Selective Inhibition of Sodium Channel Isoforms by rPnTx1, a Recombinant Spider
Toxin
Silva, A.O.1, Diniz, M.V.R.2, Peigneur, S.3, Tytgat, J.3 and Beirão, P.S.L.1
1
Departamento de Bioquímica e Imunologia, Instituto de Ciências BiológicasUniversidade Federal de Minas Gerais, Belo Horizonte, Brasil; 2Laboratório de
Biologia Molecular, Centro de Pesquisa Carlos Ribeiro Diniz, Fundação Ezequiel
Dias, Belo Horizonte, Brasil; 3Laboratory of Toxicology, University of Leuven,
Leuven, Belgium
Introduction: Phoneutria nigriventer toxin Tx1 is a peptide with 78 amino acid
residues, 14 of which are cysteines forming disulfide bridges. It was shown to
inhibit the neuronal Nav1.2 channel isoform, preferably when maintained at a
depolarized holding potential. Recombinant toxin rPnTx1, which contains two
additional amino acids (Ala-Met) at the N-terminal and one additional Gly at the Nterminal, was successfully produced in E. coli. Material and Methods: The activity
of rPnTx1 on endogenous neuronal sodium channels was measured in Dorsal
Root Ganglia neurons with the patch clamp technique, and its effect on sodium
channel isoforms was assayed in Xenopus oocytes expressing each isoform.
Results and Discussion: We observed that rPnTx1 has inhibitory activity on
sodium currents without altering the voltage-dependence of activation or steadystate inactivation, and its effect is markedly enhanced by long depolarizations.
Interesting selectivity was found when tested on relevant sodium channel
isoforms: rNaV1.2 > rNaV1.7 ≈ rNaV1.4 > rNaV1.3 > mNaV1.6 > hNaV1.8. No
significant activity was detected in the cardiac Nav1.5 and in the arthropod
isoforms (DmNaV1, BgNaV1.1a, and VdNaV1). When compared with the native
toxin, the maximal effects on Nav1.2 were 85.0 ± 0.8% and 83.3 ± 1.9% with an
IC50 of 105 ± 12 nM and 33.7 ± 2.9 nM for the natural and recombinant toxins,
respectively. To test the hypothesis that rPnTx1 acts by stabilizing the inactivated
state, Nav1.2 channels devoid of inactivation (QQQ substituted for the IFM
inactivating motif) were used. The presence of marked (74.0 ± 3.0%) inhibition
excludes this mechanism. Conclusion: We conclude that rPnTx1 is as effective
as the natural toxin on the neuronal Nav1.2 isoform, and suggest that it acts by
partial occlusion of its outer pore. Its strong effect on Nav1.7 channels is of great
interest for its role in the conduction of pain signals.
Supported by: CNPq, FAPEMIG and CAPES.
Key words: Sodium channel, Recombinant toxin, Channel inhibition, Spider toxin,
Selective inhibition