In this study, we isolated and pharmacologically characterized the first alpha-like toxin from the venom of the scarcely studied Iranian scorpion Odonthobuthus doriae. The toxin was termed OD1 and its primary sequence was determined: GVRDAYIADDKNCVYTCASNGYCNTECTKNGAESGYCQWIGRYGNACWCIKLPDEVPIRIPGKCR. Using the two-electrode voltage clamp technique, the pharmacological effects of OD1 were studied on three cloned voltage-gated Na+ channels expressed in Xenopus laevis oocytes (Na(v)1.2/beta1, Na(v)1.5/beta1, para/tipE). The inactivation process of the insect channel, para/tipE, was severely hampered by 200 nM of OD1 (EC50 = 80+/-14 nM) while Na(v)1.2/beta1 still was not affected at concentrations up to 5 microM. Na(v)1.5/beta1 was influenced at micromolar concentrations.
About one-third of the amino acid residues conserved in all scorpion long chain Na+ channel toxins are aromatic residues, some of which constitute the so-called "conserved hydrophobic surface." At present, in-depth structure-function studies of these aromatic residues using site-directed mutagenesis are still rare. In this study, an effective yeast expression system was used to study the role of seven conserved aromatic residues (Tyr5, Tyr14, Tyr21, Tyr35, Trp38, Tyr42, and Trp47) from the scorpion toxin BmK M1. Using site-directed mutagenesis, all of these aromatic residues were individually substituted with Gly in association with a more conservative substitution of Phe for Tyr5, Tyr14, Tyr35, or Trp47. The mutants, which were expressed in Saccharomyces cerevisiae S-78 cells, were then subjected to a bioassay in mice, electrophysiological characterization on cloned Na+ channels (Nav1.5), and CD analysis. Our results show an eye-catching correlation between the LD50 values in mice and the EC50 values on Nav1.5 channels in oocytes, indicating large mutant-dependent differences that emphasize important specific roles for the conserved aromatic residues in BmK M1. The aromatic side chains of the Tyr5, Tyr35, and Trp47 cluster protruding from the three-stranded β-sheet seem to be essential for the structure and function of the toxin. Trp38 and Tyr42 (located in the β2-sheet and in the loop between the β2- and β3-sheets, respectively) are most likely involved in the pharmacological function of the toxin. About one-third of the amino acid residues conserved in all scorpion long chain Na+ channel toxins are aromatic residues, some of which constitute the so-called "conserved hydrophobic surface." At present, in-depth structure-function studies of these aromatic residues using site-directed mutagenesis are still rare. In this study, an effective yeast expression system was used to study the role of seven conserved aromatic residues (Tyr5, Tyr14, Tyr21, Tyr35, Trp38, Tyr42, and Trp47) from the scorpion toxin BmK M1. Using site-directed mutagenesis, all of these aromatic residues were individually substituted with Gly in association with a more conservative substitution of Phe for Tyr5, Tyr14, Tyr35, or Trp47. The mutants, which were expressed in Saccharomyces cerevisiae S-78 cells, were then subjected to a bioassay in mice, electrophysiological characterization on cloned Na+ channels (Nav1.5), and CD analysis. Our results show an eye-catching correlation between the LD50 values in mice and the EC50 values on Nav1.5 channels in oocytes, indicating large mutant-dependent differences that emphasize important specific roles for the conserved aromatic residues in BmK M1. The aromatic side chains of the Tyr5, Tyr35, and Trp47 cluster protruding from the three-stranded β-sheet seem to be essential for the structure and function of the toxin. Trp38 and Tyr42 (located in the β2-sheet and in the loop between the β2- and β3-sheets, respectively) are most likely involved in the pharmacological function of the toxin. Scorpion neurotoxins targeting voltage-gated sodium channels are single chain polypeptides composed of 60–70 amino acids cross-linked by four disulfide bridges. They have been divided into two major classes, α- and β-toxins. Scorpion α-toxins, the most extensively studied group, can prolong the action potential by slowing the inactivation of Na+ currents with no direct effect on activation (1Couraud F. Jover E. Dobois J.M. Rochat H. Toxicon. 1982; 20: 9-16Google Scholar, 2Possani L.D. Becerril B. Delepierre M. Tytgat J. Eur. J. Biochem. 1999; 264: 287-300Google Scholar, 3Goudet C. Chi C. Tytgat J. Toxicon. 2002; 40: 1239-1258Google Scholar). According to their different pharmacological properties, the α-toxins can be further divided into three subgroups, classical α-, α-like, and insect α-toxins (4Gordon D. Martin-Eauclaire M.F. Cestele S. Kopeyan C. Carlier E. Ben Khalifa R. Pelhate M. Rochat H. J. Biol. Chem. 1996; 271: 8034-8045Google Scholar, 5Gordon D. Savarin P. Gurevitz M. Zinn-Justin S. J. Toxicol. Toxin Rev. 1998; 17: 131-159Google Scholar). The classical α-toxins (e.g. AaH II and Lqh II) are highly toxic to mammals, whereas the insect α-toxins (e.g. Lqh α insect toxin) are highly toxic to insects. The more recently characterized α-like toxins (e.g. Lqh III and BmK M1) act on both mammals and insects, but are unique in their inability to bind to rat synaptosomes despite a high toxicity by intravenous injection. Although three-dimensional structures for the classical α-toxins (6Housset D. Habersetzer-Rochat C. Astier J.P. Fontecilla-Camps J.C. J. Mol. Biol. 1994; 238: 88-103Google Scholar, 7Li H.M. Wang D.