Synthesis and characterization of Pi4, a scorpion toxin from Pandinus imperator that acts on K + channels Sarrah M’Barek 1 , Amor Mosbah 1 , Guillaume Sandoz 2 , Ziad Fajloun 1 , Timoteo Olamendi-Portugal 3 , Herve ´ Rochat 1 , Franc¸ois Sampieri 1 ,J.In ˜ aki Guijarro 4 , Pascal Mansuelle 1 , Muriel Delepierre 4 , Michel De Waard 2 and Jean-Marc Sabatier 1 1 Laboratoire International Associe ´ d’Inge ´ nierie Biomole ´ culaire et Laboratoire de Biochimie CNRS UMR 6560, IFR Jean Roche, Faculte ´ de Me ´ decine Nord, Marseille, France; 2 Laboratoire Canaux Ioniques et Signalization, Equipe mixte INSERM 99–31, CEA, Grenoble, France; 3 Institute of Biotechnology, National Autonomous University of Mexico, Cuernavaca, Mexico; 4 Unite ´ de RMN des Biomole ´ cules, De ´ pt. de Biochimie Structurale et Chimie, Institut Pasteur, CNRS URA 2185, Paris, France Pi4 is a 38-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the Chactidae scorpion Pandinus imperator. Together with maurotoxin, Pi1, Pi7 and HsTx1, Pi4 belongs to the a KTX6 subfamily of short four-disulfide-bridged scorpion toxins acting on K + channels. Due to its very low abundance in venom, Pi4 was chemically synthesized in order to better characterize its pharmacology and structural properties. An enzyme-based cleavage of synthetic Pi4 (sPi4) indicated half-cystine pair- ings between Cys6–Cys27, Cys12–32, Cys16–34 and Cys22– 37, which denotes a conventional pattern of scorpion toxin reticulation (Pi1/HsTx1 type). In vivo, sPi4 was lethal after intracerebroventricular injection to mice (LD 50 of 0.2 lg per mouse). In vitro, addition of sPi4 onto Xenopus laevis oocytes heterologously expressing various voltage-gated K + channel subtypes showed potent inhibition of currents from rat Kv1.2 (IC 50 of 8 p M )andShaker B(IC 50 of 3 n M ) channels, whereas no effect was observed on rat Kv1.1 and Kv1.3 channels. The sPi4 was also found to compete with 125 I-labeled apamin for binding to small-conductance Ca 2+ - activated K + (SK) channels from rat brain synaptosomes (IC 50 value of 0.5 l M ). sPi4 is a high affinity blocker of the Kv1.2 channel. The toxin was docked ( BIGGER program) on the Kv channel using the solution structure of sPi4 and a molecular model of the Kv1.2 channel pore region. The model suggests a key role for residues Arg10, Arg19, Lys26 (dyad), Ile28, Lys30, Lys33 and Tyr35 (dyad) in the inter- action and the associated blockage of the Kv1.2 channel. Keywords: Pi4; scorpion toxin; K + channels; half-cystine pairings; molecular docking. Pi4 is a K + channel-acting toxin that was isolated from the venom of scorpion Pandinus imperator [1]. It is a 38-mer peptide cross-linked by four disulfide bridges, and therefore belongs to the aKTX6 subfamily [2] of short-chain four- disulfide-bridged scorpion toxins acting on K + channels. The highest sequence identities of Pi4 are shared with members of this structural subfamily, i.e. 68% with Mauro- toxin (Scorpio maurus palmatus) [3,4], 66% with Pi7 (P. imperator)[1],61%withPi1(P. imperator) [5–7] and 45% with HsTx1 (Heterometrus spinnifer) [8,9]. The pri- mary structure of Pi4 also contains a variant of the consensus sequence of scorpion toxins (i.e. […]C 1 […] C 2 XXPC 3 […]C 4 […](A/S/G)XC 5 […]C 6 XC 7 […]C 8 instead of […]C 1 […]C 2 XXXC 3 […](G/A/S)XC 4 […]C 5 XC 6 […]for three-disulfide-bridged toxins) that is representative of toxins from the a KTX6 structural subclass. Both the classical and variant consensus sequences are thought to be responsible for folding of toxins according to a common a/b scaffold [10–13] independent of the toxin size and pharma- cology (except for Ca 2+ channel-acting toxins which fold according to an Ôinhibitor cystine knotÕ motif [14,15]). Recent 1 H-NMR analysis showed that synthetic Pi4 (sPi4) indeed exhibits the a/b scaffold [16]. This motif, from which arises the great functional diversity of scorpion toxins, is mainly composed of an a-helix connected to a b-sheet structure (two or three strands) by two disulfide bridges. The first report on native Pi4 by Olamendi-Portugal et al. [1] provided some preliminary data on its pharmacology: (a) it blocked completely and reversibly voltage-gated Shaker B channel expressed in Sf9 insect cells, at 100 n M toxin concentration (IC 50 value of 8 n M ), and (b) it competed with 125 I-labeled noxiustoxin (Centruroides noxius) for binding on rat brain synaptosomal membranes, with an IC 50 value of approximately 10 n M . Here, we report the first chemical synthesis of Pi4 in order to better characterize its pharmacology on the various K + channel subtypes generally recognized