Martentoxin, a novel K+‐channel‐blocking peptide: purification, cDNA and genomic cloning, and electrophysiological and pharmacological characterization

Martentoxin, a novel K+‐channel‐specific peptide has been purified and characterized from the venom of the East‐Asian scorpion (Buthus martensi Karsch). The whole cDNA precursor sequence suggested that martentoxin was composed of 37 residues with a unique sequence compared with other scorpion neurotoxins. The genomic DNA of martentoxin showed an additional intron situated unexpectedly in the 5′ UTR region, besides one located close to the C‐terminal of the signal peptide. The patch‐clamp recording found that martentoxin at the applied dose of 100 nm could strongly block large‐conductance Ca2+‐activated K+ (BKCa) currents in adrenal medulla chromaffin cells, and BKCa currents blocked by martentoxin could be fully recovered within 30 seconds after washing, which is at least 10 times faster than recovery after charybdotoxin. Meanwhile, a biosensor binding assay showed a fast association rate and a slow dissociation rate of martentoxin binding on rat brain synaptosomes. The binding of martentoxin on rat brain synaptosomes could be inhibited regularly by charybdotoxin, and gradually by toosendanin in a concentration‐dependent manner, but not by either apamin or P03 from Buthus martensi. The results thus indicate that martentoxin is a new member in the family of K+‐channel‐blocking ligands.

[1]  Zhuan Zhou,et al.  Action Potential-induced Quantal Secretion of Catecholamines from Rat Adrenal Chromaffin Cells (*) , 1995, The Journal of Biological Chemistry.

[2]  Christopher Miller.  An overview of the potassium channel family , 2000, Genome Biology.

[3]  P. Bougis,et al.  Genomic organization of the KTX2 gene, encoding a ‘short’ scorpion toxin active on K+ channels , 1997, FEBS letters.

[4]  H. Rochat,et al.  Characterization of a new leiurotoxin I‐like scorpion toxin , 1993, FEBS letters.

[5]  G. Kaczorowski,et al.  The beta subunit of the high-conductance calcium-activated potassium channel contributes to the high-affinity receptor for charybdotoxin. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Y. Ji,et al.  Biosensor binding assay of BmK AS-1, a novel Na+ channel-blocking scorpion ligand on rat brain synaptosomes. , 1999, Neuroreport.

[7]  S. Kourrich,et al.  A new class of scorpion toxin binding sites related to an A‐type K+ channel: pharmacological characterization and localization in rat brain , 2001, FEBS letters.

[8]  B. Martin,et al.  The primary structure of noxiustoxin: A K+ channel blocking peptide, purified from the venom of the scorpion Centruroides noxius Hoffmann , 1982 .

[9]  Yong-hua Ji,et al.  The blocking effect of BmP02, one novel short-chain scorpion peptide on transient outward K+ channel of adult rat ventricular myocyte , 2000, Regulatory Peptides.

[10]  M. Garcia-Calvo,et al.  Purification, characterization, and biosynthesis of margatoxin, a component of Centruroides margaritatus venom that selectively inhibits voltage-dependent potassium channels. , 1993, The Journal of biological chemistry.

[11]  G A Gutman,et al.  A unified nomenclature for short-chain peptides isolated from scorpion venoms: alpha-KTx molecular subfamilies. , 1999, Trends in pharmacological sciences.

[12]  C. Miller,et al.  Mapping hydrophobic residues of the interaction surface of charybdotoxin. , 1992, Biophysical journal.

[13]  A. Ménez,et al.  Neuromuscular effects of some potassium channel blocking toxins from the venom of the scorpion Leiurus quinquestriatus hebreus. , 1994, Toxicon : official journal of the International Society on Toxinology.

[14]  H. Rochat,et al.  Kaliotoxin, a novel peptidyl inhibitor of neuronal BK-type Ca(2+)-activated K+ channels characterized from Androctonus mauretanicus mauretanicus venom. , 1992, The Journal of biological chemistry.

[15]  C. Miller,et al.  The charybdotoxin family of K+ channel-blocking peptides , 1995, Neuron.

[16]  Yu-liang Shi,et al.  Toosendanin-induced inhibition of small-conductance calcium-activated potassium channels in CA1 pyramidal neurons of rat hippocampus , 2001, Neuroscience Letters.

[17]  L. Possani,et al.  A novel structural class of K+-channel blocking toxin from the scorpion Pandinus imperator. , 1996, The Biochemical journal.

[18]  R. Aldrich,et al.  Cloning and Functional Characterization of Novel Large Conductance Calcium-activated Potassium Channel β Subunits, hKCNMB3 and hKCNMB4* , 2000, The Journal of Biological Chemistry.