-C. Zeng Z.H. Jin L. Hu R.Q. J. Mol. Biol. 1996; 261: 415-431Google Scholar), α-like toxins (8He X.L. Li H.M. Zeng Z.H. Liu X.Q. Wang M. Wang D.-C. J. Mol. Biol. 1999; 292: 125-135Google Scholar, 9Krimm I. Gilles N. Sautiere P. Stankiewicz M. Pelhate M. Gordon D. Lancelin J.M. J. Mol. Biol. 1999; 285: 1749-1763Google Scholar), and insect α-toxins (10Tugarinov V. Kustanovich I. Zilberberg N. Gurevitz M. Anglister J. Biochemistry. 1997; 36: 2414-2424Google Scholar) have been elucidated, in-depth structure-function studies of these long chain toxins using site-directed mutagenesis are still rare, mainly because of folding problems; and the focus has often been on the charged residues in the toxins (11Zilberberg N. Froy O. Loret E. Cestele S. Arad D. Gordon D. Gurevitz M. J. Biol. Chem. 1997; 272: 14810-14816Google Scholar, 12Sun Y.-M. Liu W. Zhu R.-H. Goudet C. Tytgat J. Wang D.-C. J. Pept. Res. 2002; 60: 247-256Google Scholar). Here, we report the importance of the conserved aromatic residues in α-toxins identified by mutagenesis analysis using the α-like toxin BmK M1 as template. BmK M1 is a toxin from the venom of the scorpion Buthus martensii Karsch, which resides in eastern Asia, and is composed of 64 amino acids cross-linked by four disulfide bridges (3Goudet C. Chi C. Tytgat J. Toxicon. 2002; 40: 1239-1258Google Scholar, 7Li H.M. Wang D.-C. Zeng Z.H. Jin L. Hu R.Q. J. Mol. Biol. 1996; 261: 415-431Google Scholar). BmK M1 has been the subject of different studies: its three-dimensional structure was determined by x-ray crystallography at 1.7-Å resolution (8He X.L. Li H.M. Zeng Z.H. Liu X.Q. Wang M. Wang D.-C. J. Mol. Biol. 1999; 292: 125-135Google Scholar); the pharmacological properties of Na+ channels have recently been investigated (13Goudet C. Huys I. Clynen E. Schoofs L. Wang D.-C Waelkens E. Tytgat J. FEBS Lett. 2001; 495: 61-65Google Scholar); and gene cloning and expression of wild-type BmK M1 have also been carried out (14Xiong Y.-M. Ling M.H. Wang D.-C. Chi C.W. Toxicon. 1997; 35: 1025-1031Google Scholar, 15Shao F. Xiong Y.-M. Zhu R.-H. Ling M.H. Chi C.W. Wang D.-C. Protein Expression Purif. 1999; 17: 358-365Google Scholar). Alignment of the amino acid sequences of several α-toxins shows that seven aromatic residues, including Tyr5, Tyr14, Tyr21, Tyr35, Tyr42, Trp38/Tyr38, and Trp47/Tyr47, are notably conserved (Fig. 1A) (5Gordon D. Savarin P. Gurevitz M. Zinn-Justin S. J. Toxicol. Toxin Rev. 1998; 17: 131-159Google Scholar). In a previously performed structural analysis, a conserved hydrophobic surface (CHS) 1The abbreviations used are: CHS, conserved hydrophobic surface; rBmK, recombinant BmK; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; AaH, Androctonus australis Hector; Lqh, Leiurus quinquestriatus hebraeus. was identified (7Li H.M. Wang D.-C. Zeng Z.H. Jin L. Hu R.Q. J. Mol. Biol. 1996; 261: 415-431Google Scholar). The CHS is assumed to be part of the functional site of scorpion toxins targeting sodium channels (16Li H.M. Zhao T. Jin L. Wang M. Zhang Y. Wang D.-C. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 341-344Google Scholar, 17Fontecilla-Camps J.C. Habersetzer-Rochat C. Rochat H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7443-7447Google Scholar). Tyr5, Tyr35, and Trp47 are located on the so-called Face A surface of the toxin (CHS). Tyr14 and Tyr21 are situated on the surface opposite to Face A, called Face B. Trp38 and Tyr42 are located in the β2-sheet and in the loop between the β2- and β3-sheets, respectively (Fig. 1B). Seven of the 15 residues conserved in scorpion toxins (5Gordon D. Savarin P. Gurevitz M. Zinn-Justin S. J. Toxicol. Toxin Rev. 1998; 17: 131-159Google Scholar) are aromatic residues and have been studied in this work. Based on an efficient yeast expression system (15Shao F. Xiong Y.-M. Zhu R.-H. Ling M.H. Chi C.W. Wang D.-C. Protein Expression Purif. 1999; 17: 358-365Google Scholar), the importance of the above-mentioned aromatic residues in the scorpion toxin BmK M1 was analyzed by site-directed mutagenesis. The results from mutagenesis and expression, characterization, bioassays, and electrophysiological analysis of the mutants are reported here. Based on these findings, the important role of these conserved aromatic residues is discussed. Strains, Materials, and Animals—Plasmid pVT102U/α, Escherichia coli strain TG1, and Saccharomyces cerevisiae strain S-78 (Leu2, Ura3, Rep4) were used. Restriction endonucleases and T4 DNA ligase were obtained from Roche Applied Science (Mannheim Germany). Primers were synthesized by Sangon (Shanghai, China). Taq DNA polymerase and Klenow fragment were obtained from MBI. CM32-cellulose cation-exchange and Sephasil® peptide C18 reversed-phase (12-μm ST4.6/250) columns were from Whatman and Amersham Biosciences AB (Uppsala, Sweden), respectively. All other chemicals were at least analytical grade and were purchased from Merck or Sigma. The mice used for the bioassay were ICR mice from the Beijing Center for Experimental Animals. Site-directed Mutagenesis of BmK M1—The cDNA of BmK M1 was previously cloned (14Xiong Y.