The Shotr Chain Neurotoxin, Cobrotoxin Modulate Membrane Excitability And The Underlying Mechanisms In Small Dorsal Root Ganglia Neurons In Mice

Objective: To investigate the analgesic effect and the related ionic channelmechanisms of cobrotoxin （CbT）.Methods: Behavior tests （hot plate test, von Frey test, formalin test and acidwrithing test） were used to define the analgesic effects of CbT in mice. MolecularBiology methods were used to identificate the type of acetylcholine receptors of thecultural dorsal root ganglia neurons. The whole-cell patch clamp recording was used toinvestigate the effect of CbT on T-type Ca²⁺currents （T-currents） and A-type K+currents （A-currents） in small DRG neurons and pharmacological methods were used toclarify its signaling pathways.Results: In the present study, we found that1) Intrathecal administration of CbT （5μg/kg） produced antinociceptive effects in mechanical, thermal, and inflammatory painmodels. Moreover, these CbT-induced antinociceptions in mice could be abrogated by4-DAMP；2)In current-clamp, we observed a significant decreased firing frequency ofaction potential of small DRG neurons by³0.7%, induced by CbT, meanwhile, the APamplitude and first spike latency also significantly changed;3)The four subtypes ofmuscarinic acetylcholine receptors （MAChR） were identified on dorsal root ganglianeurons, without the expression of α1subtype of nicotine receptor;4) CbT inhibitedT-currents and increased A-currents in a dose-dependent manner, CbT at1μMreversibly inhibited T-currents by²7.4%and increase A-currents by³0.3%;5) Theseeffects were blocked by atropine, a MAChR antagonist, or4-DAMP, a selectiveM3AChR antagonist, while naloxone, an opioid receptor antagonist had no effect. In themean time, the effects also could be blocked by GDP-β-S, a selective G proteinantagonist, PTX, Gi/oantagonist, and anti-Gopeptide; Using depolarizing prepulse, we observed the absence of functional coupling between G-protein βγ subunits and T-typeCa²⁺channels. Pretreatment of PLC inhibitor U73122, PKC inhibitors GF109203X,chelerythrine chloride, PKCδ inhibitor Rottlerin, and ERK1/2inhibitors U0126,PD98059, blocked CbT-induced T-current inhibition or A-current increase, whereasinhibition of PKA （H89, PKI6-22） have no such effects.Conclusions: These results together showed that Intrathecal administration of CbTproduced antinociceptive effects, and CbT inhibited T-currents in small DRG neuronsvia an M3AChR-dependent PTX-sensitive PLC-PKC pathway, and increase A-currentsvia Gi-protein coupled M3AChR, and a sequent PLC-PKCδ, ERK1/2pathway, whichcould contribute to its physiological functions including neuronal hypoexcitability andanalgesic effects in mice.