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A novel conotoxin inhibiting vertebrate voltage-sensitive potassium channels
Toxicon 42 (2003) 43–52 www.elsevier.com/locate/toxicon
A novel conotoxin inhibiting vertebrate voltage-sensitive potassium channels Silke Kaufersteina,*, Isabelle Huysb, Hung Lamthanhc, Reto Sto¨cklind, Filipina Sottoe, Andre´ Menezc, Jan Tytgatb, Dietrich Mebsa a
Zentrum der Rechtsmedizin, University of Frankfurt, Kennedyallee 104, D-60596 Frankfurt am Main, Germany b Laboratory of Toxicology, University of Leuven, B-3000 Leuven, Belgium c Dept. d’Ingenierie et d’Etudes des Proteines, CEA/Saclay, F-91191 Gif-sur-Yvette, France d Atheris Laboratories, CH-1233 Bernex-Geneva, Switzerland e Marine Biology Section, San Carlos University, Cebu City, Philippines Received 4 March 2003; accepted 9 April 2003
Abstract Toxins from cone snail (Conus species) venoms are multiple disulfide bonded peptides. Based on their pharmacological target (ion channels, receptors) and their disulfide pattern, they have been classified into several toxin families and superfamilies. Here, we report a new conotoxin, which is the first member of a structurally new superfamily of Conus peptides and the first conotoxin affecting vertebrate Kþ channels. The new toxin, designated conotoxin ViTx, has been isolated from the venom of Conus virgo and comprises a single chain of 35 amino acids cross-linked by four disulfide bridges. Its amino acid sequence (SRCFPPGIYCTSYLPCCWGICCSTCRNVCHLRIGK) was partially determined by Edman degradation and deduced from the nucleotide sequence of the toxin cDNA. Nucleic acid sequencing also revealed a prepropeptide comprising 67 amino acid residues and demonstrated a posttranslational modification of the protein by releasing a six-residue peptide from the C-terminal. Voltage clamp studies on various ion channels indicated that the toxin inhibits the vertebrate Kþ channels Kv1.1 and Kv1.3 but not Kv1.2. The chemically synthesized product exhibited the same physiological activity and identical molecular mass (3933.7 Da) as the native toxin. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Conotoxin; Voltage-sensitive potassium channel; Voltage clamp electrophysiology; Structure; Novel conotoxin superfamily; Posttranslational modification
1. Introduction Cone snails (Conus species; suborder: Toxoglossa) represent a large genus of venomous gastropods consisting of more than 500 species. Each snail produces a complex venom containing a large number of functionally different toxins. In all Conus venoms, small peptides of 10 – 35 amino acids represent the major biologically active compounds. These peptides are often disulfide-rich and exert a wide * Corresponding author. Tel.: þ 49-69-6301-7564; fax: þ 49-696301-5882. E-mail address: [email protected] (S. Kauferstein).
range of physiological effects, e.g. targeting various receptors, voltage or ligand-gated ion channels (Olivera et al., 1990, 1991). Conus toxins have been classified into major superfamilies according to their cysteine framework (McIntosh et al., 1999). Whereas hypervariability in amino acid sequence has been observed in the regions between the cysteine residues, the signaling sequence in the precursor structure is particularly conserved (McIntosh et al., 1999; Olivera et al., 1999). The high selectivity of conotoxins for molecular targets in the nervous system has made them important pharmacological tools to identify or to distinguish receptors and ion channel subtypes or to map the functional
0041-0101/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0041-0101(03)00099-0
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surface of these receptors (Walker et al., 1999). For instance, a family of Conus peptides, a-conotoxins, targets the nicotinic acetylcholine receptor (Hopkins et al., 1995). m-Conotoxins inhibit Naþ channels of muscle membranes leaving Naþ channels of nerve membranes unaffected (Cruz et al., 1985; Moczydlowski et al., 1986). v-Conotoxins exhibit high affinity to certain subtypes of Ca2þ channels such as a conotoxin (GVIA) from Conus geographus, which specifically blocks N-type Ca2þ channels (Olivera et al., 1984; MacCleskey et al., 1987). So far, one peptide only, namely k-conotoxin PVIIA from the venom of Conus purpurascens, has been found to interact with a voltagegated Kþ channel (Terlau et al., 1996). k-Conotoxin is a 27residue, four-loop peptide with three disulfide bonds, which blocks the Kþ-conductance in oocytes expressing the cloned Shaker Kþ channel encoded from Drosophila, but no affinity to other Kþ channel subtypes, particularly to those of vertebrate origin has been found (Shon et al., 1998b; Terlau et al., 1999). The three-dimensional structure of the synthetic toxin was elucidated by NMR spectroscopy (Scanlon et al., 1997; Savarin et al., 1998). It was predicted that the determinant by which k-conotoxin PVIIA occludes the pore should include a binding diad, composed of Lys 7 and Phe 9 or Phe 23 (Savarin et al., 1998). This prediction was based on the observation that a similar critical diad is present in the determinant by which other toxins, even structurally unrelated, block voltage-gated Kþ channels (Dauplais et al., 1997; Gasparini et al., 1998). An alanine walk along the polypeptide chain of the toxin (Jacobsen et al., 2000) experimentally confirmed this prediction. In the present paper we report the isolation and characterization of a novel toxin from Conus virgo, which specifically inhibits voltage-gated vertebrate Kþ channels of the type Kv1.1 and Kv1.3. It consits of a polypeptide with 35 amino acid residues cross-linked by four disulfide bonds representing a structurally new family of conotoxins.
2. Materials and methods 2.1. Preparation of the venom extract Specimens of Conus virgo were collected in the Philippines (Olango Island and Bohol). The venom ducts were dissected from the snails, cut into small pieces and extracted with 5% acetic acid. After centrifugation of the extract, the supernantant (i.e. crude venom) was lyophilized. Dissected venom ducts were also placed in RNAlatere (Ambion Inc., Austin, USA), a tissue storage reagent which protects and stabilizes RNA. 2.2. Peptide purification The crude venom extract was fractionated by gel filtration on a Sephadex G-50 column (85 £ 1.5 cm;
Amersham Biotech, Budinghamshire, UK), which was eluted with 0.1 M ammonium acetate, pH. 6.8 at a flow rate of 0.5 ml/min. Further purification of the active fraction (as indicated by electrophysiological screening; see below) was performed by reversed-phase HPLC on a BDSHypersilw-C18-column (125 £ 4 mm; Hewlett Packard, Waldbronn, Germany) using a gradient of trifluoroacetic acid (0.1% in water, solvent A) and acetonitrile (60% in 0.1% trifluoroacetic acid, solvent B) for elution at a flow rate of 0.5 ml/min over 45 min. The effluent was monitored at 220 nm, peaks were collected, lyophilized and tested for biological activity. The active fraction was rechromatographed using the same column and gradient for elution, but over a period of 90 min, followed by chromatography of the active fraction on a Lichrocartw Purosphere column (250 £ 4 mm; Merck, Darmstadt, Germany) and eluted using a modified gradient (40% acetonitrile in 0.1% trifluoroacetic acid, solvent B, over 90 min at a flow rate of 0.3 ml/min). The coelution experiment was performed as followed. A mixture of synthetic and native ViTx were applied onto a 218TP104 HPLC C18 reversed phase column (Vydacw) equilibrated with 0.1% trifluoroacetic acid (Sigma, Belgium). After 4 min, a linear gradient from 0 to 60% acetonitrile was applied for 26 min. The flow rate was 0.75 ml/min. 2.3. Electrophysiology Capped cRNAs encoding ion channels were synthesized after linearizing the plasmids and performing the transcription with T7 polymerase by a standard protocol (Krieg and Melton, 1987). Capped cRNA of Kv1.1 (Baumann et al., 1988) was synthesized in vitro after linearizing the plasmid with PstI (New England Biolabs Inc., Beverly, MA, USA). Kv1.2 (Stu¨hmer et al., 1988) was subcloned by double digestion with BglII and EcoRI and ligated into the corresponding sites of the high-expression vector pGEMHE (Liman et al., 1992). For in vitro transcription, the cDNA was linearized with SphI. Kv1.3 (Swanson et al., 1990) was cloned into the vector pCl.neo and linearized with NotI for in vitro transcription. Oocytes from Xenopus laevis were prepared as previously decribed (Stu¨hmer, 1992). HERG (Warmke and Ganetzky, 1994; Sanguinetti et al., 1995). The HERG clone (HERG/pSP64) was first linearized with EcoRI and cRNA was synthesized using the large scale T7 mMESSAGE mMACHINE transcription kit (Ambion, USA). Kir2.1 (Kubo et al., 1993). The in vitro synthesis of cRNA encoding the IRK1 channel was as previously described (Tytgat et al., 1996). Stage V– VI Xenopus laevis oocytes were isolated by partial ovariectomy under anaesthesia (tricaine, 1 g/l). Anaesthetized animals were kept on ice during dissection. The oocytes were defolliculated by treatment with 2 mg/ml collagenase (Sigma, Belgium) in zero calcium ND-96 solution (see Solutions). Between 2 – 24 h after