-M. Ling M.H. Wang D.-C. Chi C.W. Toxicon. 1997; 35: 1025-1031Google Scholar) and inserted into pVT102U/α (15Shao F. Xiong Y.-M. Zhu R.-H. Ling M.H. Chi C.W. Wang D.-C. Protein Expression Purif. 1999; 17: 358-365Google Scholar). According to the sequence of pVT102U/α-BmK M1, two primers were designed: primer 1 (5′-CGTCTAGATAAAAGAAATTCTGTTCGG-3′, including a KEX2 protease linker and an XbaI restriction site) and primer 2 (5′-CGAAGCTTTTAATGGCATTTTCCTGGTAC-3′, with a HindIII site). The substitute residue for all aromatic residues was glycine. In addition, the more conservative substitutions of phenylalanine for Tyr5, Tyr14, Tyr35, and Trp47 were carried out. The mutagenic primers used to generate the desired mutations were as follows: Y5G, 5′-CGTCTAGATAAAAGAAATTCTGTTCGGGATGCTGGTATTGCCAAGCCCCATAACTGT; Y5F, 5′-CGTCTAGATAAAAGAAATTCTGTTCGGGATGCTTTCATTGCCAAGCCCCATAACTGT; Y14G, 5′-AACTGTGTAGGTGAATGTGCT (positive strand) and 5′-AGCACATTCACCTACACAGTT (negative strand); Y14F, 5′-AACTGTGTATTCGAATGTGCT (positive strand) and 5′-AGCACATTCGAATACACAGTT (negative strand); Y21G, 5′-AGAAATGAAGGTTGCAACGATTTATGT (positive strand) and 5′-ACATAAATCGTTGCAACCTTCATTTCT (negative strand); Y35G, 5′-AAGAGTGGCGGTTGCCAATGG (positive strand) and 5′-CCATTGGCAACCGCCACTCTT (negative strand); Y35F, 5′-AAGAGTGGCTTCTGCCAATGG (positive strand) and 5′-CCATTGGCAGAAGCCACTCTT (negative strand); W38G, 5′-TATTGCCAAGGTGTAGGTAAA (positive strand) and 5′-TTTACCTACACCTTGGCAATA (negative strand); Y42G, 5′-GTAGGTAAAGGTGGAAATGGC (positive strand) and 5′-GCCATTTCCACCTTTACCTAC (negative strand); W47G, 5′-AATGGCTGCGGTTGCATAGAG (positive strand) and 5′-CTCTATGCAACCGCAGCCATT (negative strand); and W47F, 5′-AATGGCTGCTTCTGCATAGAG (positive strand) and 5′-CTCTATGCAGAAGCAGCCATT (negative strand). Using pVT102U/α-BmK M1 (recombinant BmK (rBmK) M1) as template along with primer 2 and the mutagenic primer, mutants Y5G and Y5F were created by one-step PCR. Other mutants (Y14G, Y14F, Y21G, Y35G, Y35F, W38G, Y42G, W47G, and W47F) were obtained by three-step PCR. A pair of mutagenic primers was applied in the first or second PCR with primer 1 or 2, respectively, to create two intermediate products, which shared an identical sequence. After treatment with Klenow fragment, two intermediate products acted as primers for each other, and extension of this overlap by DNA polymerase created the full-length mutant, which had the mutation at the desired position. All PCR products were purified by gel excision. Expression and Purification of Mutants—After digestion with XbaI and HindIII, the mutated cDNA gene was inserted into plasmid pVT102U/α and transformed into E. coli TG1 competent cells. The recombinant plasmid pVT102U/α-mutant was extracted, sequenced, and transformed into S. cerevisiae S-78 using the LiCl method (18Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Google Scholar). The expression of the mutants was carried out using a described previously procedure (15Shao F. Xiong Y.-M. Zhu R.-H. Ling M.H. Chi C.W. Wang D.-C. Protein Expression Purif. 1999; 17: 358-365Google Scholar). After fermentation, the supernatant of the culture was adjusted to pH 4.2 with acetic acid. The sample was directly applied to a CM32-cellulose cation-exchange column (2.8 × 14 cm), which was equilibrated with 0.1 m sodium acetate at a flow rate of 1 ml/min. Upon reaching a steady base line, the column was washed by stepwise elution with 0.2, 0.3, and 0.5 n NaCl equilibration buffer. The 0.5 n NaCl fraction was directly applied to a Sephasil® peptide C18 reversed-phase column. Buffer A contained 0.1% trifluoroacetic acid in water; buffer B contained 0.1% trifluoroacetic acid in acetonitrile. The C18 column was eluted with a linear gradient of 0–80% buffer B for 15 column volumes. Reversed-phase chromatography was carried out using an ÄKTA purifier chromatography system (Amersham Biosciences AB). Molecular Mass Determination—The molecular masses of the purified mutants were obtained using a Finnigan LCQ ion-trap mass spectrometer (ThermoQuest, San Jose, CA) equipped with an electrospray ionization source. The spray voltage was 4.50 kV. Calculations were performed using the program provided by the manufacturer. Bioassay—Using 0.9% NaCl as a negative control and rBmK M1 as a positive control, the toxicity of the mutants was determined in mice (male, specified pathogen free level, 18–20 g of body weight). Each group consisted of 10 mice. Various doses of toxin mutants were dissolved in 0.9% NaCl and injected into the mice through the tail vein. Survival times (times between injection and death), reaction, and doses were recorded. Evaluation of toxicity was based on the determination of LD50 (the dose capable of statistically killing 50% of the mice) according to the method of Meier and Theakston (19Meier J. Theakston R.D.G. Toxicon. 1986; 24: 395-401Google Scholar). Expression in Xenopus Oocytes, Electrophysiological Recordings, and Analysis—The human Nav1.5 gene was subcloned into pSP64T (20Gellens M.E. George Jr., A.L. Chen L.Q. Chahine M. Horn R. Barchi R.L. Kallen R.