[19]  C. Chi,et al.  Genomic organization of three neurotoxins active on small conductance Ca2+‐activated potassium channels from the scorpion Buthus martensi Karsch 1 , 1999, FEBS letters.

[20]  B. Hammock,et al.  Tamulotoxin, a novel member of the potassium channel active short toxins from the venom of the Indian red scorpion Buthus tamulus , 1997 .

[21]  L. Possani,et al.  Two Novel Toxins from the Venom of the Scorpion Pandinus imperator Show that the N-terminal Amino Acid Sequence is Important for their Affinities towards Shaker B K+ Channels , 1996, Journal of Membrane Biology.

[22]  C. Park,et al.  Mapping function to structure in a channel-blocking peptide: electrostatic mutants of charybdotoxin. , 1992, Biochemistry.

[23]  M. Garcia-Calvo,et al.  Use of toxins to study potassium channels , 1991, Journal of bioenergetics and biomembranes.

[24]  K. Swartz,et al.  Inhibition of T-type voltage-gated calcium channels by a new scorpion toxin , 1998, Nature Neuroscience.

[25]  M. Navia,et al.  Purification, sequence, and model structure of charybdotoxin, a potent selective inhibitor of calcium-activated potassium channels. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[26]  B. Martin,et al.  Novel K(+)-channel-blocking toxins from the venom of the scorpion Centruroides limpidus limpidus Karsch. , 1994, The Biochemical journal.

[27]  H. Rochat,et al.  Chemical synthesis and characterization of maurotoxin, a short scorpion toxin with four disulfide bridges that acts on K+ channels. , 1996, European journal of biochemistry.

[28]  R. Vianna-Jorge,et al.  The beta subunit of the high conductance calcium-activated potassium channel. Identification of residues involved in charybdotoxin binding. , 1998, The Journal of biological chemistry.

[29]  G. Giménez-Gallego,et al.  Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus. , 1990, The Journal of biological chemistry.

[30]  P. Escoubas,et al.  Characterization of four toxins from Buthus martensi scorpion venom, which act on apamin-sensitive Ca2+-activated K+ channels. , 1997, European journal of biochemistry.

[31]  C. Lingle,et al.  Two components of calcium‐activated potassium current in rat adrenal chromaffin cells. , 1992, The Journal of physiology.

[32]  G. Kaczorowski,et al.  Characterization of high affinity binding sites for charybdotoxin in synaptic plasma membranes from rat brain. Evidence for a direct association with an inactivating, voltage-dependent, potassium channel. , 1990, The Journal of biological chemistry.

[33]  R. MacKinnon,et al.  Purification and characterization of three inhibitors of voltage-dependent K+ channels from Leiurus quinquestriatus var. hebraeus venom. , 1994, Biochemistry.

[34]  Harald Sontheimer,et al.  Cloning and Characterization of Glioma BK, a Novel BK Channel Isoform Highly Expressed in Human Glioma Cells , 2002, The Journal of Neuroscience.

[35]  Pankaj Sah,et al.  Ca2+-activated K+ currents in neurones: types, physiological roles and modulation , 1996, Trends in Neurosciences.

[36]  O. Pongs,et al.  Purification, characterization, and synthesis of three novel toxins from the Chinese scorpion Buthus martensi, which act on K+ channels. , 1997, Biochemistry.

[37]  J. Wu,et al.  Solution structure of BmP02, a new potassium channel blocker from the venom of the Chinese scorpion Buthus martensi Karsch. , 2000, Biochemistry.

[38]  Neil V Marrion,et al.  Calcium-activated potassium channels , 1998, Current Opinion in Neurobiology.

[39]  O. Froy,et al.  Dynamic Diversification from a Putative Common Ancestor of Scorpion Toxins Affecting Sodium, Potassium, and Chloride Channels , 1999, Journal of Molecular Evolution.

[40]  L. Kolmakova-Partensky,et al.  Intimations of K+ channel structure from a complete functional map of the molecular surface of charybdotoxin. , 1994 .

[41]  G. Hu,et al.  Solution structure of BmKK2, a new potassium channel blocker from the venom of chinese scorpion Buthus martensi Karsch , 2004, Proteins.

[42]  X. Mao,et al.  Molecular cloning and genomic organization of a K(+) channel toxin from the Chinese scorpion Buthus martensii Karsch. , 2001, Toxicon : official journal of the International Society on Toxinology.

[43]  Z. F. Wang,et al.  Inhibition of large-conductance Ca2+-activated K+ channels in hippocampal neurons by toosendanin , 2001, Neuroscience.

[44]  M. Saraste,et al.  FEBS Lett , 2000 .