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defolliculation, oocytes were injected with 50 nl of 1 – 100 ng/ml cRNA. The oocytes were then incubated in ND-96 solution at 18 8C for 1– 4 days. The whole cell currents of the oocytes were recorded using the two-microelectrode voltage clamp technique. Voltage and current electrodes (range: 0.4 –4 megaohms) were filled with 3 M KCl. Current recordings were sampled at 2 kHz after low pass filtering at 0.1 kHz. A pulse frequency of 0.2 Hz was chosen to maximize the recovery from inactivation of the channels. The solution was exchanged at a flow rate of 5 ml/min and the volume of the recording chamber was 100 ml. Offline analysis was done using a Pentium III processor computer. Linear components of capacity and leak current were not substracted. All experiments were performed at room temperature (19 – 22 8C). Oocytes were held at 2 90 mV and stepped to different test potentials. The bath solution was ND-96 containing (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 Hepes, pH 7.5, supplemented with 50 mg l21 gentamycine sulfate (only for incubation). Lyophilized toxins were dissolved and freshly diluted in the ND-96 solution. 2.4. Peptide sequencing N-terminal amino acid sequence analysis was performed by automated Edman degradation using an ABI Modell 47A/120A Protein– Peptide Sequencing/Analysis System and Analysis Software System (Applied Biosystems, Foster city, CA, USA). The toxin was reduced, alkylated with 4-vinylpyridine and HPLC-purified. 40 pmol to 1 nmol of the sample was loaded onto a trifluoroacetic acid-treated cartridge filter previously conditioned with BioBrene Pluse. Before each analysis, a calibration was done using a phenylthiohydantoin-amino acids standard solution. 2.5. Mass spectrometry Chromatography of the samples was performed by HPLC (C18, 150 £ 1 mm; Nucleosilw) followed by on-line liquid chromatography – electrospray-mass spectrometry. The mass spectrometer equipped with an electrospray ion source (Micromass Platform II, Micromass Altrincham, UK), was operated in positive-ionisation mode under control of the Mass Lynx data system with scanning at a mass to charge ratio range of 500– 1700 m/z at 6 s/scan cycle. 2.6. Structural studies by gene sequencing RNA from venom ducts was isolated according to standard procedures (Chomczynski and Sacci, 1987). To identify the toxin gene, 50 and 30 rapid amplification of cloned ends (RACE) experiments were carried out. 30 RACE was performend according to Frohman et al. (1989). A degenerated primer (50 -TGYTTYCCNCCNGGNATHTAYTG-30 )
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was designed according to the partial amino acid sequence as determined by Edman degradation. PCR conditions were as follows: 10 min 95 8C (1 cycle); 1 min 95 8C, 1 min 42 8C, 1 min 72 8C (30 cycles); 1 min 72 8C (1 cycle). 50 RACE was performed using cDNA produced with the SMART system (Clontech, Palo Alto, USA). Gene-specific primer sequences were as follows (IUB code; 50 ! 30 ): GSP1: CGTCCCACAGCAAATTCCCC; GSP 2: GCAAATTCCCCAACAGCAGGGG and the following PCR conditions were applied: 10 min 94 8C (1 cycle); 30 s 94 8C, 10 s 64 8C, 2 min 72 8C (30 cycles); 7 min 72 8C (1 cycle). Amplification products of the appropriate size were ligated into the T-tailed plasmid vector pGEMw-T (Promega, Madison, USA) which were then transformed into competent Escherichia coli. Plasmids containing inserts of the expected size were sequenced by the dye terminator method on ABI 310 automated sequencer. 2.7. Synthesis of ViTx conotoxin The linear peptide of ViTx conotoxin was synthesized using Fmoc (fluorenylmethyloxycarbonyl) chemistry on the ABI 430 automated peptide synthesizer (Applied Biosystems). Starting with 0.100 mmole of Fmoc-LLys(Boc)-PEG-PS-resin (PE-ABI), the peptide was constructed using a double couple cycle with TBTU (2(1H-benzotriazole1-yl)-1.1.3.3-tetramethyluronium tetrafluoroborate) as condensing reagent (Knorr et al., 1989) except for the coupling of the fifth to the sixth residue. The Pro5 – Pro6 peptide bond was revealed as a peculiar bond in this sequence. Therefore a manual coupling of the FmocPro5 to Pro6-peptide resin was carried out by using the HATU/HOAt (N-[(dimethylamino)-1H-1,2,3-triazolo[4,5b]pyridinyl-1-methylene]-N-methylmethanaminium hexafluorophosphate/1-hydroxy-7-azabenzotriazole) method (Carpino et al., 1994) before continuing the synthesis process for the last four residues on the synthesizer. The peptide was then cleaved from the resin by trifluoroacetic acid/thioanisole/m-cresol/ethanedithiol/water (90/2.5/2.5/ 2.5/2.5) for 2 h at room temperature. The crude linear peptide was precipitated, washed with cold ether and centrifuged. The pellet was dissolved in acetonitrile/water (50/50) and lyophilized. Several attempts to fold the linear peptide in an aqueous medium failed to yield the native form of ViTx. Hence, the best folding process proceeded in an aqueous-organic medium. 100 mg of crude peptide was loaded on a HPLC semi-preparative column (Vydacw, C18, 250 £ 25 mm) and eluted (10 ml/min) with a gradient from 50 to 100% of B solvent for 100 min (A solvent: 0.1% trifluoroacetic acid in water; B solvent: acetonitrile/0.1% trifluoroacetic acid in water 60/40). Fractions containing the linear peptide (MWcalc: 3941.0; MWobs: 3941.2) were mixed with 200 ml of isopropanol and 50 ml of trifluoroethanol. Then, 250 ml of ammonium bicarbonate (0.1 M; pH 8.5) and 1 mmol of reduced glutathione were added. The medium was adjusted at pH 8.5 with ammonia and left for 48 h at room temperature. After adjusting the pH to 4
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Fig. 1. Purification of the toxin ViTx. (A) Lyophilized venom from Conus virgo was dissolved in with 0.1 M ammonium acetate, pH 6.8, and applied onto a Sephadex G-50 column (85 £ 1.5 cm) using the same buffer for elution at a flow rate of 0.5 ml/min. (B). The active fraction (*) was separated by reversed-phase HPLC on a BDS-Hypersil-C18-column (125 £ 4 mm, 5 mM particle size) at a flow rate of 0.5 ml/min using a linear gradient from 0 to 60% acetonitrile in 0.1% trifluoractic acid over 45 min. (C) The active fraction as indicated in (B) was further purified on the same column with an slightly modified gradient (from 0 to 60% acetonitrile over 90 min). (D) A 218TP104 HPLC C18 reversed phase column (Vydacw), equilibrated with 0.1% TFA, was used for the coelution experiment. After 4 min, a linear gradient from 0 to 60% acetonitrile was applied for 26 min. The flow rate was 0.75 ml/min.
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with trifluoroacetic acid, the volume was diluted to 1 l with water, loaded directly onto the preparative Vydacw column, which was washed out with solvent A and eluted with the previous gradient of acetonitrile. Five fractions containing folded peptides were analyzed by analytical ˚ column, 250 £ 4.6 mm) and HPLC (Zorbax SB-C18, 300 A mass spectrometry. The most homogeneous fraction was repurified on a semipreparative column (Zorbax SB- C18, ˚ , column 250 £ 9.4 mm). The overall yield of the 300 A synthetic, folded ViTx toxin was 1 or 2% in weight of the starting crude linear peptide. Synthetic ViTx was characterized by amino acid analysis, analytical HPLC and mass spectrometry (MWcalc: 3933.7; MWobs: 3933.4) and submitted to bioassays.
