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Toxins (Basel)
2014 Feb 28;63:892-913. doi: 10.3390/toxins6030892.
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Electrophysiological characterization of Ts6 and Ts7, K⁺ channel toxins isolated through an improved Tityus serrulatus venom purification procedure.
Cerni FA
,
Pucca MB
,
Peigneur S
,
Cremonez CM
,
Bordon KC
,
Tytgat J
,
Arantes EC
.
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In Brazil, Tityus serrulatus (Ts) is the species responsible for most of the scorpion related accidents. Among the Ts toxins, the neurotoxins with action on potassium channels (α-KTx) present high interest, due to their effect in the envenoming process and the ion channel specificity they display. The α-KTx toxins family is the most relevant because its toxins can be used as therapeutic tools for specific target cells. The improved isolation method provided toxins with high resolution, obtaining pure Ts6 and Ts7 in two chromatographic steps. The effects of Ts6 and Ts7 toxins were evaluated in 14 different types of potassium channels using the voltage-clamp technique with two-microelectrodes. Ts6 toxin shows high affinity for Kv1.2, Kv1.3 and Shaker IR, blocking these channels in low concentrations. Moreover, Ts6 blocks the Kv1.3 channel in picomolar concentrations with an IC50 of 0.55 nM and therefore could be of valuable assistance to further designing immunosuppressive therapeutics. Ts7 toxin blocks multiple subtypes channels, showing low selectivity among the channels analyzed. This work also stands out in its attempt to elucidate the residues important for interacting with each channel and, in the near future, to model a desired drug.
Figure 1. Elution profile of Ts venom on CM-cellulose-52 column. Absorbance was monitored at 280 nm. (A) Classical method of fractionation. Tityus serrulatus venom (200 mg) was dispersed in 2 mL of 0.05 M NH4HCO3 and the supernatant was fractionated on a column 2.5 cm × 63.0 cm, equilibrated with 0.05 M ammonium bicarbonate, pH 7.8 (Buffer A), at 4 °C. Flow: 0.3 mL/min. The dotted line represents the beginning of the convex concentration gradient of 0.01–1 M of ammonium bicarbonate (Buffer B). (B) Improved method of fractionation using a FPLC Äkta Purifier UPC-10 system. Tityus serrulatus venom (50 mg) was dispersed in 2 mL of 0.05 M NH4HCO3 and the supernatant was fractionated on a column 1.6 cm × 100.0 cm, equilibrated with 0.05 M ammonium bicarbonate, pH 7.8 (Buffer A), at 25 °C. Flow: 0.5 mL/min. The dotted line represents the beginning of the linear concentration gradient from 0% to 100% of 0.6 M of ammonium bicarbonate (Buffer B). Absorbance was monitored at 280 nm.
Figure 2. Reversed-phase FPLC of fractions X and XIIA resulting from the improved Ts venom fractionation procedure. The fractions were purified on a C18 column (4.6 mm × 250 mm, 5 µm particles) equilibrated with 0.1% (v/v) of trifluoroacetic acid (TFA). Adsorbed proteins were eluted using a concentration gradient from 0% to 100% of solution B (80% acetonitrile in 0.1% TFA), represented by the dotted line. Flow: 0.8 mL/min. Absorbance was monitored at 214 nm, at 25 °C. (A) Fraction X; (B) Fraction XIIA.
Figure 3. Blocking effect of Ts6 on 14 cloned potassium channels. Representative traces in the absence or presence (*) of 1 µM Ts6 are shown. Voltage protocol is detailed in Section 4.2 of Material and Methods.
Figure 4. Blocking effect of toxins Ts6 and Ts7 on different types of potassium channels. (A) Ts6 blocking effect on 14 cloned potassium channels (n ≥ 3); (B) Ts7 blocking effect on 12 cloned potassium channels (n ≥ 3).
Figure 5. Functional features of Ts6 on Kv1.2 and Kv1.3 potassium channels. (A) Dose-response curve of Ts6 on Kv1.2 channels using Hill equation (for each tested concentration, n ≥ 3). (B) Dose-response curve of Ts6 on Kv1.3 channels using Hill equation (for each tested concentration, n ≥ 3). (C) Current/Voltage relationship on Kv1.2 in the absence (square) or in the presence of 1 µM Ts6 toxin (circle) (n ≥ 3). (D) Current/Voltage relationship on Kv1.3 in the absence (square) or in the presence of 1 µM Ts6 toxin (circle) (n ≥ 3).
