Estudo comparativo das toxinas PnTx2-5 e PnTx2-6 sobre canais de sódio

• Ano de defesa: 2004
• Autor(a) principal: Alessandra Cristine de Souza Matavel
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Minas Gerais; UFMG
• Palavras-chave em Português: Bioquímica e Imunologia; Phoneutria nigriventer; Bioquímica; Toxinas; Canais de sódio
• Link de acesso: http://hdl.handle.net/1843/BUBD-9W8GXY

The spider Phoneutria nigriventer is the major responsible for the araneism accidents in the Southeast of Brazil. The predominant symptoms of the envenomation indicate a general hyperexcitability. Two toxins of the venom, PnTx2-5 and PnTx2-6 have 48 amino acids each, and molecular weight 5112.3 e 5289.3 Da, respectively. The toxin PnTx2-6 is the most toxic component of the spider venom, and produces similar effects of the whole venom, modifying the sodium channels. As PnTx2-5 and PnTx2-6 diverge in 5 amino acids, we compared their effects on the sodium channel currents of GH3 cells, and compared the effects of PnTx2-6 on sodium currents of neuronal and skeletal muscle fibers. The data show that both toxins increase the time constant of inactivation of sodium current in all preparation tested, and produce a non-inactivating component. Additionally, in frog skeletal muscle PnTx2-6 decreases the sodium channel current amplitude, shifts the activation and steady-state inactivation voltage dependences to hyperpolarized potentials, and increases the time for recovery from inactivation. The PnTx2-6 affinity in skeletal muscle is 25 times lesser than that of GH3 cells. In these cells, PnTx2-6 modified the parameters related to the inactivation of sodium channels (the voltage-dependence of steady-state inactivation curve and the recovery from the sodium current), and the time to peak of the current as well. PnTx2-5 modifies the steady state inactivation curve, but does not modify the time to peak nor the recovery from inactivation of the sodium current. The toxins do not modify the amplitude of the sodium channel in GH3 cells. The main difference PnTx2-6 and PnTx2-5 on GH3 cells was their affinities: the K0,5 of PnTx2-6 is 3 times smaller than PnTx2-5. In addition, PnTx2-5 binds reversibly and can be washed from its binding site. When the cell is strongly depolarized, this toxin unbind faster, However, PnTx2-6 is not displaced from its binding site in ours experimental condition, even after strong depolarization The circular dichroism analysis of Pntx2-6 suggests that its secondary structure is formed predominantly by -sheet and random coil, with a small proportion -helix. The tryptophan fluorescence spectra show that these residues are exposed on PnTx2-6. One tryptophan and one tyrosine are replaced in PnTx2-5 when compared to PnTx2-6, and these amino acids may be involved in the binding of the toxin to the sodium channel, and their absence may account for the difference of affinities.