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gel filtration (Fig. 1A). The major fraction containing peptides in a range of 1.5– 5 kDa was further separated into numerous fractions by reversed phase HPLC on a BDSHypersil-C18-column (Fig. 1B). Of the fractions tested on Kv1.1 and Kv 1.3 channels, one exhibited blocking activity (data not shown) and was rechromatographed on the same column using a lower gradient (Fig. 1C). It was finally purified on a HPLC Lichrocartw Purosphere column eluted with a modified acetonitrile gradient. Coelution of the synthetic and native toxin onto the C18 HPLC column (Vydacw) resulted in a single symmetric peak (Fig. 1D). Homogeneity of the purified toxin, named ViTx, was confirmed by electrospray ionization mass spectrometry (ES-MS) with a measured molecular mass of 3933.3 Da. 3.2. Amino acid sequence
3. Results 3.1. Toxin purification and characterization In a first set of experiments, the venom extract from Conus virgo exhibited blocking activity when tested on Kþ channels of the type Kv1.1 and Kv1.3 expressed in Xenopus oocytes, but no activity was observed on Kv1.2 channels. Initial fractionation of the crude venom was performed by
The N-terminal amino acid sequence of the reduced and pyridylethylated peptide was determined until position 27 (SRCFPPGIYCTSYLPCCWGICCSTCRN). Due to the low concentration of the toxin in the crude venom and its poor yield during purification, the complete sequence could not be established by Edman degradation. Therefore attempts were made to elucidate the toxins’ primary structure by sequencing its gene isolated from cDNA.
Fig. 2. Sequence encoding the prepropeptide of ViTx. The bottom line shows the combined nucleotide sequences of two clones encoding the precursor of ViTx, while the top line shows the deduced amino acids. The proposed start and stop signals are indicated by an aterisk, while the toxin coding region is underlined. The proposed posttranslational cleavage site is indicated by an arrow. The clones from the cDNA prepared from the venom duct of Conus virgo were achieved using PCR as described under Materials and methods.
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Fig. 3. Macroscopic Kþcurrent through Kv1.1, Kv1.2 and Kv1.3 channels expressed in Xenopus oocytes. Currents were obtained using the twomicroelectrode voltage clamp technique. Oocytes were held at 290 mV and stepped to a test potential of 0 mV. The effect of the concentration of the native ViTx is shown. Application of 4 mM native ViTx leads to approximately full block of the Kv1.1 channels. Addition of 0.8 mM native ViTx to the bath solution leads to approximately 36% blockage of the Kv1.3 Kþ currents, but has no effect on the Kv1.2 Kþ currents. Block induced by synthetic ViTx was identical.
3.3. Isolation of cDNA clones For identification of cDNA clones of ViTx gene fragments, degenerated primers were synthesized corresponding to position 3 – 12 and 15 – 22 (reverse direction) of the partial amino acid sequence and used as gene-specific primers in the 30 RACE (rapid amplification of cDNA ends). A cDNA fragment consisting of 313 bp was obtained, cloned and sequenced. From this nucleotide sequence, specific primers were designed to obtain overlapping 30 and 50 RACE products. Amplification with these primers in the 50 RACE experiment produced a specific product of 175 bp. The 50 and 30 RACE sequences were assembled to achieve the complete cDNA sequence of ViTx. The nucleotide sequence of the prepropeptide and of the deduced amino acid sequence is shown in Fig. 2. The 67 amino acid precursor of ViTx has a N-terminal-signal sequence, which is followed by 41 amino acids including the toxin’s sequence. Two amino acids, Lys – Arg near the C-terminus appear to serve as a proteolytic cleavage site for posttranslational processing of the peptide chain. According to mass-spectrometry data (MW: 3933.3), the toxin consists of a chain of 35 amino acids with 8 cysteines and is folded by four disulfide bonds. Lysine should represent the C-terminus after a sixresidue peptide has been released by proteolytic cleavage. The sequence was confirmend by chemical synthesis of the peptide.
the linear ViTx peptide in an aqueous buffer failed to yield the native form of ViTx in several attempts. Then, a mixture of organic solvents (e.g. acetonitrile, 2-propanol, trifluoroethanol) and water was found to be efficient for the folding process.
3.4. Chemical synthesis To verify the sequence from ViTx, the peptide was synthesized in a carboxylic form at the C-terminal side and folded as described under Section 2. The final yield of active peptide was 1 or 2% in weight of the starting crude peptide. The synthetic peptide elicited the same biological activity as the native peptide purified from the venom when tested on the vertebrate Kþ channels (Fig. 3). In addition the mass of the synthetic peptide, as determined by ES-MS (MW: 3933.6 Da), agreed with the mass calculated for the native toxin ViTx (MW: 3933.7). The folding process of
Fig. 4. Current/voltage ðItest =Vtest Þ curve for the block induced by ViTx. Oocytes were held at 290 mV and stepped to test potentials varying between 270 and þ 20 mV (mean ^ SD, n ¼ 3). Presence of 0.8 mM ViTx (X) reduced the Kþ currents of Kv1.1 (A) and Kv1.3 (B) to about 66 and 63% of the control ðoÞ value, respectively, in an almost non-reversible manner. There is no voltage-dependent block observed.
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3.5. Electrophysiological studies Initial voltage clamp experiments using venom fractions had suggested that the toxin ViTx inhibits homomeric vertebrate Kþ channels Kv1.1 (rat) and Kv1.3 (human), but not Kv1.2 (rat) expressed in Xenopus oocytes. Detailed electrophysiological studies with purified and synthetic toxin confirmend this observation. Native ViTx inhibited the currents through Kv1.1 and Kv1.3 channels but had no effect on Kv1.2 channels (Fig. 3). An identical effect has been observed with synthetic ViTx. Application of 0.8 mM synthetic peptide produced inhibtion of 33.4% of the Kþ current through Kv1.1 channels and of 36.1% of the Kþ current through Kv1.3 channels, respectively (data not shown). This blockage induced by ViTx exhibited no voltage-dependence, as the extent of blockage was not different in the range of test potentials from 220 to þ20 mV (Fig. 4). No blocking effect on the Kv1.2 Kþ current was observed (Fig. 3 and 5), e.g. the percentage of block after application of 2 mM of ViTx was about 0% and about 50% for Kv1.2 and Kv1.3 channels, respectively. Only a small recovery of Kþ currents was achieved by washing-out. The Kd values were 1.59 ^ 0.14 mM and 2.09 ^ 0.11 mM for Kv1.1 and Kv1.3 channels, respectively, deduced from the dose-response curve as shown in Fig. 5. Furthermore, ViTx binding did not alter channel gating. The maximum conductance ðgmax Þ curve for Kv1.3 (Fig. 6) fitted well with a single, first-order Boltzmann function. In a control experiment the half maximum potential ðV1=2 Þ was 219.8 ^ 1.4 mV. In the presence of
Fig. 6. Effects of ViTx gating. Maximal conductance-voltage ðgmax 2 Vtest Þ relationship for Kv1.3 in the absence ðoÞ or presence of 1 mM ViTx (X). The conductance-voltage curve was fitted with a single, first order Boltzmann function ðItail ¼ Imax =ð1 þ expððV1=2 Þ=sÞÞ; where Itail is the tail current at 250 mV, Imax is the maximum tail current, and s the slope factor of the voltage dependence. No ViTx-induced shift in the half-maximum potential of activation ðV1=2 Þ was observed (vertical dashed line). A similar effect was observed for Kv1.1 channels.
1 mM ViTx, a V1=2 value of 220.8 ^ 2.7 mV was obtained, which is not significantly different. A similar effect was observed for Kv1.1 channels. ViTx had no effect on other channels, such as HERG-type channels, Naþ hH1 channels and Kir-type channels (IRK1) (Fig. 7). Among the Kv1-type channels, the peptide seems to be a selective probe for Kv1.1 and Kv1.3 channels.
4. Discussion
Fig. 5. Dose-response curve for the block produced by ViTx. The Kd values were calculated according to the fits of the data points using the Hill equation: I ¼ Imax =½1 þ ðKd =½TÞh ; where I represents the current percentage, Imax is the maximal current percentage (both measured at 0 mV), Kd is the concentration of the toxin that evokes the half-maximal response, ½T is the concentration of the toxin, and h the Hill coefficient. The Hill coefficients were, respectively, 1.6 and 1.7 mV for Kv1.1 and Kv1.3 channels. The Kd value for Kv1.1 (B) was 1.59 ^ 0.14 mM and 2.09 ^ 0.11 mM for Kv1.3 (X), respectively. For Kv1.2 (O) no inhibition was observed in the concentration range indicated.
