Antinociceptive Effect And Ion Channel Mechanisms Of AGAP,a Novel Recombinant Neurotoxic Polypeptide Derived From Buthus Martensii Karsch

Antitumor-analgesic peptide(AGAP),a recombinant neurotoxic polypeptide derived from Buthus Martensii Karsch,had an antinociceptive activity with medicinal value.A previous study showed that AGAP exhibits strong analgesic activities in the hot-plate and writhing tests in mice.In this study,we further investigated the analgesic effect and possible mechanisms of AGAP.Firstly,we investigated the analgesic effect of AGAP in formalin test in mice.Our results showed that intravenously administered AGAP(0.25 mg/kg-1.0 mg/kg)had strong analgesic effect against formalin in both two phases in dose-dependent manners.This revealed that AGAP had an antinociceptive activity.In order to clarify the possible mechanisms of analgesic effect of AGAP,a method for acutely isolated dorsal root ganglia(DRG)neurons from adult rats was established.The whole cell patch clamp technique was used to record currents of voltage-gated sodium,calcium and potassium channels in rat small DRG neurons.Our results demonstrated that the methods were simple and easy to obtain and identify DRG neurons.Electrophysiological properties of DRG neurons were typical and clear,and it can be applied for the further research.In this study,we used whole cell patch clamp technique to investigate the effects of AGAP on voltage-gated ion channel currents in rat small DRG neurons.The results showed that AGAP(3 nM-1000 nM)inhibited the sodium currents in small diameter DRG neurons in a concentration-dependent manner.The concentration of half-maximal block(IC50)was 200.7 nM.In addition,1000 nM AGAP could(1)inhibit the current-voltage relationship(?-? curve)of sodium channels in a voltage-dependent manner and negatively shift the activation;(2)reduce the tetrodotoxin-resistant(TTX-R)sodium currents by 42.8%in small diameter DRG neurons;(3)potently inhibited Nav1.8 currents by 59.4%,and negatively shifted the activation and inactivation kinetics;(4)reduced the Nav1.9 currents by 33.7%,but had no significant effect on activation and inactivation kinetics.The effects of AGAP on the currents and dynamics of high-voltage activation(HVA)and Low-voltage activation(LVA)calcium channels in rat small DRG neurons were studied.The results showed that AGAP(3 nM-3000 nM)inhibited HVA and LVA calcium currents in concentration-dependent manners.The concentration-responsive-ness measured in small DRG neurons revealed an IC50 value of 89.6 nM and 71.9 nM,respectively.AGAP decreased the currents of HVA and LVA calcium channels in voltage-,time-dependent manners.AGAP had no significant effect on the dynamic functions of HVA and LVA calcium channels.1000 nM AGAP potently inhibited N-and L-type calcium currents by 78.2%and 57.3%,respectively.The effect of AGAP on the potassion currents in rat small DRG neurons were also studied by using whole cell patch clamp technique.The results showed that 300 nM AGAP had slight effect on transient outward potassium current,but had no significant effect on delayed rectifier potassium current in small diameter DRG neurons.In order to further investigate the analgesic effects mechanisms of AGAP in neuropathic pain,the method of chronic constriction injury(CCI)of the sciatic nerve model was established.As the ipsilateral and contralateral L4-6 small DRG neurons of CCI model for the study object,the effects of AGAP on currents and dynamics of Nav1.8 and Nav1.9 were studied.The results showed that AGAP potently inhibited Nav1.8 currents in concentration-dependence manners in an ex vivo DRG-nerve preparation isolated from CCI model.In addition,1000 nM AGAP(1)inhibited ?-?relationships of Nav1.8 currents in voltage-dependent manners,and negatively shifted the steady-state activation and inactivation kinetics of Nav 1.8 channels;(2)inhibited?-? relationships of Nav1.9 currentsvoltage-dependent manners,but had no significant effect on the steady-state activation and inactivation kinetics of Nav1.9 channels.In conclusion,our results suggested AGAP had strong analgesic activation.It is the first demonstrated that the analgesic effect mechanisms of AGAP might relate with the regulation of sodium and calcium currents,contrubuted to reducing neuronal excitability.