by toxins of the a KTX6 subfamily, i.e. insect Shaker B, rat SK, Kv1.1, Kv1.2 and Kv1.3. We also investigated the disulfide bridge Correspondence to J M. Sabatier, Laboratoire de Biochimie CNRS UMR 6560, et Laboratoire International Associe ´ d’Inge ´ nierie Biomole ´ culaire, IFR Jean Roche, Faculte ´ de Me ´ decine Nord, Bd Pierre Dramard, 13916 Marseille Cedex 20, France. Fax: +33 491 657595, Tel.: + 33 491 698852, E-mail: sabatier.jm@jean-roche.University-mrs.fr Abbreviations: HsTx1, toxin 1 from the scorpion Heterometrus spin- nifer; Kv channel, mammalian voltage-gated K + channel; Pi1, Pi4, Pi7, toxin 1, 4 or 7 from the scorpion Pandinus imperator; SK channel, small-conductance Ca 2+ -activated K + channel; sPi4, synthetic Pi4. (Received 7 April 2003, revised 11 June 2003, accepted 8 July 2003) Eur. J. Biochem. 270, 3583–3592 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03743.x organization of sPi4 as two distinct patterns of reticulation have been described for these toxins hitherto, which are herein referred to as the maurotoxin (C 1 –C 5 ,C 2 –C 6 ,C 3 –C 4 and C 7 –C 8 ) [3] and Pi1/HsTx1 (C 1 –C 5 ,C 2 –C 6 ,C 3 –C 7 and C 4 –C 8 ) [7,9] types. Because sPi4 was found to be active at a picomolar concentration range on rat Kv1.2 channel, we detailed the interaction of the toxin with the latter channel by in silico docking experiments. The 1 H-NMR solution structure of sPi4 [16] and a molecular model of the rat Kv1.2 channel pore region (S5–H5–S6 portion) were used for this purpose. The data were used to generate a functional map of Pi4 towards this channel, highlighting some key residues of this interaction such as those belonging to the toxin functional dyad (a well-defined pair of residues that is thought to be crucial for toxin blocking efficacy towards the voltage-gated K + channels). Experimental procedures Materials N-a-Fmoc- L -amino acids, 4-hydroxymethylphenyloxy (HMP) resin, and reagents used for solid-phase chemical synthesis of Pi4 were purchased from Perkin-Elmer. Organic solvents were analytical grade products from SDS. Enzymes (trypsin, and chymotrypsin) were obtained from Boehringer Mannheim. Solid-phase synthesis of sPi4 The sPi4 was synthesized by the solid-phase method [17] using an automated peptide synthesizer (model 433A, Applied Biosystems Inc.). Peptide chains were assembled by conventional stepwise synthesis on 0.3 molar equivalents of HMP resin (1% cross-linked; 0.69 molar equivalents of amino group g) using 1 mmol of N-a-fluorenylmethyloxy- carbonyl (Fmoc) amino acid derivatives [7,18]. The side- chain protecting groups of sPi4 trifunctional residues were: tert-butyl (t-Bu) for Ser, Thr, Tyr, Asp, and Glu; trityl (Trt) for Cys, Asn, and Gln; pentamethylchroman (Pmc) for Arg, and tert-butyloxycarbonyl (Boc) for Lys. N-a-Amino groups were deprotected by treatments with 18% and 20% (v/v) piperidine/N-methylpyrrolidone for 3 and 8 min, respectively. The peptide resin was washed with N-methyl- pyrrolidone (5 · 1 min), then Fmoc-amino acid derivatives were coupled (20 min) as their hydroxybenzotriazole active esters (OBt) in N-methylpyrrolidone (3.3-fold excess). After complete peptide chain assembly and removal of the N-terminal Fmoc group, the peptide-resin (c. 2.1 g) was treated under stirring for 3 h at room temperature with a mixture of 88% trifluoroacetic acid/5% H 2 O/5% thioani- sole/2% ethanedithiol (v/v) in the presence of crystalline phenol (2.5 g) in a final volume of 30 mLÆg )1 of peptide resin. The peptide mixture was filtered to remove the resin, and the filtrate was precipitated and washed twice with cold diethyl ether. After centrifugation at 2800 g for 12 min, the supernatant was discarded and the crude peptide was dissolved in H 2 O and lyophilized. Oxidative folding of the reduced peptide was performed by dissolving the lyophilized peptide ( 1m M final concentration) in 0.2 M Tris/HCl buffer, pH 8.4 and gentle stirring under air for 72 h at 25 °C. The sPi4 was purified to homogeneity by semipreparative reverse-phase HPLC (Perkin-Elmer, C 18 Aquapore ODS 20 lm, 250 · 10 mm) by means of a 60 min linear gradient of 0.08% trifluoroacetic acid/0% to 35% acetonitrile (v/v) in 0.1% trifluoroacetic acid/H 2 O (v/v) at a flow rate of 6mLÆmin )1 (k ¼ 230 nm). The correct identity and the high degree of homogeneity of sPi4 were established by: (a) analytical C 18 reverse-phase HPLC (Chromolith RP18, 5 lm, 4.6 · 100 mm) using a 40-min linear gradient of 0.08% trifluoroacetic acid/0–60% acetonitrile (v/v) in 0.1% (v/v) trifluoroacetic acid/H 2 O (v/v) at a flow rate of 1 mLÆmin )1 ; (b) amino acid