Figure 6. Blocking effect of Ts7 on 12 cloned potassium channels. Representative traces in the absence or presence (*) of 1 µM Ts7 are shown. Voltage protocol is detailed in Section 4.2 of Material and Methods.
Figure 7. Multiple sequence alignment and Kv block effect of α-KTx toxins including Ts6 and Ts7. The aligment and percentage of identity Id (%) of 11 primary structures were created by ClustalW2. The figure was generated by ESPript and adapted in CorelDrawn13. UniProt accession numbers are followed by the toxins names. The highly conserved residues are in red. Cysteine residues are highlighted in pink. The amino acid residues in blue indicate low consensus and those not conserved are in black. The functional dyads are demarcated (*).
Abbas,
A new Kaliotoxin selective towards Kv1.3 and Kv1.2 but not Kv1.1 channels expressed in oocytes.
2008, Pubmed,
Xenbase
Abbas,
A new Kaliotoxin selective towards Kv1.3 and Kv1.2 but not Kv1.1 channels expressed in oocytes.
2008,
Pubmed
,
Xenbase
Abdel-Mottaleb,
A common "hot spot" confers hERG blockade activity to alpha-scorpion toxins affecting K+ channels.
2008,
Pubmed
,
Xenbase
Abdel-Mottaleb,
OdK2, a Kv1.3 channel-selective toxin from the venom of the Iranian scorpion Odonthobuthus doriae.
2008,
Pubmed
,
Xenbase
Anderson,
Charybdotoxin block of single Ca2+-activated K+ channels. Effects of channel gating, voltage, and ionic strength.
1988,
Pubmed
Arantes,
A simplified procedure for the fractionation of Tityus serrulatus venom: isolation and partial characterization of TsTX-IV, a new neurotoxin.
1989,
Pubmed
Avdonin,
Mechanisms of maurotoxin action on Shaker potassium channels.
2000,
Pubmed
,
Xenbase
Banerjee,
Structure of a pore-blocking toxin in complex with a eukaryotic voltage-dependent K(+) channel.
2013,
Pubmed
Batista,
Two novel toxins from the Amazonian scorpion Tityus cambridgei that block Kv1.3 and Shaker B K(+)-channels with distinctly different affinities.
2002,
Pubmed
Beeton,
Potassium channels, memory T cells, and multiple sclerosis.
2005,
Pubmed
Beeton,
Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for therapy of autoimmune diseases.
2005,
Pubmed
Bergeron,
Scorpion toxins specific for potassium (K+) channels: a historical overview of peptide bioengineering.
2012,
Pubmed
Blanc,
Solution structure of TsKapa, a charybdotoxin-like scorpion toxin from Tityus serrulatus with high affinity for apamin-sensitive Ca(2+)-activated K+ channels.
1997,
Pubmed
Blaustein,
Polypeptide toxins from the venoms of Old World and New World scorpions preferentially block different potassium channels.
1991,
Pubmed
Brew,
Seizures and reduced life span in mice lacking the potassium channel subunit Kv1.2, but hypoexcitability and enlarged Kv1 currents in auditory neurons.
2007,
Pubmed
Chen,
ImKTx1, a new Kv1.3 channel blocker with a unique primary structure.
2011,
Pubmed
Cologna,
Tityus serrulatus scorpion venom and toxins: an overview.
2009,
Pubmed
Cologna,
Purification and characterization of Ts15, the first member of a new α-KTX subfamily from the venom of the Brazilian scorpion Tityus serrulatus.
2011,
Pubmed
Coronas,
Disulfide bridges and blockage of Shaker B K(+)-channels by another butantoxin peptide purified from the Argentinean scorpion Tityus trivittatus.
2003,
Pubmed
Corzo,
A selective blocker of Kv1.2 and Kv1.3 potassium channels from the venom of the scorpion Centruroides suffusus suffusus.
2008,
Pubmed
Dauplais,
On the convergent evolution of animal toxins. Conservation of a diad of functional residues in potassium channel-blocking toxins with unrelated structures.
1997,
Pubmed
Eccles,
Tityustoxin-K alpha, from scorpion venom, blocks voltage-gated, non-inactivating potassium current in cultured central neurons.
1994,
Pubmed
Edman,
A protein sequenator.
1967,
Pubmed
Ellis,
Interaction of a toxin from the scorpion Tityus serrulatus with a cloned K+ channel from squid (sqKv1A).
2001,
Pubmed
,
Xenbase
Felipe,
Targeting the voltage-dependent K(+) channels Kv1.3 and Kv1.5 as tumor biomarkers for cancer detection and prevention.
