Effect of ANEPIII, a novel recombinant neurotoxic polypeptide, on sodium channels in primary cultured rat hippocampal and cortical neurons. Regul Pept
Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, PR China. Regulatory Peptides
(Impact Factor: 1.83).
09/2010; 164(2-3):105-12. DOI: 10.1016/j.regpep.2010.05.010
Previous studies have shown that the recombinant neurotoxic polypeptide BmK ANEP (ANEPIII) displayed good anti-neuroexcitation activity as demonstrated by pharmacological tests of the blockade of chemical-induced convulsive seizures. In order to search for further anticonvulsant mechanism of action of ANEPIII, the effects of ANEPIII on sodium channels were assessed using the whole-cell patch clamp recordings in primary cultures of rat hippocampal and cortical neurons. ANEPIII decreased the sodium currents in a voltage-dependent manner, which appeared as a shift of the current-voltage relation to positive potentials. The effect was reversible after washing. The concentration-responsiveness measured in hippocampal and cortical neurons revealed an IC(50) value of 124.6 nM and 192.7 nM, respectively. Furthermore, ANEPIII 1000 nM significantly shifted the activation curves of sodium current in hippocampal and cortical neurons to more positive potentials and the recovery from inactivation of sodium current was significantly slower. Voltage-dependent inactivation curves of sodium channels in hippocampal and cortical neurons did not change in the presence of 1000 nM ANEPIII. Thus, our results demonstrated that ANEPIII in submicromolar concentrations was a voltage-dependent, reversible blocker of sodium current in hippocampal and cortical neurons. It is concluded that these phenomena may explain, at least in part, the anti-neuroexciting properties of this peptide.
Available from: sciencedirect.com
- "Recording electrodes were filled with an internal solution containing (in mM): 140 CsCl, 10 EGTA, 0.1 CaCl 2 , 2 MgCl 2 , 10 HEPES, 2 ATP, and pH 7.2 adjusted with Tris. Patch-clamp electrodes were pulled with a P-97 puller, and had a resistance of 3–5 MX . Peak current amplitudes were measured before and after a 3–5-min incubation with AGAP. "
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ABSTRACT: A previous study showed that antitumor-analgesic peptide (AGAP), a novel recombinant polypeptide, which had been expressed in Escherichia coli, exhibits analgesic and antitumor effects in mice. In the present study, we investigated the underlying analgesic mechanism of AGAP. The effect of AGAP on voltage-gated calcium channels (VGCCs) was assessed in acutely isolated rat dorsal root ganglia (DRG) neurons using the whole-cell patch clamp technique. The results showed that AGAP potently inhibited VGCCs, especially high-voltage activated (HVA) calcium channels. AGAP inhibited HVA and T-type calcium currents in a dose-dependent manner, but had no significant effect on their dynamic functions in rat small-diameter DRG neurons. AGAP inhibited N- and L-type calcium currents at 78.2% and 57.3%, respectively. Thus, the present study demonstrates that AGAP affects calcium currents through the inhibition of N-, L- and T-type channels in DRG neurons, explaining the potential mechanisms of antinociception.
Available from: Tao Tan
- "VGCCs and subsequently modulate the Ca 2+ entry into neurons. The facilitation of Ca 2+ entries into nerve terminals by modulating the opening of VGCCs may further increase the amount of the released excitatory amino acids, which increase the excitability of the neurons (Li et al., 2010). There is a residual after-effect of consecutive daily applications of rTMS. "
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