composition after acidolysis [6 M HCl/2% phenol (w/v), 20 h, 118 °C, N 2 atmosphere); (c) Edman sequencing; and (d) molecular mass analysis by MALDI-TOF mass spectrometry. Assignment of sPi4 half-cystine pairings The sPi4 (800 lg) was incubated with a mixture of trypsin and chymotrypsin at 10% (w/w) each, in 0.2 M Tris/HCl buffer, pH 7.4 (14 h, 37 °C). The resulting peptides were separated by analytical reverse-phase HPLC (Chromolith RP18, 5 lm, 4.6 · 100 mm) with a 60-min linear gradient of 0.08% (v/v) trifluoroacetic acid/0–40% acetonitrile in 0.1% (v/v) trifluoroacetic acid/H 2 O at a flow rate of 1mLÆmin )1 (k ¼ 230 nm), and freeze-dried prior to ana- lysis. The peptides were treated by acidolysis (6 M HCl/ phenol) and their amino acid contents were determined (Beckman, System 6300 amino acid analyzer). The peptides were also analyzed by mass spectrometry (RP-DE Voyager, Perseptive Biosystems), and Edman sequencing using a gas-phase microsequencer (Applied Biosystems 470A). In standard HPLC conditions for analyzing phenylthio- hydantoin (PTH) amino acid derivatives, diPTH-cystine eluted at a retention time of 9.8 min. Lethal activity of sPi4 in mice The sPi4 was tested in vivo for neurotoxicity by determining the 50% lethal dose (LD 50 ) after intracerebroventricular injection of 20 g C57/Bl6 mice (animal testing agreement number 006573 granted by the department Sante ´ et Protection Animales, Ministe ` re de l’Agriculture et de la Peˆ che). Groups of four to six mice per dose were injected with 5 lL sPi4 solution containing 0.1% bovine serum albumin and 0.9% sodium chloride (w/v). Binding of sPi4 on SK channels from rat brain synaptosomes Rat brain synaptosomes (P2 fraction) were prepared according to Gray and Whittaker [19]. Protein content was assayed by a modified Lowry method. [ 125 I]Apamin (2000 CiÆmmol )1 ) was obtained as described [20]. Aliquots of 50 lL0.1n M [ 125 I]apamin were added to 400 lLof synaptosome suspension (0.4 mg proteinÆmL )1 ). The sam- ples were incubated for 1 h at 0 °Cwith50lL of one of a series of concentrations of sPi4 or apamin (10 )2 )10 )13 M )in 500 lL final volume. The incubation buffer was 25 m M Tris/HCl, 10 m M KCl, pH 7.2. The samples were centri- fuged and the resulting pellets were washed three times with 1 mL of the same buffer. Bound radioactivity was counted (Packard Crystal II). Reported values represent the means 3584 S. M’Barek et al. (Eur. J. Biochem. 270) Ó FEBS 2003 of triplicate experiments. Nonspecific binding was evaluated in the presence of an excess (10 n M ) of unlabeled apamin and was shown to be less than 8% of the total binding. Electrophysiology Oocyte preparation. Xenopus laevis oocytes (stages V and VI) were recovered and prepared for cRNA injection and electrophysiological recordings as described [21]. The oocyte follicular cell layers were removed by enzyme treatment using 2 mgÆmL )1 collagenase IA (Sigma) in classical Barth’s medium [in m M :88NaCl,1KCl,0.82MgSO 4 , 0.33 Ca(NO 3 ) 2 ,0.41CaCl 2 , 2.4 NaHCO 3 , 15 N-2-hydroxy- ethylpiperazine-N¢-ethanesulphonic acid (Hepes), pH 7.4 with NaOH]. The plasmids were cleaved with SmaI (Shaker B), Not1(ratKv1.1),Xba1 (rat Kv1.2) and EcoR1 (rat Kv1.3). The linearized plasmids were transcribed by means of a T7 or SP6 mMessage mMachine kit (Ambion). The cRNAs (1 lgÆlL )1 ) were kept frozen in H 2 Oat)80 °C. The cells were microinjected 2 days later with 50 nL of cRNA (0.2 lgÆlL )1 Shaker B, rat Kv1.1, Kv1.2, or Kv1.3 channels). To favor ion channel expression, cells were incubated at 16 °C in a defined nutrient oocyte medium [22] 2 to 6 days before current recordings. Electrophysiological recordings. A standard two-micro- electrode technique was used to record oocyte currents (20– 22 °C). The current and voltage electrodes were filled with 140 m M KCl and had resistance values ranging between 0.5 and 1 MX. The recordings of potassium currents were performed using a voltage-clamp amplifier (GeneClamp 500, Axon Instruments, Foster City, CA, USA) interfaced with a 16-bit AD/DA converter (Digidata 1200 A, Axon Instruments) for acquisition and voltage protocol applica- tion. Current records were sampled at 10 kHz and low pass- filtered at 2 kHz using an eight-pole Bessel filter. The data were stored on a computer for subsequent analysis. The extracellular recording solution contained (in m M ): 88 NaCl, 10 KCl, 2 MgCl 2 , 0.5 CaCl 2 , 0.5 niflumic acid, 5 Hepes, pH 7.4 (NaOH). Leak and capacitive currents were subtracted on-line by a P/4 protocol. Residual capacitive artifacts were blanked for display purposes. The sPi4 solutions were perfused