2012,
Pubmed
Ferreira,
Peptide T, a novel bradykinin potentiator isolated from Tityus serrulatus scorpion venom.
1993,
Pubmed
Fletcher,
Vesicle-associated membrane protein (VAMP) cleavage by a new metalloprotease from the Brazilian scorpion Tityus serrulatus.
2010,
Pubmed
Frénal,
Exploring structural features of the interaction between the scorpion toxinCnErg1 and ERG K+ channels.
2004,
Pubmed
Fulton,
Contribution of Kv1.2 voltage-gated potassium channel to D2 autoreceptor regulation of axonal dopamine overflow.
2011,
Pubmed
Gallego,
Transient outward potassium channel regulation in healthy and diabetic hearts.
2009,
Pubmed
Garcia,
Purification and characterization of three inhibitors of voltage-dependent K+ channels from Leiurus quinquestriatus var. hebraeus venom.
1994,
Pubmed
,
Xenbase
Ghanshani,
Up-regulation of the IKCa1 potassium channel during T-cell activation. Molecular mechanism and functional consequences.
2000,
Pubmed
Goldstein,
A point mutation in a Shaker K+ channel changes its charybdotoxin binding site from low to high affinity.
1992,
Pubmed
,
Xenbase
Gomez,
Separation of toxic components from the brazillian scorpion Tityus serrulatus venom.
1966,
Pubmed
Gouet,
ESPript: analysis of multiple sequence alignments in PostScript.
1999,
Pubmed
He,
Current pharmacogenomic studies on hERG potassium channels.
2013,
Pubmed
Heginbotham,
The aromatic binding site for tetraethylammonium ion on potassium channels.
1992,
Pubmed
Holaday,
NMR solution structure of butantoxin.
2000,
Pubmed
Hopkins,
Toxin and subunit specificity of blocking affinity of three peptide toxins for heteromultimeric, voltage-gated potassium channels expressed in Xenopus oocytes.
1998,
Pubmed
,
Xenbase
Hu,
Functional blockade of the voltage-gated potassium channel Kv1.3 mediates reversion of T effector to central memory lymphocytes through SMAD3/p21cip1 signaling.
2012,
Pubmed
Jouirou,
Toxin determinants required for interaction with voltage-gated K+ channels.
2004,
Pubmed
Khabiri,
Charybdotoxin unbinding from the mKv1.3 potassium channel: a combined computational and experimental study.
2011,
Pubmed
Kharrat,
Maurotoxin, a four disulfide bridge toxin from Scorpio maurus venom: purification, structure and action on potassium channels.
1997,
Pubmed
,
Xenbase
Koo,
Blockade of the voltage-gated potassium channel Kv1.3 inhibits immune responses in vivo.
1997,
Pubmed
Lam,
The Lymphocyte Potassium Channels Kv1.3 and KCa3.1 as Targets for Immunosuppression.
2011,
Pubmed
Larkin,
Clustal W and Clustal X version 2.0.
2007,
Pubmed
Lebrun,
A four-disulphide-bridged toxin, with high affinity towards voltage-gated K+ channels, isolated from Heterometrus spinnifer (Scorpionidae) venom.
1997,
Pubmed
,
Xenbase
Lin,
Voltage-gated potassium channels regulate calcium-dependent pathways involved in human T lymphocyte activation.
1993,
Pubmed
Liu,
Structural and functional role of the extracellular s5-p linker in the HERG potassium channel.
2002,
Pubmed
,
Xenbase
Lowe,
A portable device for the electrical extraction of scorpion venom.
2011,
Pubmed
MacKinnon,
Charybdotoxin block of Shaker K+ channels suggests that different types of K+ channels share common structural features.
1988,
Pubmed
,
Xenbase
Middleton,
Substitution of a single residue in Stichodactyla helianthus peptide, ShK-Dap22, reveals a novel pharmacological profile.
2003,
Pubmed
,
Xenbase
Mouhat,
The 'functional' dyad of scorpion toxin Pi1 is not itself a prerequisite for toxin binding to the voltage-gated Kv1.2 potassium channels.
2004,
Pubmed
,
Xenbase
Naranjo,
A strongly interacting pair of residues on the contact surface of charybdotoxin and a Shaker K+ channel.
1996,
Pubmed
,
Xenbase
Novello,
TsTX-IV, a short chain four-disulfide-bridged neurotoxin from Tityus serrulatus venom which acts on Ca2+-activated K+ channels.