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 554-558Google Scholar). For in vitro transcription, pSP64T/Nav1.5 was first linearized by XbaI. Using the large-scale SP6 mMESSAGE mMACHINE transcription kit (Ambion Inc.), capped cRNAs were synthesized from the linearized plasmids. The in vitro synthesis of cRNA encoding histone H1 and the isolation of Xenopus oocytes were done as described previously (21Liman E.R. Tytgat J. Hess P. Neuron. 1992; 9: 861-871Google Scholar). Oocytes were injected with 50 nl of Nav1.5 cRNA solution at a concentration of 1 ng/nl using a Drummond microinjector. Whole cell currents from oocytes were recorded using the two-micro-electrode voltage clamp technique between 1 and 3 days after injection. Voltage and current electrodes were filled with 3 m KCl. Resistances of both electrodes were kept as low as possible (∼0.1–0.2 megaohms). Experiments were performed using a GeneClamp 500 amplifier (Axon Instruments, Inc.) controlled by a pClamp data acquisition system (Axon Instruments, Inc.). Currents were sampled at 10 kHz and filtered at 5 kHz using a four-pole low-pass Bessel filter. Digital leak subtraction of the current records was carried out using a P/2 protocol. The bath solution composition was 96 mmol/liter NaCl, 2 mmol/liter KCl, 1.8 mmol/liter CaCl2, 2 mmol/liter MgCl2, and 5 mmol/liter HEPES (pH 7.4). This solution was supplemented with 50 mg/liter gentamycin sulfate for incubation of the oocytes. All experiments were performed at room temperature (20–22 °C). Circular Dichroism Measurements—Samples used for analyses were dissolved in 20 mm phosphate buffer (pH 7) at a concentration of 1.0 mg/ml. Circular dichroism spectra were recorded on a Jasco J-720 spectropolarimeter. Spectra were run at 25 °C from 250 to 200 nm using a quartz cell 0.5 mm in length. Data were collected at 0.5-nm intervals with a scan rate of 50 nm/min. All CD spectra resulted from averaging four scans. The final spectrum was corrected by subtracting the corresponding base-line spectrum obtained under identical conditions. Spectra were smoothed by the instrument's software. The secondary structure content was estimated by standard Jasco CD analysis. Mutation, Expression, and Purification—Single point mutants Y14G, Y14F, Y21G, Y35G, Y35F, W38G, Y42G, W47G, and W47F were created by three-step PCR. Mutants Y5G and Y5F were produced by one-step PCR. The target gene was expressed using the pVT102U/α vector. Tricine/SDS-PAGE analyses of yeast cultures demonstrated that mutants Y5G, Y5F, Y14G, Y14F, Y21G, Y35G, Y35F, W38G, Y42G, and W47F were expressed and secreted into the medium. Mutant Y5G could not be used in the following characterization because its expression level was in a trace amount. Mutant W47G could not be expressed at all. The expression levels of the five glycine mutants Y14G, Y21G, Y35G, W38G, and Y42G were ∼1–2 mg/liter of culture medium. For three of the mutants with the conservative phenylalanine substitution (Y5F, Y14F, and Y35F), the expression levels were ∼3 mg/liter, comparable to that of unmodified rBmK M1 (∼3 mg/liter). Remarkably, the amount of W47F in the culture medium was 9–10 mg/liter, which is about three times the value of rBmK M1 (Table I). Apparently, the conservative phenylalanine mutations of Tyr5 and Trp47 changed the expression levels of mutants Y5G and W47G from a trace or nothing at all to the normal level or to even a high level.Table IOverview of the results for the 11 BmK M1 mutantsToxinExpressionToxicity (LD50)Relative toxicityEC50mg/litermg/kg%μMWild-type rBmK M1∼30.531000.50Y5GTraceW47GNoneY21G1-21.01521.0Y35G1-216.0435.0W38G1-2>25<2>100Y42G1-2>25<2>100Y14G1-2>25<2>100Y14F≈30.75713.4Y35F≈30.97550.7W47F9-102.34233.1Y5F≈313.444>100 Open table in a new tab Expressed mutants were purified by a simple and efficient protocol. One liter of culture (10 g/liter yeast extract, 20 g/liter bacteriological peptone, 20 g/liter glucose, pH after sterilization of 6.5) was harvested and initially purified by chromatography on a CM32 cation-exchange column. The next step of purification was carried out on a C18 column. The elution peaks corresponding to target mutants were pooled and lyophilized. The Tricine/SDS-polyacrylamide gels and the mass spectra showed a high purity of the final products. As an example, the entire purification process of Y42G is shown at the following stages: Tricine/SDS-PAGE before and after purification (Fig. 2A), reversed-phase chromatography (Fig. 2B), mass spectrometry (Fig. 2C). Molecular Mass—The molecular masses of the purified variants were measured with the Finnigan LCQ ion-trap mass spectrometer. The individual peaks showed that the molecular masses of mutants Y5F, Y14G, Y14F, Y21G, Y35G, Y35F, W38G, Y42G, and W47F were 7403, 7312, 7403, 7315, 7312, 7403, 7289, 7312, and 7380 Da, respectively (Y42G is shown as an example in Fig. 2C). This corresponded well with the estimated molecular masses of the mutants: 7404, 7313, 7404, 7313, 7313, 7404, 7290, 7313, and 7380 Da, respectively. Conformational Analysis—The CD spectra of rBmK M1 and its mutants in the UV range of 250–200 nm are shown in Fig. 3. Compared with native BmK M1, the CD spectra of Y14G and Y35G dramatically changed (Fig. 3A), indicating that there are apparent changes in the secondary structures of these two mutants. The secondary structure estimation (J-700 for Windows Secondary Structure Estimation, Version 1.10.00) indicates that mutation Y14G interrupts both the α-helix and β-sheet, whereas mutation Y35G interrupts only the β-sheet. In both cases, the estimated random coils show a significant increase. For Y21G, W38G, and Y42G, the CD spectra show that the secondary structures have almost not changed compared with the native toxin (Fig. 3A). It seems that the loss of the aromatic side chains in these mutants does not alter the general structure of the toxin. Regarding mutants with the conservative phenylalanine substitution, the CD spectra show small alterations for Y5F, Y14F, and Y35F, but large changes for W47F compared with wild-type BmK M1 (Fig. 3B). Bioassay—The mice showed typical symptoms of envenomation after injection with rBmK M1. The LD50 determined by the method of Meier and Theakston (19Meier J. Theakston R.D.G. Toxicon. 