in the recording chamber at a flow rate of 2 mLÆmin )1 using a ValveBank4 apparatus (Auto- mate Scientific Inc.). Bovine serum albumin (0.1%) was added to the recording and perfusion solutions to prevent toxin loss to the plastic chamber and tubules and nonspecific binding onto the cell. Data analysis was performed using the software PCLAMP 6.0.3 (Axon Instruments, Foster City, CA, USA). Results are presented as mean ± SEM. Molecular modeling of rat Kv1 channel subtypes Molecular modeling of rat Kv1 channel subtypes was based on the crystal structure of KcsA solved at 2.0 A ˚ resolution (PDB accession number 1K4C) [23]. The amino acid sequences from residues 323–422 (corresponding to domains S5–H5–S6) of the rat voltage-gated K + channel a-subunits [24] were aligned with region 26–125 of KcsA. The sequence identities between the Kv-type ion channel regions and KcsA are approximately 30%. For the regions between the two transmembrane segments S5 and S6, the sequence identities with KcsA are approximately 50%. Based on the high degree of similarity, the S5–H5–S6 regions of rat Kv1.1, Kv1.2, and Kv1.3 channels (Swiss-Prot accession numbers P10499, A33814 and P15384, respect- ively) were modeled by homology methods. Sequences were aligned with clustal [25], the residue mutations were introduced in the KcsA channel structure with the TURBO - FRODO software [26], and the structures of the Kv channels thus obtained were minimized by using CNS [27]. The final molecular models of the S5–H5–S6 regions of the rat Kv1.1, Kv1.2 and Kv1.3 channels adopt 3D structures which are similar to that of the KcsA channel (not shown). The transmembrane a-helices are comprised between residues 322–344 (S5) and 383–413 (S6). The pore region contains one a-helix between residues 360–370. Of note, the ion conducting pathway region is formed by four S5–H5–S6 domains. The quality of all molecular models was assessed using the programs WHAT IF [28] and PROCHECK [29]. In each case, the stereochemical quality and the Ramachandran [30] scores were good and similar to that of the template. Docking of Pi4 on rat Kv1.2 channel The experimental 3D structure of sPi4 in solution, as recently solved by 1 H-NMR [16], was used in docking experiments together with the molecular models we gener- ated for rat Kv channel subtypes (S5–H5–S6 domains). The molecular interaction simulations were performed using BIGGER , a docking program [31]. The algorithm used by BIGGER performs a complete and systematic search for surface complementarity (both geometry complementarity and amino acid residue pairwise affinities are considered) between two potentially interacting molecules, and enables an implicit treatment of molecular flexibility. In each case, a population of 1000 candidate protein–protein-docked geometries was selected by BIGGER . In a subsequent step, the docked structures were ranked using an interaction scoring function, which combines several interaction terms that are thought to be relevant for the stabilization of protein complexes: geometric packing of the surfaces, electrostatic interactions, desolvation energy, and pairwise propensities of the residue sidechains to contact across the molecular interface. In the ab initio simulations, the entire molecular surface was searched using absolutely no additional infor- mation regarding the binding sites. Among the 1000 candidate protein–protein-docked geometries selected, the five best scoring Pi4–Kv1.2 channel complexes were further treated with the TURBO - FRODO software, taking also into account the proposed functional maps of voltage-gated K + channel-acting scorpion toxins [32–38]. Finally, a rigid body minimization was used to minimize the selected complexes. The best energy solutions, corresponding to the most favorable Pi4–Kv1.2 channel complexes, were selected. The de visu analysis was carried out using the TURBO - FRODO software, and the geometric quality of the structures was assessed by PROCHECK 3.3 [39]. Results and discussion Pi4 (Fig. 1) shares its highest sequence identities with scorpion toxins belonging to the a KTX6 subfamily: maurotoxin (68%) [3,4], Pi7 (66%) [1], Pi1 (61%) [5–7], Ó FEBS 2003 Synthesis and pharmacology of Pi4 (Eur. J. Biochem. 