The data presented above demonstrate the presence of a peptide in a cone snail venom which interacts with vertebrate voltage-gated Kþ channels, e.g. of the Kv1.1 and Kv1.3 type. Electrophysiological characterization of the purified toxin was successfully achieved, however, elucidation of its structure by Edman degradation was incomplete due to the very low concentration of this peptide in the venom of Conus virgo. Cloning and sequencing of gene fragments obtained by PCR revealed that the peptide comprises a single chain of 35 amino acids cross-linked by four disulfide bridges. Chemical synthesis of ViTx produced, even in low yield, a quantity of toxin that enabled its biological characterization. Solid phase synthesis of the ViTx linear peptide did not display anymore difficulties other than the coupling of residue Pro5 to Pro6. The linear peptide was obtained in a reasonable yield (30 – 40%). The main step governing the overall yield seemed to be the folding step. First, difficulties were encountered in folding the linear peptide in an aqueous medium. This was probably due to the hydrophobic profile of the peptide according to Kyte and Doolittle (1982). Therefore, aqueous-organic solvent conditions (isopropanol/water
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Fig. 7. Effect of 2 mM ViTx on Kir-type channels and hERG channels expressed in oocytes. (A) Current recordings under two-microelectrode voltage clamp in the presence of 98 mM [Kþ]o. The holding potential ðVhold Þ was 0 mV. Traces in the absence (left) and presence (right) of 2 mM ViTx were elicited every 5 s by steps to Vtest of 2160 to 60 mV for 1 s. (B) hERG currents, in control (left) and in the presence of 2 mM ViTx (right), were evoked by application of 2 s test pulses ranging from 250 to þ 70 mV in 10 mV increments from a holding potential of 290 mV. Peak tail currents ðItail Þ were measured at a potential of 270 mV.
1/1) as described by Daly et al. (1999) were used. The presence of low trifluoroethanol concentration in the folding medium could assist the folding of secondary structure as suggest by Reiersen and Rees (2000) in contrast to the absence of trifluoroethanol (data not shown). Second, at least five HPLC peaks corresponding to different folded isomass peptides including the correctly folded toxin were observed (data not shown). Inhibitory activity to the Kþ channels of the synthetic peptide was identical to that of the native toxin. The new toxin has some homology (39%) to a peptide from the venom of Conus betulinus (SWISSPROT-databank, access no. AF20866), however, nothing is known about the biological activity of this peptide. Therefore, ViTx represents a new type of conotoxins, structurally and
pharmacologically, being the first known to act on vertebrate Kþ channels. Despite its specificity, the affinity of ViTx to Kv1.1 and Kv1.3 channels appears to be moderate, when compared with that of other toxins, e.g. scorpion or snake venom toxins (Possani et al. 1999; Smith et al., 1997). In the doseresponse curve as shown in Fig. 5, the half-maximum inhibitory concentration (IC50) of ViTx is in the micromolar range (1.59 and 2.09 mM for Kv1.1 Kv1.3 channels, respectively), whereas the k-conotoxin PVIIA, which blocks the Shaker Kþ channel, is effective in nanomolar concentration (IC50 is about 70 nM). This low binding affinity may be due to the particular structural features of ViTx. However, we can not exclude that this novel toxin performes higher affinities to other targets that remain to be
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elucidated; but it is interesting to note that ViTx displays selectivity among Kv1-type channels. For the k-conotoxin PVIIA it has been proposed that the toxin excerts its blocking activity by positioning a lysine into the vestibule of the channel pore (Savarin et al., 1998). However, the only lysine residue in ViTx is the last residue located at the C-terminus, which could make it rather unlikely that it is involved in binding to a channel structure. However, in the absence of knowledge of the 3D structure of the toxin and of the individual role of its amino acids, the presence of a typical dyad remains to be clarified.
Acknowledgements We thank Olaf Pongs for providing the cDNA for the Kv1.2 channel, Maria L. Garcia for the Kv1.3 clone, R.G. Kallen for the hH1 clone; Philippe Favreau (Atheris Laboratories, Geneva) and Robert Thai (CEA-Saclay) Denis Hochstrasser (University Hospital), Robin Offord (University Medical Center) and Keith Rose (GeneProt Inc.) in Geneva for their assistance in MS-characterization. The hERG clone was generously donated by Mark Keating, HHMI, University of Utah, Salt Lake City, UT, USA. The IRK1 cDNA clone was kindly provided by L. Y. Jan. Isabelle Huys is Research Assistent of the Flemish Fund for Scientific Research (F.W.O.-Vlaanderen). Part of this work was supported by a grant of the German Research Agency (DFG) and of the Ministry of Research and Technology (BMBF) to D. M.
References Baumann, A., Gruppe, A., Ackermann, A., Pongs, O., 1988. Structure of the voltage-dependent potassium channel is highly conserved from Drosophila to vertebrate central nervous systems. EMBO J. 7, 2457–2463. Carpino, L.A., El-Faham, A., Minor, C., Albericio, F., 1994. Advantageous applications of azabenzotriazole (triazolopyridine)-based coupling reagents to solid-phase peptide synthesis. J. Chem. Soc., Chem. Commun., 201–203. Chomczynski, P., Sacci, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate –phenol– chloroform extraction. Anal. Biochem. 162, 156–159. Cruz, L.J., Gray, W.R., Olivera, B.M., Zeikus, R.D., Kerr, L., Yoshikami, D., Moczydlowski, E., 1985. Conus geographus toxins that discriminate between neuronal and muscle sodium channels. J. Biol. Chem. 260, 9280–9288. Daly, N.L., Love, S., Alewood, P.F., Craik, D.J., 1999. Chemical synthesis and folding pathways of large cyclic polypeptides: studies of the cystine knot polypeptide kalata B1. Biochemistry 38, 10606–10614. Dauplais, M., Lecoq, A., Song, J., Cotton, J., Jamin, N., Gilquin, B., Roumestand, C., Vita, C., De Medeiros, C.L.C., Rowan, E.G., Harvey, A.L., Menez, A., 1997. On the convergent evolution of animal toxins. Conservation of a diad of functional residues in
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potassium channel-blocking toxins with unrelated structures. J. Biol. Chem. 272, 4302–4309. Frohman, M.A., Dush, M.K., Martin, G.R., 1989. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998–9002. Gasparini, S., Danse, J.-M., Lecoq, A., Pinkasfeld, S., Zinn-Justin, S., Young, L.C., de Medeiros, C.L.C., Rowan, E.G., Harvey, A.L., Menez, A., 1998. Delineation of the functional site of a-Dendrotoxin. The functional topographies of dendrotoxins are different but share a conserved core with those of other Kv1 potassium channel-blocking toxins. J. Biol. Chem. 273, 25393–25403. Hopkins, C., Grilley, M., Miller, M., Shon, K.J., Cruz, L.J., Gray, W.R., Dykert, J., Rivier, J., Yoshikami, D., Olivera, B.M., 1995. A new family of Conus peptides targeted to the nicotinic acetylcholine receptor. J. Biol. Chem. 270, 22361–22367. Jacobsen, R.B., Koch, E.D., Lange-Malecki, B., Stocker, M., Verhey, J., Van Wagoner, R.M., Vyazovkina, A., Olivera, B.M., Terlau, H., 2000. Single amino acid substitutions in kappaconotoxin PVIIA disrupt interaction with the Shaker Kþ channel. J. Biol. Chem. 275, 24639–24644. Knorr, R., Trzeciak, A., Bannwarth, W., Gillessen, D., 1989. New coupling reagents in peptide chemistry. In: Jung, G., Bayer, E. (Eds.), Peptides, Proceedings of the 20th European Peptide Symposium, W. de Gruyter, Berlin, pp. 37 –39. Krieg, P.A., Melton, D.A., 1987. In vitro RNA synthesis with SP6 RNA polymerase. Methods Enzymol. 