1986; 24: 395-401Google Scholar) was ∼0.53 mg/kg, which is consistent with that of native BmK M1 (16Li H.M. Zhao T. Jin L. Wang M. Zhang Y. Wang D.-C. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 341-344Google Scholar). Excluding W47G and Y5G, which were not expressed and expressed only in trace amounts, respectively, the other nine mutants (Y5F, Y14G, Y14F, Y21G, Y35G, Y35F, W38G, Y42G, and W47F) were used for bioassays. Each purified mutant was injected into the mice through the tail vein at different doses to determine the LD50 value (Fig. 4 and Table I). Mutants Y14G, W38G, and Y42G showed no detectable toxicity even at a dose of 25 mg/kg, which is 47 times the LD50 of rBmK M1 (Table I). Mutant Y35G lost most of its toxicity (LD50 = 16.04 mg/kg, which is 30 times the LD50 of rBmK M1). In contrast, the LD50 of Y21G was only twice that of rBmK M1 (Fig. 4 and Table I). For the phenylalanine mutants, the toxicities of Y14F (71%) and Y35F (55%) were in the same order as that of unmodified rBmK M1. The toxicity of W47F displayed a certain decrease (23%). The toxicity of Y5F was dramatically reduced to 4% in comparison with unmodified rBmK M1 (Fig. 4 and Table I). Effect of rBmK M1 and Its Aromatic Amino Acid Mutants on Voltage-gated Na+Channels—Fig. 5 displays the effects of rBmK M1 and mutants Y14G, Y35G, W38G, Y42G, Y21G, Y14F, Y35F, Y5F, and W47F on Nav1.5 Na+ channels expressed in Xenopus laevis oocytes. The currents displayed were evoked by a depolarization step to –20 mV from a holding potential of –90 mV. The current traces recorded after the addition of the toxin reveal that rBmK M1 induced a slowing of the inactivation process of Na+ currents. This effect appeared a few seconds after the addition of the toxin and continued until reaching a steady state after 4–5 min. Under control conditions, the inactivation kinetics of Nav1.5 currents were rapid, and almost no remaining currents were visible at the end of the traces, after 25 ms. The toxin-induced slowing of inactivation was evaluated by a single exponential fit (pClamp Version 8) of the current decay after the peak. The time window for each fit was manually set from the peak current to the end of the trace (25 ms). Under steady-state conditions, the time constant of inactivation (τ) calculated by a single exponential fit increased from 1.4 ± 0.2 ms (n = 33) under control conditions to 4.4 ± 1.2 ms (n = 6) after the addition of 1 μm rBmK M1 and to 5.8 ± 0.8 ms (n = 3) after the addition of 5 μm rBmK M1. This represents an increase of ∼414% in the time constant τ in the presence of 5 μm rBmK M1. As shown in Fig. 5, all of the glycine mutants except Y21G were less efficient even at high concentrations (30 and 50 μm) in slowing the inactivation kinetics of Nav1.5 channels compared with the wild-type toxin. Mutants Y14G and Y42G of rBmK M1 were the least effective in slowing the inactivation of the channel. The time constants of inactivation were 2.1 ± 0.3 ms (n = 3) and 1.7 ± 0.3 ms (n = 4) after the addition of 30 μm Y14G and 50 μm Y42G, respectively, corresponding to 150 and 121% of the time constants under control conditions, respectively. The addition of 30 μm W38G increased the time constant of inactivation to 2.6 ± 0.4 ms (n = 3), corresponding to 186% of the control value. Y35G was the second most effective mutant, increasing the τ value to 4.2 ± 1.3 ms (n = 3) at a concentration of 30 μm, corresponding to 300% of the control value. Y21G was the most effective mutant, increasing the τ value to 4.7 ± 0.4 ms (n = 3) at a concentration of 5 μm, corresponding to 335% of the control value. As shown in Fig. 5, all of the phenylalanine mutants except Y5F had about the same efficacy in Nav1.5 as rBmK M1. The time constants of inactivation were 5.4 ± 0.6 ms (n = 4), 7.1 ± 0.4 ms (n = 4), and 5.8 ± 0.6 ms (n = 3) after the addition of 10 μm W47F, 7.5 μm Y35F, and 10 μm Y14F, respectively, corresponding to 385, 507, and 414% of the time constants under control conditions, respectively. Y5F was the least effective in slowing the inactivation of the Na+ channel. The time constant of inactivation was only 2.3 ± 0.6 ms (n = 3) after the addition of 100 μm, corresponding to 165% of the control value. The effects of rBmK M1 and some of its mutants on the peak Na+ current and time to peak were somewhat variable (oocyte-dependent) and not further analyzed. The slowing of inactivation induced by rBmK M1 and its mutants was concentration-dependent (Fig. 6). The EC50 values of rBmK M1, Y21G, Y35G, Y35F, Y14F, and W47F were determined by a sigmoidal fit of the τ-V relationship as displayed in Fig. 6. The EC50 values determined for rBmK M1, Y21G, Y35G, Y35F, Y14F, and W47F were 0.50 ± 0.03, 1.02 ± 0.15, 5.05 ± 0.36, 0.71 ± 0.09, 3.36 ± 0.48, and 3.1 ± 0.3 μm, respectively. The EC50 value determined in this study for rBmK M1 is slightly higher than the EC50 value determined for the native BmK M1 toxin (0.2 μm) in one of our previous studies (13Goudet C. Huys I. Clynen E. Schoofs L. Wang D.