270) 3585 and HsTx1 (45%) [8,9]. This subclass of K + channel blockers contains short-chain toxins, from 34 to 38 amino acid residues, cross-linked by four disulfide bridges (instead of the three disulfide bridges generally observed in other K + channel-acting scorpion toxins). To better characterize Pi4, we chemically synthesized the toxin by using the solid-phase technique [17]. Production of synthetic Pi4 (sPi4) The Pi4 backbone assembly was achieved stepwise on 0.3 mmol HMP resin by means of an optimized Fmoc/t-Bu chemical strategy [18]. We found that the amount of target peptide linked to the resin was 0.21 mmol, which indicates a 70% yield of peptide assembly. Accordingly, a relative homogeneity of the crude reduced peptide was observed after final acidolytic treatment, as assessed by analytical C 18 reverse-phase HPLC (Fig. 2). The crude peptide was folded/ oxidized for 72 h in alkaline conditions using a standard oxidative buffer [7], and the main oxidized species (sPi4) was purified to >99% homogeneity by preparative C 18 reverse- phase HPLC (Fig. 2). An amino acid analysis of this purified product showed amino acid ratios that were consistent with the values deduced from the Pi4 primary structure (Fig. 3). The mass spectrometry analysis (MALDI-TOF technique) of the peptide gave an experi- mental M r (M + H) + of 4180.7, which is similar to the M r (M + H) + of 4180.9 calculated for Pi4 from its sequence. The identity and homogeneity of sPi4 were also verified by Edman sequencing (data not shown). The total yield of sPi4 synthesis (which combines yields of peptide assembly, final acidolytic treatment, oxidative folding and peptide purifi- cation), was approximately 3% (9 lmol). Disulfide bridge pattern of sPi4 In order to assign half-cystine pairings, sPi4 was treated by a mixture of enzymes (trypsin and chymotrypsin). The resulting peptide fragments were separated by HPLC, then characterized by amino acid analysis, Edman sequencing and mass spectrometry (Fig. 4). The results obtained from these techniques unambiguously map the half-cystine pair- ings as Cys6–Cys27, Cys12–Cys32, Cys16–Cys34 and Cys22–Cys37. Therefore, the disulfide bridge organization of sPi4 is of the conventional type being C 1 –C 5 ,C 2 –C 6 ,C 3 – C 7 and C 4 –C 8 , a pattern identical to that of Pi1 or HsTx1 but different from that of maurotoxin. Of note, the disulfide bridges of natural Pi4 have been determined by NMR and structure calculations, as well as by Edman sequencing/mass spectrometry identification of peptides obtained by proteo- lysis of natural Pi4 [16]. Both approaches have indicated half-cystine pairings between Cys6–Cys27, Cys12–Cys32, Cys16–Cys34 and Cys22–Cys37 for the natural toxin, consistent with the disulfide bridge arrangement found experimentally for sPi4. As expected, analysis of 2D 1 H- NMR spectra of natural Pi4 and sPi4 [16] indicates that both peptides have the same structure. Biological properties of sPi4 In our bioassays, we tested sPi4 rather than its natural counterpart as the latter is present in too low abundance in the venom of scorpion P. imperator to allow a detailed analysis of its structural and pharmacological properties. In vivo, sPi4 injected intracerebroventricularly produced Fig. 1. Amino acid sequence (one-letter code) of Pi4 and comparison with other related scorpion toxin sequences. The amino acid sequences of Pi4 (P. imperator) [1], maurotoxin (S. maurus palmatus) [4], Pi7 and Pi1 (P. imperator) [1,5–7], and HsTx1 (H. spinnifer)[8]were aligned according to the eight half-cystine residues. The positions of half-cystine residues are highlighted in gray boxes and numbered from N- to C-terminus. The asterisk indicates a C-terminal carboxylami- dated extremity. Fig. 2. Analytical C 18 reverse-phase HPLC profiles of Pi4 at different stages of its chemical synthesis. (A) The crude reduced peptide after final trifluoroacetic acid treatment. (B) The crude peptide after 72 h folding/oxidation. (C) The purified folded/oxidized peptide, sPi4. For conditions, see Experimental procedures. 3586 S. M’Barek et al. (Eur. J. Biochem. 270) Ó FEBS 2003 lethal effects in mice, with an LD 50 value of 0.2 lgper mouse. This activity is identical to that of Pi1, but 2.5-fold less potent than that of maurotoxin. The lethal effects found for sPi4 are about 10-fold less potent than those produced by K + channel-acting scorpion toxins reticulated by three disulfide bridges [40,41]. The sPi4-induced neurotoxic symptoms resembled those of other K + channel-acting scorpion toxins suggesting that it targets some K + channels. In vitro, we first tested the ability of sPi4 to compete with 125 I-labeled apamin for binding on rat brain synaptosomes. Figure 5 illustrates an sPi4-induced, concentration-depend- ent, inhibition of 125 I-labeled apamin binding, with an IC 50 value of 0.5 ± 0.2 l M . At a similar concentration, unlabe- led apamin produced a complete inhibition (IC 100 )of 125 I- labeledapaminbinding,withanIC 50 value of 6 ± 3 p M . The half-effect of sPi4 occurred at a 100- or 10 000-fold higher concentration than that required for maurotoxin [3,4,42] or Pi1 [7], respectively, indicating a low, but significant, affinity interaction of sPi4 with rat brain apamin-sensitive SK channels. Of note, HsTx1 was reported to be inactive for binding on these SK channels [8], whereas Pi7 binding capability on the latter has not been described. The blocking efficacy of sPi4 was also investigated by electrophysiology on different subtypes of K + channels that were heterologously expressed in Xenopus laevis oocytes. We focused on K + channels recognized by toxins from the a KTX6 subfamily, i.e. Shaker B, rat Kv1.1, Kv1.2 and Kv1.3 subtypes, and studied the putative sPi4-induced dose- dependent inhibition of currents associated with these channels. Figure 6A shows that the application of 100 n M sPi4 blocked over 80% of Shaker B currents. The dose– response curves for sPi4 current inhibition were obtained for Shaker B (Fig. 6B), rat Kv1.2 (Fig. 6C), rat Kv1.1 and Kv1.3 channels (Fig. 6D). The IC 50 values of sPi4 were 3.0 ± 2.2 n M for Shaker B(n ¼ 45) and 8 ± 5 p M for rat Kv1.2 (n ¼ 55) channels, whereas it had no detectable effect at concentrations up to 10 l M on rat Kv1.1 (n ¼ 10) and Kv1.3 (n ¼ 10) channels. It is noteworthy that the maximal extent of blockage of K + currents by sPi4 was approxi- mately 80% (Shaker B) or 60% (rat Kv1.2) of total currents. Similar partial current blocks (from 50% to 80% of total currents) of these channels have been described for several scorpion toxins and their analogs, including Pi1 and maurotoxin [7,42–45]. This phenomenon remains difficult to explain, but an incomplete permeability block, possibly associated to an imperfect ion channel pore occlusion, can Fig. 4. Assignment of sPi4 half-cystine pairings. (A) Characterization of the sPi4-derived peptides that were generated by enzyme-based cleavage of sPi4 (see Experimental procedures). Retention times in HPLC (left column) and identified half-cystine pairings (right column) of the peptides are shown. (B) Complete disulfide bridge organization of sPi4 as experimentally established by proteolysis of the synthetic toxin. The half-cystine residues are numbered according to their positions in the Pi4 amino acid sequence. The half-cystine connections are represented by solid lines. Fig. 5. Binding of sPi4 on apamin-sensitive SK channels from rat brain synaptosomes. Concentration-dependent inhibition of binding of [ 125 I]apamin to rat brain synaptosomes by either unlabeled apamin (d) or sPi4 (s) in a competition assay. B 0 is the binding of [ 125 I]apamin without any other ligand, and B is the binding in the presence of the indicated concentrations of competitor. Abscissa axis is the logarithm of the molar concentration of competitor. Nonspecific binding, less than 8% total binding, was subtracted for the calculation of ratios. When absent, error bars are within symbol size. The data were fitted to the equation y ¼ y o + a/[1+exp(–(x ) IC 50 )/b)]. The resulting IC 50 values are 0.5 ± 0.2 l M (sPi4) and 6 ± 3 p M (unlabeled apamin). Fig. 3. Physicochemical characterization of sPi4. Aminoacidcontent (uncorrected values) of sPi4 after hydrolysis (118 °C, 20 h, N 2 atmo- sphere) with 6 M HCl in the presence of 2% (w/v) phenol. The deduced amino acid composition is shown in parenthesis. Deduced and experimental relative molecular masses are indicated. Ó FEBS 2003 Synthesis and pharmacology of Pi4 (Eur. J. Biochem. 270) 3587 tentatively be proposed. Similar phenomena have also been reported in a number of cases [42,43,45]. From the experimental data obtained both in vivo and in vitro,sPi4 appears to be pharmacologically more closely related to Pi1 than to maurotoxin, Pi7 or HsTx1. Indeed, the two toxins apparently share the same lethal effects and selectivity profile towards the tested K + channel subtypes (SK, Shaker Band rat Kv1.2 channels), although their binding properties or blocking efficacies towards these channels are clearly distinct. At the structural level, this should obviously rely on marked differences of amino acid sequences between both toxins, which guide the number and/or spatial positioning of key functional residues that participate in the interaction with the ion channel pore protein. These data strengthen the idea of a multipoint interaction between scorpion toxins and their target ion channels. As sPi4 blocked rat Kv1.2 channel at low picomolar concentrations, we examined the interaction between Pi4 and this channel at the molecular level, using the docking program BIGGER [31]. Docking experiments To perform computed docking experiments, the structure of sPi4 in solution recently solved from 1 H-NMR data [16] was used, and specific models of rat voltage-gated K + channels (S5–H5–S6 domains) [24] were generated. According to the docking simulation (Fig. 7A–C), the toxin–ion channel complex is stabilized by four salt bridges between the sidechains of Glu332 of each rat Kv1.2 a-subunit (Kv1.2 channel is composed of four a-subunits) and Arg10, Arg19, Lys30 and Lys33 of Pi4. The Lys26 sidechain of Pi4 enters into the ion channel pore and is surrounded by the four Asp357 carbonyl oxygen atoms of the P-domain selectivity filter. Residue Tyr35 of Pi4 is involved in a hydrophobic cluster of aromatic residues consisting of Trp344, from one of the four Kv1.2 a-subunits, and Trp345 and Tyr355 of an adjacent a-subunit. The phenol ring of Tyr35 additionally forms an hydrogen bond with the N e of the Trp344 indole ring. Some hydrophobic interactions are also likely to occur between Ile28 of Pi4 and Val361 of the Kv1.2 a-subunit. For comparison, Pi1 was also docked on the Kv1.2 channel (data not shown). Similar types of low- energy interactions were found but involving Arg5, Arg12, Lys24 (dyad), Ile26, Arg28, Lys31 and Tyr33 (dyad) residues of Pi1. However, in the case of Pi1, toxin positioning over the channel was different, with a slight rotation over the channel as compared to Pi4, its Tyr33 being in contact with the cluster of aromatic residues belonging to the same Kv1.2 a-subunit. Of note, Pi4 (or Pi1) did not give good scores when assayed for docking on rat Kv1.1 and Kv1.3 channels (data not shown), in agreement with its lack of bioactivity on these channels. Functional maps of Pi4 and Pi1 towards rat Kv1.2 channels Results from docking experiments allow us to propose functional maps for both Pi4 and Pi1 regarding their Fig. 6. Blocking efficacy of sPi4 towards the voltage-gated K + channel subtypes. (A) Oocytes expressing Shaker BK + channels were recorded under two-electrode voltage clamp. K + currents were obtained by depolarization from a holding potential of )90 mV to +70 mV. Left panel: Shaker B K + control currents during superfusion of 100 n M of sPi4 illustrating over 80% block; right panel: K + currents during superfusion of 100 n M sPi4, illustrating over 60% block. The dose–response curves for sPi4 current inhibition were performed for: (B) Shaker B, (C) rat Kv1.2, and (D) rat Kv1.1 (s)andKv1.3(d) channels. The solid lines through the data are obtained from the equation y ¼ y o + a/[1+exp(–(x ) IC 50 )/b)]. The IC 50 values of sPi4 were 3.0 ± 2.2 n M for Shaker B(n ¼ 45) and 8 ± 5 p M for rat Kv1.2 (n ¼ 55) channels. No significant effects on rat Kv1.1 (n ¼ 10) and Kv1.3 (n ¼ 10) channels were detected at sPi4 concentrations up to 10 l M . Data points are the mean ± SEM. When absent, error bars are within symbol size. All inhibitions were determined by inducing currents by depolarizations at +70 mV. 3588 S. M’Barek et al. (Eur. J. Biochem. 270) Ó FEBS 2003 recognition and blockage of rat Kv1.2 channels Fig. 8A,B). These maps suggest an important contribution of Arg10, Arg19, Lys26, Ile28, Lys30, Lys33 and Tyr35 residues for Pi4, as well as of Arg5, Arg12, Lys24, Ile26, Arg28, Lys31 and Tyr33 residues for Pi1. The functional dyads are attributed to Lys26 and Tyr35 for Pi4 [1], and Lys24 and Tyr33 for Pi1 [7]. Therefore, as mentioned by Olamendi- Portugal et al. [1], the substitution of the functional Lys26 in Pi4 for an Arg26 in the structurally homologous Pi7 might be a key natural Ôpoint mutationÕ responsible for the lack of activity of Pi7 on Kv channels. We suggest a two- step pictorial view of Pi4 binding in which the toxin ring of basic residues (ring composed of Arg10, Arg19, Lys30 and Lys33) plays a crucial role (via electrostatic forces) in the recognition, interaction and correct positioning of Pi4 on the Kv1.2 channels, and then a tighter interaction takes place through both hydrophobic forces and hydrogen bonding between Tyr35 (dyad) and the aromatic cluster consisting of Trp344, Trp345* and Tyr355* (Fig. 7B, legend), and between Ile28 and Val361. The Lys26 (dyad) sidechain enters the ion channel pore and is stabilized by the four Asp357 carbonyl oxygen atoms of the Kv1.2 a-subunits; the Lys sidechain presumably acts by blocking K + ion flux through the pore, and might thus be involved in the toxin blocking efficacy. Presence of the ring of basic residues in other toxins active on Kv1.2 channels To examine the potential importance of the ring of basic residues in the recognition and interaction of Pi4 with