155, 397–441. Kubo, Y., Baldwin, T.J., Jan, Y.N., Jan, L.Y., 1993. Primary structure and functional expression of a mouse inward rectifier potassium channel. Nature 362, 127–133. Kyte, J., Doolittle, R.F., 1982. A simple method for displaying the hydrophobic character of a protein. J. Mol. Biol. 157, 105–132. Liman, E.R., Tytgat, J., Hess, P., 1992. Subunit stoichiometry of a mammalian Kþ channel determined by construction of multimeric cDNAs. Neuron 9, 861– 871. MacCleskey, E.W., Fox, A.P., Feldman, D.H., Cruz, L.J., Olivera, B.M., Tsien, R.W., Yoshikami, D., 1987. v-Conotoxin: direct and persistent blockade of specific types of calcium channels in neurons but not muscles. Proc. Natl. Acad. Sci., USA 84, 4327–4331. McIntosh, M.J., Santos, A.D., Olivera, B.M., 1999. Conus peptides targeted to specific nicotinic acetylcholine receptor subtypes. Annu. Rev. Biochem. 68, 59–88. Moczydlowski, E., Olivera, B.M., Gray, W.R., Strichartz, G.R., 1986. Discrimination of muscle and neuronal Na-channel subtypes by binding competition between [3H] saxitoxin and mu-conotoxin. Proc. Natl. Acad. Sci., USA 83, 5321– 5325. Olivera, B.M., McIntosh, J.M., Cruz, L.J., Luque, F.A., Gray, W.R., 1984. Purification and sequence of a presynaptic peptide toxin from Conus geographus venom. Toxicon 23, 277 –282. Olivera, B.M., Rivier, J., Craig, C., Ramilo, C., Corpuez, G.P., Abogadie, F.E., Mena, E., Woodward, S.R., Hillyard, R., Cruz, L.J., 1990. Diversity of Conus neuropeptides. Science 249, 257 –263. Olivera, B.M., Rivier, J., Scott, J.K., Hillyard, D.R., Cruz, L.J., 1991. Conotoxins. J. Biol. Chem. 266, 22067–22070. Olivera, B.M., Walker, C., Cartier, G.E., Hooper, D., Santos, A.D., Schoenfeld, R., Shetty, R., Watkins, M., Bandyopadhyay, B., Hillyard, D.R., 1999. Speciation of cone snails and interspecific
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hyperdivergence of their venom peptides: potential evolutionary significance of introns. Ann. N Y. Acad. Sci. 870, 223–237. Possani, L.D., Selisko, B., Gurrola, G.B., 1999. Structure and function of scorpion toxins affecting Kþ channels. Perspect. Drug Discov. Des. 15/16, 15–40. Reiersen, H., Rees, A.R., 2000. Trifluoroethanol may form a solvent matrix for assisted hydrophobic interactions between peptide side chains. Protein Engng. 13, 739– 743. Sanguinetti, M.C., Jiang, C., Curran, M.E., Keating, M.T., 1995. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell 81, 299–307. Savarin, P., Guenneugues, M., Gilquin, B., Lamthanh, H., Gasparini, S., Zinn-Justin, S., Me´nez, A., 1998. Three-dimensional structure of k-conotoxin PVIIA, a novel potassium channel-blocking toxin from cone snails. Biochemistry 37, 5407–5416. Scanlon, M.J., Naranjo, D., Thomas, L., Alewood, D., Kent, S.B.H., 1997. Solution structure and proposed binding mechanism of a novel potassium channel toxin k-conotoxin PVIIA. Structure 5, 1585–1597. Shon, K., Stocker, M., Terlau, H., Stu¨hmer, W., Jacobsen, R., Walker, C., Grilley, M., Watkins, M., Hillyard, D.R., Gray, W.R., Olivera, B., 1998b. k-Conotoxin PVIIA inhibiting the Shaker Kþ channel. J. Biol. Chem. 273, 33–38. Smith, L., Reich, P.F., Wang, F.C., Parcej, D.N., Schmidt, J., Olson, M.A., Dolly, J.O., 1997. Site-directed mutagenesis of dendrotoxin k reveals amino acids critical for its interaction with neuronal Kþ channels. Biochemistry 36, 7690– 7696.
Stu¨hmer, W., 1992. Electrophysiological recording from Xenopus oocytes. Methods Enzymol. 207, 319–339. Stu¨hmer, W., Stocker, M., Sakmann, B., Seeburg, P., Baumann, A., Grupe, A., Pongs, O., 1988. Potassium channels expressed from rat brain cDNA have delayed rectifier properties. FEBS Lett. 242, 199–206. Swanson, R., Marshall, J., Smith, J.S., Williams, J.B., Boyle, M.B., Folander, K., Luneau, C.J., Antanavage, J., Oliva, C., Buhrow, S.A., 1990. Cloning and expression of cDNA and genomic clones encoding three delayed rectifier potassium channels in rat brain. Neuron 4, 929–939. Terlau, H., Shon, K., Grilley, M., Stocker, M., Stu¨hmer, W., Olivera, B., 1996. Strategy for rapid immobilization of prey by a fish-hunting marine snail. Nature 381, 148–151. Terlau, H., Boccaccio, A., Olivera, B.M., Conti, F., 1999. The block of Shaker Kþ channels by k-conotoxin PVIIA is state dependent. J. Gen. Physiol. 114, 125 –140. Tytgat, J., Buyse, G., Eggermont, J., Droogmans, G., Nilius, B., Daenens, P., 1996. Do voltage-gated Kv1.1 and inward rectifier Kir2.1 potassium channels form heteromultimers? FEBS Lett. 390, 280–284. Walker, C., Steel, D., Jacobsen, R.B., Liranzan, M.B., Cruz, L.J., Hooper, D., Shetty, R., DelaCruz, R.C., Nielsen, j.S., Zhou, L., Bandyopadhyay, P., Craig, A., Olivera, B.M., 1999. The Tsuperfamily of conotoxins. J. Biol. Chem. 274, 30664–30671. Warmke, J.W., Ganetzky, B., 1994. A family of potassium channel genes related to eag in Drosophila and mammals. Proc. Natl. Acad. Sci. USA 91, 3438–3442.
A novel conotoxin inhibiting vertebrate voltage-sensitive potassium channels Silke Kaufersteina,*, Isabelle Huysb, Hung Lamthanhc, Reto Sto¨cklind, Filipina Sottoe, Andre´ Menezc, Jan Tytgatb, Dietrich Mebsa a
Zentrum der Rechtsmedizin, University of Frankfurt, Kennedyallee 104, D-60596 Frankfurt am Main, Germany b Laboratory of Toxicology, University of Leuven, B-3000 Leuven, Belgium c Dept. d’Ingenierie et d’Etudes des Proteines, CEA/Saclay, F-91191 Gif-sur-Yvette, France d Atheris Laboratories, CH-1233 Bernex-Geneva, Switzerland e Marine Biology Section, San Carlos University, Cebu City, Philippines Received 4 March 2003; accepted 9 April 2003
Abstract Toxins from cone snail (Conus species) venoms are multiple disulfide bonded peptides. Based on their pharmacological target (ion channels, receptors) and their disulfide pattern, they have been classified into several toxin families and superfamilies. Here, we report a new conotoxin, which is the first member of a structurally new superfamily of Conus peptides and the first conotoxin affecting vertebrate Kþ channels. The new toxin, designated conotoxin ViTx, has been isolated from the venom of Conus virgo and comprises a single chain of 35 amino acids cross-linked by four disulfide bridges. Its amino acid sequence (SRCFPPGIYCTSYLPCCWGICCSTCRNVCHLRIGK) was partially determined by Edman degradation and deduced from the nucleotide sequence of the toxin cDNA. Nucleic acid sequencing also revealed a prepropeptide comprising 67 amino acid residues and demonstrated a posttranslational modification of the protein by releasing a six-residue peptide from the C-terminal. Voltage clamp studies on various ion channels indicated that the toxin inhibits the vertebrate Kþ channels Kv1.1 and Kv1.3 but not Kv1.2. The chemically synthesized product exhibited the same physiological activity and identical molecular mass (3933.7 Da) as the native toxin. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Conotoxin; Voltage-sensitive potassium channel; Voltage clamp electrophysiology; Structure; Novel conotoxin superfamily; Posttranslational modification
1. Introduction Cone snails (Conus species; suborder: Toxoglossa) represent a large genus of venomous gastropods consisting of more than 500 species. Each snail produces a complex venom containing a large number of functionally different toxins. In all Conus venoms, small peptides of 10 – 35 amino acids represent the major biologically active compounds. These peptides are often disulfide-rich and exert a wide * Corresponding author. Tel.: þ 49-69-6301-7564; fax: þ 49-696301-5882. E-mail address: [email protected] (S. Kauferstein).