-C Waelkens E. Tytgat J. FEBS Lett. 2001; 495: 61-65Google Scholar). Y21G was comparable to native rBmK M1. Y35G was ∼30% less efficient in slowing the inactivation kinetics of Nav1.5 channels compared with wild-type rBmK M1. The EC50 value of Y35G was at least 10 times higher than that of rBmK M1. The EC50 values of the phenylalanine mutants were comparable to that of rBmK M1, except Y5F. As shown in Fig. 6, the EC50 values of Y14G, Y42G, W38G, and Y5F could not be determined because the highest concentrations used did not reach a maximal effect in the dose-response curve. When the three-dimensional structures of the scorpion toxins CsE V3 and AaH II were elucidated ∼20 years ago, the CHS, mainly including Tyr5, Tyr35, and Trp47/Tyr47, was proposed to be responsible for the pharmacological effect of these toxins (17Fontecilla-Camps J.C. Habersetzer-Rochat C. Rochat H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7443-7447Google Scholar, 22Fontecilla-Camps J.C. Alamassy R.J. Suddath F.L. Watt D.D. Bugg C.E. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 6496-6500Google Scholar, 23Fontecilla-Camps J.C. Almassy R.J. Suddath F.L. Bugg C.E. Toxicon. 1982; 20: 1-7Google Scholar). Although the CHS is found in all scorpion toxin structures known today, this assumption required experimental identification. The individual residues in this cluster (e.g. Trp45 in AaH II and Tyr49 in Lqh α insect toxin) have been assessed by chemical modification (24Kharrat R. Darbon H. Rochat H. Granier C. Eur. J. Biochem. 1989; 181: 381-390Google Scholar) and mutagenesis analysis (11Zilberberg N. Froy O. Loret E. Cestele S. Arad D. Gordon D. Gurevitz M. J. Biol. Chem. 1997; 272: 14810-14816Google Scholar, 25Zilberberg N. Gordon D. Pelhate M. Adams M.E. Norris T. Zlotkin E. Gurevitz M. Biochemistry. 1996; 35: 10215-10222Google Scholar) and shown to play an important role in bioactivity. In this study, seven aromatic residues, including three amino acids of this cluster, were analyzed by site-directed mutagenesis. Correlating the high impact substitution of glycine to the more conservative mutation of phenylalanine, our results clearly indicate that these conserved aromatic residues are specifically involved in either or both pharmacological function and structural stability. Aromatic Residues Possibly Involved in Pharmacological Function—The bioassay showed that the toxicity of W38G and Y42G was dramatically reduced (Table I). In concordance, electrophysiological analysis showed that W38G and Y42G were the least effective in slowing the inactivation of the sodium channel. The EC50 could not be determined because the highest concentration available could not induce a maximal effect in the dose-response curves (Figs. 5 and 6). Simultaneously, the CD spectra of these two mutants show the least alteration in comparison with that of unmodified BmK M1 (Fig. 3A). These results can be used to speculate that the conserved aromatic residues Trp38 and Tyr42 are involved in the functional performance of the toxin. Tyr42 is located in the loop between the β2- and β3-sheets (Fig. 7). Considering that this loop is remarkably different in sequence and structure between α- and β-toxins (7Li H.M. Wang D.-C. Zeng Z.H. Jin L. Hu R.Q. J. Mol. Biol. 1996; 261: 415-431Google Scholar, 17Fontecilla-Camps J.C. Habersetzer-Rochat C. Rochat H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7443-7447Google Scholar), this aromatic residue may be related to the preference for the distinct target site of α-toxins. Aromatic Residues Possibly Involved in Structural Stability—The non-expression of W47G and the extremely unstable expression of Y5G indicate that these two residues are essential for the general structure of the toxin. The polypeptide chain of the toxin cannot be folded correctly without these aromatic side chains. Y35G could be expressed in an amount comparable to the wild type, but its toxicity was reduced dramatically (Table I). In agreement with the obtained LD50 value in mice, the corresponding EC50 value of Y35G in Nav1.5 was 10 times higher than that of rBmK M1. The CD spectrum also changed dramatically (Fig. 3A), indicating an alteration of the secondary structure of Y35G. Interestingly, the conservative phenylalanine substitution mutants were expressed very well. Y5F and Y35F were present in the culture medium in an amount of ∼3 mg/liter, comparable to rBmK M1. The expression level of W47F was high (9–10 mg/liter) in comparison with rBmK M1 (Table I). Compared with rBmK M1, alterations in the CD spectra were milder for Y5F and Y35F, but severe for W47F (Fig. 3B). In addition, the bioassay, in concordance with the electrophysiological characterization, showed that the bioactivities of all of the phenylalanine