1999,
Pubmed
Oyama,
Probing the pH-dependent structural features of alpha-KTx12.1, a potassium channel blocker from the scorpion Tityus serrulatus.
2005,
Pubmed
Papazian,
Cloning of genomic and complementary DNA from Shaker, a putative potassium channel gene from Drosophila.
1987,
Pubmed
Pardo-Lopez,
Mapping the binding site of a human ether-a-go-go-related gene-specific peptide toxin (ErgTx) to the channel's outer vestibule.
2002,
Pubmed
Pennington,
Chemical synthesis and characterization of ShK toxin: a potent potassium channel inhibitor from a sea anemone.
1995,
Pubmed
Pessini,
A hyaluronidase from Tityus serrulatus scorpion venom: isolation, characterization and inhibition by flavonoids.
2001,
Pubmed
Pimenta,
Covalent structure and some pharmacological features of native and cleaved alpha-KTx12-1, a four disulfide-bridged toxin from Tityus serrulatus venom.
2003,
Pubmed
Pongs,
Shaker encodes a family of putative potassium channel proteins in the nervous system of Drosophila.
1988,
Pubmed
Possani,
Scorpion toxins specific for Na+-channels.
1999,
Pubmed
Possani,
Peptides and genes coding for scorpion toxins that affect ion-channels.
2000,
Pubmed
Possani,
Purification and properties of mammalian toxins from the venom of Brazilian Scorpion Tityus serrulatus Lutz and Mello.
1977,
Pubmed
Rates,
From the stretcher to the pharmacy's shelf: drug leads from medically important brazilian venomous arachnid species.
2011,
Pubmed
Robbins,
Kv1.1 and Kv1.2: similar channels, different seizure models.
2012,
Pubmed
Rodrigues,
Tityustoxin-K(alpha) blockade of the voltage-gated potassium channel Kv1.3.
2003,
Pubmed
,
Xenbase
Rodríguez de la Vega,
Current views on scorpion toxins specific for K+-channels.
2004,
Pubmed
Rogowski,
Tityustoxin K alpha blocks voltage-gated noninactivating K+ channels and unblocks inactivating K+ channels blocked by alpha-dendrotoxin in synaptosomes.
1994,
Pubmed
Sampaio,
Isolation and characterization of toxic proteins from the venom of the Brazilian scorpion Tityus serrulatus.
1983,
Pubmed
Shepherd,
Distinct modifications in Kv2.1 channel via chemokine receptor CXCR4 regulate neuronal survival-death dynamics.
2012,
Pubmed
Teixeira,
Sequence and structure-activity relationship of a scorpion venom toxin with nitrergic activity in rabbit corpus cavernosum.
2003,
Pubmed
Tempel,
Sequence of a probable potassium channel component encoded at Shaker locus of Drosophila.
1987,
Pubmed
Tytgat,
Effect of fluoxetine on a neuronal, voltage-dependent potassium channel (Kv1.1).
1997,
Pubmed
,
Xenbase
Tytgat,
A unified nomenclature for short-chain peptides isolated from scorpion venoms: alpha-KTx molecular subfamilies.
1999,
Pubmed
Verano-Braga,
Tityus serrulatus Hypotensins: a new family of peptides from scorpion venom.
2008,
Pubmed
Werkman,
Tityustoxin-K alpha, a structurally novel and highly potent K+ channel peptide toxin, interacts with the alpha-dendrotoxin binding site on the cloned Kv1.2 K+ channel.
1993,
Pubmed
Wulff,
Voltage-gated potassium channels as therapeutic targets.
2009,
Pubmed
Wulff,
The voltage-gated Kv1.3 K(+) channel in effector memory T cells as new target for MS.
2003,
Pubmed
Xie,
A new Kv1.2 channelopathy underlying cerebellar ataxia.
2010,
Pubmed
Yoshida,
Voltage-dependent metabolic regulation of Kv2.1 channels in pancreatic beta-cells.
2010,
Pubmed
Zhu,
Molecular diversity and functional evolution of scorpion potassium channel toxins.
2011,
Pubmed
,
Xenbase
Zoccal,
Tityus serrulatus venom and toxins Ts1, Ts2 and Ts6 induce macrophage activation and production of immune mediators.
2011,
Pubmed
Zoccal,
Ts6 and Ts2 from Tityus serrulatus venom induce inflammation by mechanisms dependent on lipid mediators and cytokine production.
2013,
Pubmed