Kv1.2 channels, we focused on two scorpion toxins, Pi2 (P. im- perator) [6,46] and TsTXa (Tityus serrulatus) [47], that are also classified as high affinity blockers of Kv1.2 channels (both being active at the picomolar concentration range) and of known 3D structures [48,49]. The two toxins possess well-defined b-sheet-associated functional dyads, i.e. Lys27 and Tyr36 (TsTXa) and Lys24 and Phe33 (Pi2). Of note, the usual aromatic Tyr is replaced by an aromatic Phe in the case of Pi2, which is thought to interact, via its phenyl ring, with the aromatic cluster of the Kv1.2 a-subunit as well. Pi2 and TsTXa also exhibit a four-membered ring of basic residues similar to that of Pi4 or Pi1. It is clear that more structure–activity relationship studies on these toxins are needed to validate the idea of a possible key role of such a ring in the toxin binding on Kv1.2 channels. Conclusions From a number of previous reports on different scorpion toxins that act on Kv-type channels, it appears that the toxin Fig. 7. Docking of sPi4 on rat voltage-gated Kv1.2 channel (pore region). (A) Side view ( TURBO - FRODO software) depicting the interaction of sPi4 [1] (structure solved by 1 H-NMR) with rat voltage-gated Kv1.2 channel (molecular model of the S5–H5–S6 domains) [24]. For clarity, Ca peptide backbones of only two out of the four S5–H5–S6 a-subunits of the Kv1.2 channel are presented (deep blue). The Ca peptide backbone of sPi4 is shown in green. Only the sidechains of amino acid residues that are involved in the sPi4–Kv1.2 channel interaction are displayed. Basic, acidic and aromatic residues are shown in light blue, red and purple, respectively. The residues are numbered according to their positions within the Pi4 and rat Kv1.2 a-subunit amino acid sequences [1,24]. (B) Magnified side view showing the interactions of sPi4 with the rat Kv1.2 channel. For sPi4, only the sidechains of residues involved in this interaction are depicted. Also, only interacting residues from the Kv1.2 a-subunits are pictured in their exact 3D positions, according to the ion channel molecular model (see Fig. 7A for details). The asterisks indicate that the corresponding residues belong to distinct a-subunits. (C) Top view showing the docking of sPi4 on rat voltage-gated Kv1.2 channel (pore region). Only interacting residues are presented with their corresponding sidechains (see Fig. 7A for details). The four a-subunits (S5–H5–S6 domains) forming the Kv1.2 channel are noted from A to D. Ó FEBS 2003 Synthesis and pharmacology of Pi4 (Eur. J. Biochem. 270) 3589 b-sheet structure plays a premium role in binding to these channels [32,41,50,51]. Amongst the residues belonging to the b-sheet, the key contribution of a pair of well-defined basic and aromatic residues, referred to as the functional dyad, which we attributed to Lys26 and Tyr35 in the case of Pi4, has been shown. The docking of Pi4 (or Pi1) on rat Kv1.2 channels further provides additional insights into the structural basis of this recognition/interaction. Indeed, an unexpected contribution of a ring composed of four basic residues belonging to various faces of the toxin has been highlighted, which supports the idea of a multipoint interaction between Pi4 and this ion channel. It is interesting to note that this ring of basic residues also exists in other potent Kv1.2 channel-acting scorpion toxins, such as Pi1 [7], Pi2 [6,46], and TsTXa [47,49]. At the level of rat Kv1.2 channel, a key functional residue appears to be Glu332 of the a-subunit, a residue absent in Kv1.1 and Kv1.3 a-subunits [24]. In the context of the channel, the four Glu332 from the four a-subunits are thought to interact, via salt bridges, with the four residues from the toxin ring of basic residues. The production of some selected Pi4 analogs, notably those with an altered ring of basic residues, will help to test experi- mentally the Pi4 functional map deduced from the docking experiments. Because the latter also gave some insights that might potentially explain the selectivity of the Pi4 action on voltage-gated Kv1.2 channels, the docking approach will be used to design Pi4 analogs that exhibit some changes in pharmacological selectivity or affinity towards the K + channel subtypes. The completeness of Kv1.2 pore occlusion by Pi4 is a parameter that can tentatively be improved by selective mutation of some Pi4 residues. It is worth noting that the actual docking simulation of Pi4 is informative but remains insufficient to reasonably explain, at a molecular level, the partial Kv1.2 pore occlusion. 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