range of physiological effects, e.g. targeting various receptors, voltage or ligand-gated ion channels (Olivera et al., 1990, 1991). Conus toxins have been classified into major superfamilies according to their cysteine framework (McIntosh et al., 1999). Whereas hypervariability in amino acid sequence has been observed in the regions between the cysteine residues, the signaling sequence in the precursor structure is particularly conserved (McIntosh et al., 1999; Olivera et al., 1999). The high selectivity of conotoxins for molecular targets in the nervous system has made them important pharmacological tools to identify or to distinguish receptors and ion channel subtypes or to map the functional
0041-0101/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0041-0101(03)00099-0
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surface of these receptors (Walker et al., 1999). For instance, a family of Conus peptides, a-conotoxins, targets the nicotinic acetylcholine receptor (Hopkins et al., 1995). m-Conotoxins inhibit Naþ channels of muscle membranes leaving Naþ channels of nerve membranes unaffected (Cruz et al., 1985; Moczydlowski et al., 1986). v-Conotoxins exhibit high affinity to certain subtypes of Ca2þ channels such as a conotoxin (GVIA) from Conus geographus, which specifically blocks N-type Ca2þ channels (Olivera et al., 1984; MacCleskey et al., 1987). So far, one peptide only, namely k-conotoxin PVIIA from the venom of Conus purpurascens, has been found to interact with a voltagegated Kþ channel (Terlau et al., 1996). k-Conotoxin is a 27residue, four-loop peptide with three disulfide bonds, which blocks the Kþ-conductance in oocytes expressing the cloned Shaker Kþ channel encoded from Drosophila, but no affinity to other Kþ channel subtypes, particularly to those of vertebrate origin has been found (Shon et al., 1998b; Terlau et al., 1999). The three-dimensional structure of the synthetic toxin was elucidated by NMR spectroscopy (Scanlon et al., 1997; Savarin et al., 1998). It was predicted that the determinant by which k-conotoxin PVIIA occludes the pore should include a binding diad, composed of Lys 7 and Phe 9 or Phe 23 (Savarin et al., 1998). This prediction was based on the observation that a similar critical diad is present in the determinant by which other toxins, even structurally unrelated, block voltage-gated Kþ channels (Dauplais et al., 1997; Gasparini et al., 1998). An alanine walk along the polypeptide chain of the toxin (Jacobsen et al., 2000) experimentally confirmed this prediction. In the present paper we report the isolation and characterization of a novel toxin from Conus virgo, which specifically inhibits voltage-gated vertebrate Kþ channels of the type Kv1.1 and Kv1.3. It consits of a polypeptide with 35 amino acid residues cross-linked by four disulfide bonds representing a structurally new family of conotoxins.
2. Materials and methods 2.1. Preparation of the venom extract Specimens of Conus virgo were collected in the Philippines (Olango Island and Bohol). The venom ducts were dissected from the snails, cut into small pieces and extracted with 5% acetic acid. After centrifugation of the extract, the supernantant (i.e. crude venom) was lyophilized. Dissected venom ducts were also placed in RNAlatere (Ambion Inc., Austin, USA), a tissue storage reagent which protects and stabilizes RNA. 2.2. Peptide purification The crude venom extract was fractionated by gel filtration on a Sephadex G-50 column (85 £ 1.5 cm;
Amersham Biotech, Budinghamshire, UK), which was eluted with 0.1 M ammonium acetate, pH. 6.8 at a flow rate of 0.5 ml/min. Further purification of the active fraction (as indicated by electrophysiological screening; see below) was performed by reversed-phase HPLC on a BDSHypersilw-C18-column (125 £ 4 mm; Hewlett Packard, Waldbronn, Germany) using a gradient of trifluoroacetic acid (0.1% in water, solvent A) and acetonitrile (60% in 0.1% trifluoroacetic acid, solvent B) for elution at a flow rate of 0.5 ml/min over 45 min. The effluent was monitored at 220 nm, peaks were collected, lyophilized and tested for biological activity. The active fraction was rechromatographed using the same column and gradient for elution, but over a period of 90 min, followed by chromatography of the active fraction on a Lichrocartw Purosphere column (250 £ 4 mm; Merck, Darmstadt, Germany) and eluted using a modified gradient (40% acetonitrile in 0.1% trifluoroacetic acid, solvent B, over 90 min at a flow rate of 0.3 ml/min). The coelution experiment was performed as followed. A mixture of synthetic and native ViTx were applied onto a 218TP104 HPLC C18 reversed phase column (Vydacw) equilibrated with 0.1% trifluoroacetic acid (Sigma, Belgium). After 4 min, a linear gradient from 0 to 60% acetonitrile was applied for 26 min. The flow rate was 0.75 ml/min. 2.3. Electrophysiology Capped cRNAs encoding ion channels were synthesized after linearizing the plasmids and performing the transcription with T7 polymerase by a standard protocol (Krieg and Melton, 1987). Capped cRNA of Kv1.1 (Baumann et al., 1988) was synthesized in vitro after linearizing the plasmid with PstI (New England Biolabs Inc., Beverly, MA, USA). Kv1.2 (Stu¨hmer et al., 1988) was subcloned by double digestion with BglII and EcoRI and ligated into the corresponding sites of the high-expression vector pGEMHE (Liman et al., 1992). For in vitro transcription, the cDNA was linearized with SphI. Kv1.3 (Swanson et al., 1990) was cloned into the vector pCl.neo and linearized with NotI for in vitro transcription. Oocytes from Xenopus laevis were prepared as previously decribed (Stu¨hmer, 1992). HERG (Warmke and Ganetzky, 1994; Sanguinetti et al., 1995). The HERG clone (HERG/pSP64) was first linearized with EcoRI and cRNA was synthesized using the large scale T7 mMESSAGE mMACHINE transcription kit (Ambion, USA). Kir2.1 (Kubo et al., 1993). The in vitro synthesis of cRNA encoding the IRK1 channel was as previously described (Tytgat et al., 1996). Stage V– VI Xenopus laevis oocytes were isolated by partial ovariectomy under anaesthesia (tricaine, 1 g/l). Anaesthetized animals were kept on ice during dissection. The oocytes were defolliculated by treatment with 2 mg/ml collagenase (Sigma, Belgium) in zero calcium ND-96 solution (see Solutions). Between 2 – 24 h after
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defolliculation, oocytes were injected with 50 nl of 1 – 100 ng/ml cRNA. The oocytes were then incubated in ND-96 solution at 18 8C for 1– 4 days. The whole cell currents of the oocytes were recorded using the two-microelectrode voltage clamp technique. Voltage and current electrodes (range: 0.4 –4 megaohms) were filled with 3 M KCl. Current recordings were sampled at 2 kHz after low pass filtering at 0.1 kHz. A pulse frequency of 0.2 Hz was chosen to maximize the recovery from inactivation of the channels. The solution was exchanged at a flow rate of 5 ml/min and the volume of the recording chamber was 100 ml. Offline analysis was done using a Pentium III processor computer. Linear components of capacity and leak current were not substracted. All experiments were performed at room temperature (19 – 22 8C). Oocytes were held at 2 90 mV and stepped to different test potentials. The bath solution was ND-96 containing (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 Hepes, pH 7.5, supplemented with 50 mg l21 gentamycine sulfate (only for incubation). Lyophilized toxins were dissolved and freshly diluted in the ND-96 solution. 2.4. Peptide sequencing N-terminal amino acid sequence analysis was performed by automated Edman degradation using an ABI Modell 47A/120A Protein– Peptide Sequencing/Analysis System and Analysis Software System (Applied Biosystems, Foster city, CA, USA). The toxin was reduced, alkylated with 4-vinylpyridine and HPLC-purified. 40 pmol to 1 nmol of the sample was loaded onto a trifluoroacetic acid-treated cartridge filter previously conditioned with BioBrene Pluse. Before each analysis, a calibration was done using a phenylthiohydantoin-amino acids standard solution. 2.5. Mass spectrometry Chromatography of the samples was performed by HPLC (C18, 150 £ 1 mm; Nucleosilw) followed by on-line liquid chromatography – electrospray-mass spectrometry. The mass spectrometer equipped with an electrospray ion source (Micromass Platform II, Micromass Altrincham, UK), was operated in positive-ionisation mode under control of the Mass Lynx data system with scanning at a mass to charge ratio range of 500– 1700 m/z at 6 s/scan cycle. 2.6. Structural studies by gene sequencing RNA from venom ducts was isolated according to standard procedures (Chomczynski and Sacci, 1987). To identify the toxin gene, 50 and 30 rapid amplification of cloned ends (RACE) experiments were carried out. 30 RACE was performend according to Frohman et al. (1989). A degenerated primer (50 -TGYTTYCCNCCNGGNATHTAYTG-30 )
