced, and the cells were transiently transfected with cDNA plasmids encoding one type of Ca2+ channel α subunit [α1A (Starr et al., 1991), α1C (Bourinet et al., 1994), or α1E (Soong et al., 1993)] together with auxiliary subunits β1b and α2 δ (Zamponi et al., 1997) and CD8 marker plasmid at a molar ratio 1:1:1:0.25 using Lipofectamine (Invitrogen) following the manufacturer’s instructions. After 12 h, the cells were washed with fresh medium, stored at 37◦ C and
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allowed to recover for an additional 36–72 h before electrophysiology recordings. Transiently transfected cells were selected for expression of CD8 by adherence of Dynabeads (Dynal, Great Neck, NY).
Generation of Stable Cell Lines Expressing N- and T-type Ca Channels HEK cells were transfected using linearized cDNA encoding either α1G (McRory et al., 2001) alone or α1B (Dubel et al., 1992 together with β1b and α2 δ using standard Ca-phosphate precipitation method, The stable cell lines were enzymatically dissociated using 0.25% trypsin in DMEM containing 1 mM EDTA (trypsin/EDTA), plated on 35-mm culture dishes, and allowed to recover 24–36 h before recordings.
Electrophysiological Recordings Macroscopic currents on the HEK cells were recorded using the wholecell patch-clamp technique. The external recording solution contained (in mM): 2 BaCl2 , 1 MgCl2 , 10 HEPES, 40 TEACl, 92 CsCl, and 10 glucose, pH = 7.4. The internal pipette solution contained (in mM): 120 CsCl, 1 CaCl2 , 11 EGTA, 10 HEPES, 2 Mg-ATP, pH = 7.2. Whole-cell currents were recorded using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA), controlled and monitored with a personal computer running pClamp software version 6.03 (Axon Instruments). Patch pipettes (borosilicate glass BF150-86-10; Sutter Instruments, Novato, CA), were pulled using a Narishige (Tokyo, Japan) puller, with typical resistances of 3–5 M (when filled with internal solution). The bath was connected to the ground via a 3 M KCl Agar-bridge. Voltage errors due to series resistance were kept to
