NEW INSIGHTS ON THE MECHANISM OF ACTION OF w-Phonetoxin-IIA

GOUVEA DOS SANTOS R.(1,2), VAN RENTERGHEM C(1), MARTIN-MOUTOT N(1), MANSUELLE P(3), SAMPIERI F(3), CORDEIRO MN(4), DINIZ CR(4), DE LIMA ME(5), SEAGAR M(1)

(1)INSERM U464, Marseille, France, (2)Radiobiologia, CDTN, BH, Brazil, (3)CNRS UMR 6560, Marseille, France, (4)FUNED, BH, Brazil, (5)Departamento de Bioquimica ICB, UFMG, Brazil.

w-phonetoxin IIA (w-PtxIIA) is a potent neurotoxin from Phoneutria nigriventer venom evoking flaccid paralysis and death after intracerebro ventricular injection in mouse. In order to shed more light on its mechanism of action, we have used patch clamp methods in cell lines expressing recombinant Ca_v2.1, Ca_v2.2 and Ca_v2.3 channels and studied the biochemical parameters of binding of the radiolabeled w-PtxIIA on native and recombinant Ca⁺² channels.

P24C4 fraction, obtained from the C4 column RP-HPLC, was fractionated by reverse phase HPLC. Analysis of the purified fraction was recorded on a MALDI-TOF Perseptive Voyager Elite spectrometer and a 8363 kDa peptide was detected. After amino acid sequencing the purity of the peptide was confirmed and it was identified as  w-PtxIIA. Calcium currents generated by Cav2.1 and Cav2.2 were blocked totally and almost irreversibly by 3 nMw-PtxIIA (t_{off > 230 and > 330 min, respectively), while Cav2.3 showed partial and readily reversible inhibition (}t_{off= 85 s).}  w-PtxIIA was radiolabeled with ¹²⁵I using lactoperoxidase method and its binding on native (rat brain synaptosomal membranes) and recombinant calcium channels was studied. Binding assays demonstrated that rat brain synaptosomes display multiple classes of binding sites for ¹²⁵I- w-PtxIIA. High affinity binding of ¹²⁵I-w-PtxIIA (Kd= 50 pM) was totally displaced by w-PtxIIA(Ki = 100 pM), but only partially by w-conotoxin GVIA (25% inhibition) and w-conotoxin MVIIC (50% inhibition at 0.3 mM). ¹²⁵I- w-PtxIIA bound to the recombinant Ca_V 2.1 and Ca_V2.2  channels, expressed on BHK cells, with high affinity (160 and 50 pM, respectively). The non-saturable (0.4 nM) interaction of ¹²⁵I- w-PtxIIA with Ca_V2.3 channels is in good agreement with the low affinity observed in the eletrophysiological  experiments (Kd= 67 nM).

To summarize, w-PtxIIA binds to a single high affinity binding site on rat brain synaptosomes and this interaction results in the Ca_V2.1, Ca_V 2.2 and Ca_V2.3 calcium currents blockade.

Supported by: CAPES/COFECUB, CDTN/CNEN

CORRESPONDENCE TO:

Raquel Gouvea dos Santos, Rua Botucatu, 85, Belo Horizonte, MG, CEP: 31140-300, Brasil, Email: santosr@urano.cdtn.br