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was designed according to the partial amino acid sequence as determined by Edman degradation. PCR conditions were as follows: 10 min 95 8C (1 cycle); 1 min 95 8C, 1 min 42 8C, 1 min 72 8C (30 cycles); 1 min 72 8C (1 cycle). 50 RACE was performed using cDNA produced with the SMART system (Clontech, Palo Alto, USA). Gene-specific primer sequences were as follows (IUB code; 50 ! 30 ): GSP1: CGTCCCACAGCAAATTCCCC; GSP 2: GCAAATTCCCCAACAGCAGGGG and the following PCR conditions were applied: 10 min 94 8C (1 cycle); 30 s 94 8C, 10 s 64 8C, 2 min 72 8C (30 cycles); 7 min 72 8C (1 cycle). Amplification products of the appropriate size were ligated into the T-tailed plasmid vector pGEMw-T (Promega, Madison, USA) which were then transformed into competent Escherichia coli. Plasmids containing inserts of the expected size were sequenced by the dye terminator method on ABI 310 automated sequencer. 2.7. Synthesis of ViTx conotoxin The linear peptide of ViTx conotoxin was synthesized using Fmoc (fluorenylmethyloxycarbonyl) chemistry on the ABI 430 automated peptide synthesizer (Applied Biosystems). Starting with 0.100 mmole of Fmoc-LLys(Boc)-PEG-PS-resin (PE-ABI), the peptide was constructed using a double couple cycle with TBTU (2(1H-benzotriazole1-yl)-1.1.3.3-tetramethyluronium tetrafluoroborate) as condensing reagent (Knorr et al., 1989) except for the coupling of the fifth to the sixth residue. The Pro5 – Pro6 peptide bond was revealed as a peculiar bond in this sequence. Therefore a manual coupling of the FmocPro5 to Pro6-peptide resin was carried out by using the HATU/HOAt (N-[(dimethylamino)-1H-1,2,3-triazolo[4,5b]pyridinyl-1-methylene]-N-methylmethanaminium hexafluorophosphate/1-hydroxy-7-azabenzotriazole) method (Carpino et al., 1994) before continuing the synthesis process for the last four residues on the synthesizer. The peptide was then cleaved from the resin by trifluoroacetic acid/thioanisole/m-cresol/ethanedithiol/water (90/2.5/2.5/ 2.5/2.5) for 2 h at room temperature. The crude linear peptide was precipitated, washed with cold ether and centrifuged. The pellet was dissolved in acetonitrile/water (50/50) and lyophilized. Several attempts to fold the linear peptide in an aqueous medium failed to yield the native form of ViTx. Hence, the best folding process proceeded in an aqueous-organic medium. 100 mg of crude peptide was loaded on a HPLC semi-preparative column (Vydacw, C18, 250 £ 25 mm) and eluted (10 ml/min) with a gradient from 50 to 100% of B solvent for 100 min (A solvent: 0.1% trifluoroacetic acid in water; B solvent: acetonitrile/0.1% trifluoroacetic acid in water 60/40). Fractions containing the linear peptide (MWcalc: 3941.0; MWobs: 3941.2) were mixed with 200 ml of isopropanol and 50 ml of trifluoroethanol. Then, 250 ml of ammonium bicarbonate (0.1 M; pH 8.5) and 1 mmol of reduced glutathione were added. The medium was adjusted at pH 8.5 with ammonia and left for 48 h at room temperature. After adjusting the pH to 4
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Fig. 1. Purification of the toxin ViTx. (A) Lyophilized venom from Conus virgo was dissolved in with 0.1 M ammonium acetate, pH 6.8, and applied onto a Sephadex G-50 column (85 £ 1.5 cm) using the same buffer for elution at a flow rate of 0.5 ml/min. (B). The active fraction (*) was separated by reversed-phase HPLC on a BDS-Hypersil-C18-column (125 £ 4 mm, 5 mM particle size) at a flow rate of 0.5 ml/min using a linear gradient from 0 to 60% acetonitrile in 0.1% trifluoractic acid over 45 min. (C) The active fraction as indicated in (B) was further purified on the same column with an slightly modified gradient (from 0 to 60% acetonitrile over 90 min). (D) A 218TP104 HPLC C18 reversed phase column (Vydacw), equilibrated with 0.1% TFA, was used for the coelution experiment. After 4 min, a linear gradient from 0 to 60% acetonitrile was applied for 26 min. The flow rate was 0.75 ml/min.
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with trifluoroacetic acid, the volume was diluted to 1 l with water, loaded directly onto the preparative Vydacw column, which was washed out with solvent A and eluted with the previous gradient of acetonitrile. Five fractions containing folded peptides were analyzed by analytical ˚ column, 250 £ 4.6 mm) and HPLC (Zorbax SB-C18, 300 A mass spectrometry. The most homogeneous fraction was repurified on a semipreparative column (Zorbax SB- C18, ˚ , column 250 £ 9.4 mm). The overall yield of the 300 A synthetic, folded ViTx toxin was 1 or 2% in weight of the starting crude linear peptide. Synthetic ViTx was characterized by amino acid analysis, analytical HPLC and mass spectrometry (MWcalc: 3933.7; MWobs: 3933.4) and submitted to bioassays.
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gel filtration (Fig. 1A). The major fraction containing peptides in a range of 1.5– 5 kDa was further separated into numerous fractions by reversed phase HPLC on a BDSHypersil-C18-column (Fig. 1B). Of the fractions tested on Kv1.1 and Kv 1.3 channels, one exhibited blocking activity (data not shown) and was rechromatographed on the same column using a lower gradient (Fig. 1C). It was finally purified on a HPLC Lichrocartw Purosphere column eluted with a modified acetonitrile gradient. Coelution of the synthetic and native toxin onto the C18 HPLC column (Vydacw) resulted in a single symmetric peak (Fig. 1D). Homogeneity of the purified toxin, named ViTx, was confirmed by electrospray ionization mass spectrometry (ES-MS) with a measured molecular mass of 3933.3 Da. 3.2. Amino acid sequence
3. Results 3.1. Toxin purification and characterization In a first set of experiments, the venom extract from Conus virgo exhibited blocking activity when tested on Kþ channels of the type Kv1.1 and Kv1.3 expressed in Xenopus oocytes, but no activity was observed on Kv1.2 channels. Initial fractionation of the crude venom was performed by
The N-terminal amino acid sequence of the reduced and pyridylethylated peptide was determined until position 27 (SRCFPPGIYCTSYLPCCWGICCSTCRN). Due to the low concentration of the toxin in the crude venom and its poor yield during purification, the complete sequence could not be established by Edman degradation. Therefore attempts were made to elucidate the toxins’ primary structure by sequencing its gene isolated from cDNA.
Fig. 2. Sequence encoding the prepropeptide of ViTx. The bottom line shows the combined nucleotide sequences of two clones encoding the precursor of ViTx, while the top line shows the deduced amino acids. The proposed start and stop signals are indicated by an aterisk, while the toxin coding region is underlined. The proposed posttranslational cleavage site is indicated by an arrow. The clones from the cDNA prepared from the venom duct of Conus virgo were achieved using PCR as described under Materials and methods.
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Fig. 3. Macroscopic Kþcurrent through Kv1.1, Kv1.2 and Kv1.3 channels expressed in Xenopus oocytes. Currents were obtained using the twomicroelectrode voltage clamp technique. Oocytes were held at 290 mV and stepped to a test potential of 0 mV. The effect of the concentration of the native ViTx is shown. Application of 4 mM native ViTx leads to approximately full block of the Kv1.1 channels. Addition of 0.8 mM native ViTx to the bath solution leads to approximately 36% blockage of the Kv1.3 Kþ currents, but has no effect on the Kv1.2 Kþ currents. Block induced by synthetic ViTx was identical.
3.3. Isolation of cDNA clones For identification of cDNA clones of ViTx gene fragments, degenerated primers were synthesized corresponding to position 3 – 12 and 15 – 22 (reverse direction) of the partial amino acid sequence and used as gene-specific primers in the 30 RACE (rapid amplification of cDNA ends). A cDNA fragment consisting of 313 bp was obtained, cloned and sequenced. From this nucleotide sequence, specific primers were designed to obtain overlapping 30 and 50 RACE products. Amplification with these primers in the 50 RACE experiment produced a specific product of 175 bp. The 50 and 30 RACE sequences were assembled to achieve the complete cDNA sequence of ViTx. The nucleotide sequence of the prepropeptide and of the deduced amino acid sequence is shown in Fig. 2. The 67 amino acid precursor of ViTx has a N-terminal-signal sequence, which is followed by 41 amino acids including the toxin’s sequence. Two amino acids, Lys – Arg near the C-terminus appear to serve as a proteolytic cleavage site for posttranslational processing of the peptide chain. According to mass-spectrometry data (MW: 3933.3), the toxin consists of a chain of 35 amino acids with 8 cysteines and is folded by four disulfide bonds. Lysine should represent the C-terminus after a sixresidue peptide has been released by proteolytic cleavage. The sequence was confirmend by chemical synthesis of the peptide.
the linear ViTx peptide in an aqueous buffer failed to yield the native form of ViTx in several attempts. Then, a mixture of organic solvents (e.g. acetonitrile, 2-propanol, trifluoroethanol) and water was found to be efficient for the folding process.