mutants (although in different degrees) were significantly higher than those of the mutants that lost their aromatic side chains by substitution with glycine (Table I). Hence, by constructing and thoroughly comparing glycine and phenylalanine mutants of conserved aromatic residues in BmK M1, we have shown that the aromatic side chains of W47F and Y35F are indispensable for maintaining the structure and pharmacological function of the toxin. Interestingly, these residues do not have to be Trp47 or Tyr35 because the aromatic side chain is the primordial component. Also indicated by this study is the fact that residue 5 has to be a tyrosine because the phenylalanine mutant displayed a very low bioactivity. The three-dimensional structures of BmK M1 and other α-toxins reveal that Tyr5, Tyr35, and Trp47 are located on the three-stranded β-sheet: β1-, β2-, and β3-strands, respectively. The aromatic rings of these residues are positioned orthogonally one to the other in a so-called "herringbone" arrangement (Fig. 7), which was identified as the lowest energy configuration of relatively solvent-exposed aromatic rings (26Burley S.K. Petsko G.A. Adv. Protein Chem. 1988; 39: 125-189Google Scholar). In this way, this aromatic cluster plays an important role in the stabilization of the three-stranded β-sheet. It is plausible to infer that, due to the loss of the interactions between these aromatic rings, the β-sheet will be interrupted and maybe even disintegrated. Trp47 is situated at the center of the cluster (Fig. 7), and its aromatic ring resides in the vicinity of the side chains of both Tyr5 and Tyr35 (distances of 3.5–4 Å). It can be hypothesized that the disruption of the herringbone arrangement due to the loss of the aromatic side chains in W47G and Y5G makes the mutants unable to express. This conclusion is supported by the fact that all of the phenylalanine mutants were very well expressed. W47F, Y35F, and Y14F also displayed the essential bioactivity (Table I). Instead of the non-expression for W47G, mutant W47F was expressed at a high level with a relative toxicity of 23%. However, the CD spectrum reveals a severe conformational change (Fig. 3B). In short, the glycine and phenylalanine mutants clearly indicate that, with the exception of Tyr5, the presence of the aromatic side chain is sufficient and important for the unique structure and the pharmacological function. Tyr5 seems to be a unique and irreplaceable amino acid. The CD spectrum shows a milder alteration for this mutant. Inspecting the three-dimensional structure, there is a contact through a hydrogen bond between the functional -OH group of Tyr5 and the side chain of Lys58/Arg58, which can be found in all long chain scorpion toxins (6Housset D. Habersetzer-Rochat C. Astier J.P. Fontecilla-Camps J.C. J. Mol. Biol. 1994; 238: 88-103Google Scholar, 8He X.L. Li H.M. Zeng Z.H. Liu X.Q. Wang M. Wang D.-C. J. Mol. Biol. 1999; 292: 125-135Google Scholar). By consequence, an aromatic ring without that -OH group cannot maintain the general structure as shown by the CD spectrum and can interrupt the subtle tertiary arrangement between the N-terminal part and the C-terminal segment, which in turn affects the pharmacological function. Therefore, the importance of Tyr5 is not only in the aromatic ring, but also in the functional -OH group; and as a consequence, Tyr5 is highly conserved among α-toxins. Aromatic Residues in Other Sites—Tyr14 and Tyr21 are located on Face B (Fig. 1B), which is roughly opposite to Face A, where the CHS is situated. Y14G was almost nontoxic compared with rBmK M1 (Table I). In agreement, its effect on voltage-gated sodium channels was also the least (Figs. 5 and 6). The CD spectrum was seriously altered compared with that of unmodified BmK M1 (Fig. 3A), indicating a conformational change due to the loss of the aromatic side chain in this position. The results show that Tyr14 is essential for stabilizing the unique conformation of the toxin and is involved in the interaction with the voltage-gated sodium channel. To obtain a more thorough insight in this matter, a more conservative phenylalanine substitution for Tyr14 was constructed. Y14F was expressed in an amount comparable to that of Y14G. However, in contrast to Y14G (no detectable toxicity and EC50 > 100 μm), Y14F possesses a high bioactivity (71% relative toxicity and EC50 = 3.36 ± 0.48 μm) (Table I). These data reveal that it is the aromatic side chain of Tyr14 that mainly contributes to the proper conformation and in turn affects the pharmacological function of the toxin. This residue protrudes from the loop between the β1-sheet and the α-helix (Fig. 7). The structure shows that its aromatic ring interacts with the side chain of Ile6, which is also a crucial conserved residue in α-toxins. The hydrophobic interactions between these two residues on the surface may play an important role in stabilizing the unique conformation of this loop so as to influence the β-sheet and the α-helix of the toxin. The effect of mutation Y21G was milder on bioactivity, on the EC50 in Nav1.5, and on the CD spectrum (Fig. 3A and Table I), indicating that the aromatic residue Tyr21 is putatively not a crucial determinant for the structure and pharmacological function of the toxin.
