A four-disulphide-bridged toxin, with high affinity towards voltage-gated K+ channels, isolated from Heterometrus spinnifer (Scorpionidae) venom

Abstract

A new toxin, named HsTX1, has been identified in the venom of Heterometrus spinnifer (Scorpionidae), on the basis of its ability to block the rat Kv1.3 channels expressed in Xenopus oocytes. HsTX1 has been purified and characterized as a 34-residue peptide reticulated by four disulphide bridges. HsTX1 shares 53% and 59% sequence identity with Pandinus imperator toxin1 (Pi1) and maurotoxin, two recently isolated four-disulphide-bridged toxins, whereas it is only 32-47% identical with the other scorpion K+ channel toxins, reticulated by three disulphide bridges. The amidated and carboxylated forms of HsTX1 were synthesized chemically, and identity between the natural and the synthetic amidated peptides was proved by mass spectrometry, co-elution on C18 HPLC and blocking activity on the rat Kv1.3 channels. The disulphide bridge pattern was studied by (1) limited reduction-alkylation at acidic pH and (2) enzymic cleavage on an immobilized trypsin cartridge, both followed by mass and sequence analyses. Three of the disulphide bonds are connected as in the three-disulphide-bridged scorpion toxins, and the two extra half-cystine residues of HsTX1 are cross-linked, as in Pi1. These results, together with those of CD analysis, suggest that HsTX1 probably adopts the same general folding as all scorpion K+ channel toxins. HsTX1 is a potent inhibitor of the rat Kv1.3 channels (IC50 approx. 12 pM). HsTX1 does not compete with 125I-apamin for binding to its receptor site on rat brain synaptosomal membranes, but competes efficiently with 125I-kaliotoxin for binding to the voltage-gated K+ channels on the same preparation (IC50 approx. 1 pM).

Vm24, a Natural Immunosuppressive Peptide, Potently and Selectively Blocks Kv1.3 Potassium Channels of Human T Cells

“…Vm24 is similar to other natural peptides described in the literature with respect to its high-affinity block of Kv1.3 channels. Among these peptides, the affinities of ShK [estimated IC 50 of 0.9 pM (Middleton et al, 2003) or 11 pM (Kalman et al, 1998)], OSK1 [␣-KTx3.7, K d ϭ 14 pM (Mouhat et al, 2005)], HgTx1 [␣-KTx2.5, K d ϭ 86 pM (Koschak et al, 1998) ], and HsTx1 [␣-KTx6.3, K d ϭ 12 pM (Lebrun et al, 1997)] for Kv1.3 are remarkable. However, these peptides are all less selective for Kv1.3 than is Vm24; some of the peptides ϩ lymphocytes were activated by CD3 and CD28 engagement in the presence (CD3/ CD28ϩVm24, black histograms) or absence (CD3/CD28, gray histograms) of different doses of Vm24; after 24-h stimulation, the level of CD25 expression on the cell surface was measured with flow cytometry.…”

K+ Channel Modulators for the Treatment of Neurological Disorders and Autoimmune Diseases

“…The scorpion toxins are particularly numerous and include charybdotoxin (IC 50 ) 3 nM), 301,302 48 noxiustoxin (IC 50 ) 1 nM), 306 Heterometrus spinnifer toxin 1 (IC 50 ) 12 pM). 307 hongotoxin (IC 50 ) 86 pM), 308 Pandinus imperator toxin 1 and 2 (IC 50 ) 11 nM and 50 pM, respectively), 309 anuroctoxin (IC 50 ) 730 pM), 310 and Orthochirus scrobiculosus toxin 1 (OSK1, IC 50 ) 14 pM). 311 The most potent and selective toxin inhibitor of K V 1.3 is currently the OSK1 derivative OSK1-Lys 16 Asp 20 , which blocks K V 1.3 with an IC 50 value of 3 pM and shows >300-fold selectivity over closely related channels.…”

Hg1, Novel Peptide Inhibitor Specific for Kv1.3 Channels from First Scorpion Kunitz-type Potassium Channel Toxin Family

“…In past years, many peptides isolated from various animal venoms could potently inhibit Kv1.3 channels. These toxins usually had 30 -40 residues cross-linked by three disulfide bridges, such as Odk2, KTX, OSK1, AOSK1, HsTx1, and ShK (7)(8)(9)(10). Until now, the screening and design of novel potent and selective Kv1.3 inhibitors derived from animal toxins remain an attractive prospect for disease diagnosis and treatment (11)(12)(13).…”