3.4. Chemical synthesis To verify the sequence from ViTx, the peptide was synthesized in a carboxylic form at the C-terminal side and folded as described under Section 2. The final yield of active peptide was 1 or 2% in weight of the starting crude peptide. The synthetic peptide elicited the same biological activity as the native peptide purified from the venom when tested on the vertebrate Kþ channels (Fig. 3). In addition the mass of the synthetic peptide, as determined by ES-MS (MW: 3933.6 Da), agreed with the mass calculated for the native toxin ViTx (MW: 3933.7). The folding process of
Fig. 4. Current/voltage ðItest =Vtest Þ curve for the block induced by ViTx. Oocytes were held at 290 mV and stepped to test potentials varying between 270 and þ 20 mV (mean ^ SD, n ¼ 3). Presence of 0.8 mM ViTx (X) reduced the Kþ currents of Kv1.1 (A) and Kv1.3 (B) to about 66 and 63% of the control ðoÞ value, respectively, in an almost non-reversible manner. There is no voltage-dependent block observed.
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3.5. Electrophysiological studies Initial voltage clamp experiments using venom fractions had suggested that the toxin ViTx inhibits homomeric vertebrate Kþ channels Kv1.1 (rat) and Kv1.3 (human), but not Kv1.2 (rat) expressed in Xenopus oocytes. Detailed electrophysiological studies with purified and synthetic toxin confirmend this observation. Native ViTx inhibited the currents through Kv1.1 and Kv1.3 channels but had no effect on Kv1.2 channels (Fig. 3). An identical effect has been observed with synthetic ViTx. Application of 0.8 mM synthetic peptide produced inhibtion of 33.4% of the Kþ current through Kv1.1 channels and of 36.1% of the Kþ current through Kv1.3 channels, respectively (data not shown). This blockage induced by ViTx exhibited no voltage-dependence, as the extent of blockage was not different in the range of test potentials from 220 to þ20 mV (Fig. 4). No blocking effect on the Kv1.2 Kþ current was observed (Fig. 3 and 5), e.g. the percentage of block after application of 2 mM of ViTx was about 0% and about 50% for Kv1.2 and Kv1.3 channels, respectively. Only a small recovery of Kþ currents was achieved by washing-out. The Kd values were 1.59 ^ 0.14 mM and 2.09 ^ 0.11 mM for Kv1.1 and Kv1.3 channels, respectively, deduced from the dose-response curve as shown in Fig. 5. Furthermore, ViTx binding did not alter channel gating. The maximum conductance ðgmax Þ curve for Kv1.3 (Fig. 6) fitted well with a single, first-order Boltzmann function. In a control experiment the half maximum potential ðV1=2 Þ was 219.8 ^ 1.4 mV. In the presence of
Fig. 6. Effects of ViTx gating. Maximal conductance-voltage ðgmax 2 Vtest Þ relationship for Kv1.3 in the absence ðoÞ or presence of 1 mM ViTx (X). The conductance-voltage curve was fitted with a single, first order Boltzmann function ðItail ¼ Imax =ð1 þ expððV1=2 Þ=sÞÞ; where Itail is the tail current at 250 mV, Imax is the maximum tail current, and s the slope factor of the voltage dependence. No ViTx-induced shift in the half-maximum potential of activation ðV1=2 Þ was observed (vertical dashed line). A similar effect was observed for Kv1.1 channels.
1 mM ViTx, a V1=2 value of 220.8 ^ 2.7 mV was obtained, which is not significantly different. A similar effect was observed for Kv1.1 channels. ViTx had no effect on other channels, such as HERG-type channels, Naþ hH1 channels and Kir-type channels (IRK1) (Fig. 7). Among the Kv1-type channels, the peptide seems to be a selective probe for Kv1.1 and Kv1.3 channels.
4. Discussion
Fig. 5. Dose-response curve for the block produced by ViTx. The Kd values were calculated according to the fits of the data points using the Hill equation: I ¼ Imax =½1 þ ðKd =½TÞh ; where I represents the current percentage, Imax is the maximal current percentage (both measured at 0 mV), Kd is the concentration of the toxin that evokes the half-maximal response, ½T is the concentration of the toxin, and h the Hill coefficient. The Hill coefficients were, respectively, 1.6 and 1.7 mV for Kv1.1 and Kv1.3 channels. The Kd value for Kv1.1 (B) was 1.59 ^ 0.14 mM and 2.09 ^ 0.11 mM for Kv1.3 (X), respectively. For Kv1.2 (O) no inhibition was observed in the concentration range indicated.
The data presented above demonstrate the presence of a peptide in a cone snail venom which interacts with vertebrate voltage-gated Kþ channels, e.g. of the Kv1.1 and Kv1.3 type. Electrophysiological characterization of the purified toxin was successfully achieved, however, elucidation of its structure by Edman degradation was incomplete due to the very low concentration of this peptide in the venom of Conus virgo. Cloning and sequencing of gene fragments obtained by PCR revealed that the peptide comprises a single chain of 35 amino acids cross-linked by four disulfide bridges. Chemical synthesis of ViTx produced, even in low yield, a quantity of toxin that enabled its biological characterization. Solid phase synthesis of the ViTx linear peptide did not display anymore difficulties other than the coupling of residue Pro5 to Pro6. The linear peptide was obtained in a reasonable yield (30 – 40%). The main step governing the overall yield seemed to be the folding step. First, difficulties were encountered in folding the linear peptide in an aqueous medium. This was probably due to the hydrophobic profile of the peptide according to Kyte and Doolittle (1982). Therefore, aqueous-organic solvent conditions (isopropanol/water
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Fig. 7. Effect of 2 mM ViTx on Kir-type channels and hERG channels expressed in oocytes. (A) Current recordings under two-microelectrode voltage clamp in the presence of 98 mM [Kþ]o. The holding potential ðVhold Þ was 0 mV. Traces in the absence (left) and presence (right) of 2 mM ViTx were elicited every 5 s by steps to Vtest of 2160 to 60 mV for 1 s. (B) hERG currents, in control (left) and in the presence of 2 mM ViTx (right), were evoked by application of 2 s test pulses ranging from 250 to þ 70 mV in 10 mV increments from a holding potential of 290 mV. Peak tail currents ðItail Þ were measured at a potential of 270 mV.
1/1) as described by Daly et al. (1999) were used. The presence of low trifluoroethanol concentration in the folding medium could assist the folding of secondary structure as suggest by Reiersen and Rees (2000) in contrast to the absence of trifluoroethanol (data not shown). Second, at least five HPLC peaks corresponding to different folded isomass peptides including the correctly folded toxin were observed (data not shown). Inhibitory activity to the Kþ channels of the synthetic peptide was identical to that of the native toxin. The new toxin has some homology (39%) to a peptide from the venom of Conus betulinus (SWISSPROT-databank, access no. AF20866), however, nothing is known about the biological activity of this peptide. Therefore, ViTx represents a new type of conotoxins, structurally and
pharmacologically, being the first known to act on vertebrate Kþ channels. Despite its specificity, the affinity of ViTx to Kv1.1 and Kv1.3 channels appears to be moderate, when compared with that of other toxins, e.g. scorpion or snake venom toxins (Possani et al. 1999; Smith et al., 1997). In the doseresponse curve as shown in Fig. 5, the half-maximum inhibitory concentration (IC50) of ViTx is in the micromolar range (1.59 and 2.09 mM for Kv1.1 Kv1.3 channels, respectively), whereas the k-conotoxin PVIIA, which blocks the Shaker Kþ channel, is effective in nanomolar concentration (IC50 is about 70 nM). This low binding affinity may be due to the particular structural features of ViTx. However, we can not exclude that this novel toxin performes higher affinities to other targets that remain to be
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elucidated; but it is interesting to note that ViTx displays selectivity among Kv1-type channels. For the k-conotoxin PVIIA it has been proposed that the toxin excerts its blocking activity by positioning a lysine into the vestibule of the channel pore (Savarin et al., 1998). However, the only lysine residue in ViTx is the last residue located at the C-terminus, which could make it rather unlikely that it is involved in binding to a channel structure. However, in the absence of knowledge of the 3D structure of the toxin and of the individual role of its amino acids, the presence of a typical dyad remains to be clarified.
Acknowledgements We thank Olaf Pongs for providing the cDNA for the Kv1.2 channel, Maria L. Garcia for the Kv1.3 clone, R.G. Kallen for the hH1 clone; Philippe Favreau (Atheris Laboratories, Geneva) and Robert Thai (CEA-Saclay) Denis Hochstrasser (University Hospital), Robin Offord (University Medical Center) and Keith Rose (GeneProt Inc.) in Geneva for their assistance in MS-characterization. The hERG clone was generously donated by Mark Keating, HHMI, University of Utah, Salt Lake City, UT, USA. The IRK1 cDNA clone was kindly provided by L. Y. Jan. Isabelle Huys is Research Assistent of the Flemish Fund for Scientific Research (F.W.O.-Vlaanderen). Part of this work was supported by a grant of the German Research Agency (DFG) and of the Ministry of Research and Technology (BMBF) to D. M.
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