Abstract Photoactivatable drugs targeting ligand-gated ion channels open up new opportunities for light-guided therapeutic interventions. Photoactivable toxins targeting ion channels have the potential to control excitable cell activities with low invasiveness and high spatiotemporal precision. As proof-of-concept, we develop HwTxIV-Nvoc, a UV light-cleavable and photoactivatable peptide that targets voltage-gated sodium (Na V ) channels and validate its activity in vitro in HEK293 cells, ex vivo in brain slices and in vivo on mice neuromuscular junctions. We find that HwTxIV-Nvoc enables precise spatiotemporal control of neuronal Na V channel function under all conditions tested. By creating multiple photoactivatable toxins, we demonstrate the broad applicability of this toxin-photoactivation technology.
Throughout millions of years of evolution, nature has supplied various organisms with a massive arsenal of venoms to defend themselves against predators or to hunt prey. These venoms are rich cocktails of diverse bioactive compounds with divergent functions, extremely effective in immobilizing or killing the recipient. In fact, venom peptides from various animals have been shown to specifically act on ion channels and other cellular receptors, and impair their normal functioning. Because of their key role in the initiation and propagation of electrical signals in excitable tissue, it is not very surprising that several isoforms of voltage-activated sodium channels are specifically targeted by many of these venom peptides. Therefore, these peptide toxins provide tremendous opportunities to design drugs with a higher efficacy and fewer undesirable side effects. This review puts venom peptides from spiders, scorpions and cone snails that target voltage-activated sodium channels in the spotlight, and addresses their potential therapeutical applications. Keywords: Sodium channel, toxin, spider, scorpion, conus
Significance The venom of Australian funnel-web spiders contains δ-hexatoxins (δ-HXTXs) that exert fatal neurotoxic effects in humans by inhibiting inactivation of voltage-gated sodium channels, but their precise ecological role remains unclear. Sequencing of venom-gland transcriptomes from 10 funnel-web species uncovered 22 δ-HXTXs. Evolutionary analysis revealed extreme conservation of these toxins, despite their ancient origin. We isolated the lethal δ-HXTX from venom of the Sydney funnel-web spider and showed that it induces pain in mice, suggesting a role in predator deterrence. Although humans are not the target of δ-HXTXs, these toxins likely evolved to deter vertebrate predators commonly encountered by these spiders, such as bandicoots, birds, and lizards. Thus, the lethal potency of δ-HXTXs against humans is an unfortunate evolutionary coincidence.
Abstract Voltage-gated sodium (Na V ) channels are essential for the transmission of pain signals in humans making them prime targets for the development of new analgesics. Spider venoms are a rich source of peptide modulators useful to study ion channel structure and function. Here we describe β/δ-TRTX-Pre1a, a 35-residue tarantula peptide that selectively interacts with neuronal Na V channels inhibiting peak current of hNa V 1.1, rNa V 1.2, hNa V 1.6, and hNa V 1.7 while concurrently inhibiting fast inactivation of hNa V 1.1 and rNa V 1.3. The DII and DIV S3-S4 loops of Na V channel voltage sensors are important for the interaction of Pre1a with Na V channels but cannot account for its unique subtype selectivity. Through analysis of the binding regions we ascertained that the variability of the S1-S2 loops between Na V channels contributes substantially to the selectivity profile observed for Pre1a, particularly with regards to fast inactivation. A serine residue on the DIV S2 helix was found to be sufficient to explain Pre1a’s potent and selective inhibitory effect on the fast inactivation process of Na V 1.1 and 1.3. This work highlights that interactions with both S1-S2 and S3-S4 of Na V channels may be necessary for functional modulation, and that targeting the diverse S1-S2 region within voltage-sensing domains provides an avenue to develop subtype selective tools.
'/%3e%3c/defs%3e%3cpath fill='%23012169' d='M0 0h10080v5040H0z'/%3e%3cpath stroke='%23fff' stroke-width='.6' d='m0 0 6 3m0-3L0 3' clip-path='url(%23b)' transform='scale(840)'/%3e%3cpath stroke='%23e4002b' stroke-width='.4' d='m0 0 6 3m0-3L0 3' clip-path='url(%23c)' transform='scale(840)'/%3e%3cpath stroke='%23fff' stroke-width='840' d='M2520 0v2520M0 1260h5040'/%3e%3cpath stroke='%23e4002b' stroke-width='504' d='M2520 0v2520M0 1260h5040'/%3e%3cg fill='%23fff'%3e%3cuse xlink:href='%23d' x='2520' y='3780'/%3e%3cuse xlink:href='%23a' x='7560' y='4200'/%3e%3cuse xlink:href='%23a' x='6300' y='2205'/%3e%3cuse xlink:href='%23a' x='7560' y='840'/%3e%3cuse xlink:href='%23a' x='8680' y='1869'/%3e%3cuse xlink:href='%23e' x='8064' y='2730'/%3e%3c/g%3e%3c/svg%3e)
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)