COVALENT STRUCTURE AND SOME PHARMACOLOGICAL FEATURES OF NATIVE AND CLEAVED a-KTX_12-1, A FOUR DISULFIDE-BRIDGED TOXIN FROM Tityus serrulatus VENOM

Pimenta, A.M.C.(1,3), Mansuelle, P.(1), Diniz, C.R.(2), De Lima, M.E.(3), Martin-Eauclaire M.F.(1)

(1)Laboratoire de Biochimie - Ingénierie des Protéines, UMR 6560 - IFR Jean Roche, Marseille, France, (2)Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, MG, Brazil, (3)Laboratório de Venenos e Toxinas Animais, Departamento de Bioquímica e Imunologia, ICB, UFMG, Belo Horizonte, Minas Gerais, Brasil

In searching for new molecules able to block voltage-activated K⁺-channels currents, a screening was carried out on rat brain synaptosomal preparations using ¹²⁵I-Kaliotoxin (¹²⁵I-KTx) in competition tests as an activity marker. A toxin with four disulfide bridges from Tityus serrulatus venom, named a-KTx_12-1, was found able to compete with ¹²⁵I-KTx with an IC₅₀ of 46 nM. The obtained amino-acid sequence and molecular mass are identicals to previously described butantoxin and differs by only one residue at C-terminal region to TsTX-IV, a toxin that was found able to inhibit Ca²⁺-activated K⁺ channels of high conductance. Enzymatic cleavages in native peptide followed by mass spectrometry peptide mapping analysis were used to determine the disulfide bridges pattern of a-KTx_12-1. Also, after the cleavage of the first six N-terminal residues, including the unusual disulfide bridge which forms a N-terminus ring, the potency of the cleaved peptide was found to decrease at least 50 fold compared to the native toxin on the same preparation. Although activity on voltage-activated K⁺-channels is found toward the C-terminal portion of KTx, it is clear, in light of these results, that at least in case of a-KTx_12-1 the N-terminal portion plays an important role on its potency.

Financial support: CAPES, COFECUB, CNPq, INSERM, CNRS and FAPEMIG.

CORRESPONDENCE TO:

Adriano Monteiro de Castro Pimenta, Rua Marechal Hermes, 810, Apto. 302, Belo Horizonte, MG, CEP: 30430-030, Brasil, Email: apimenta@icb.ufmg.br