Inhibition Of T-type Ca²⁺ Channels By Cobratoxin In Small Dorsal Root Ganglia Neurons In Mice And The Signaling Mechanisms Involved.

Objective: To investigate the analgesic effect and the related ionic channel mechanisms of cobratoxin, a long-chain postsynapticα-neurotoxin isolated from Thailand cobra (Naja naja kaouthia) venom. .Methods: The whole-cell patch clamp recording was used to investigate the effect of cobratoxin on T-type Ca²⁺ currents (T-currents) in small DRG neurons and pharmacological methods (mAchR, PLC, PKC, PKA etc.) was used to clarify its signaling pathways. Behavior tests (hot plate test, von Frey test, formalin test and acid writhing test) were used to define the analgesic effects of cobratoxin in mice.Results: We found that cobratoxin inhibited T-currents in a dose-dependent manner in mouse small dorsal root ganglia (DRG) neurons. cobratoxin at 0.5μM reversibly inhibited T-currents by ²2.3%. This inhibitory effect was blocked by atropine or scopolamine, a muscarinic acetylcholine receptor (mAChR) antagonist, or tropicamide, a selective M4 mAChR antagonist, while naloxone, an opioid receptor antagonist had no effect. Intracellular infusion of GDP-β-S or pretreatment of the cells with pertussis toxin (PTX) completely blocked the inhibitory effects of cobratoxin. Using depolarizing prepulse, we observed the absence of functional coupling between G-proteinβγsubunits and T-type Ca²⁺ channels. Pretreatment of the cells with H89, a protein kinase A (PKA) inhibitor, or intracellular application of PKI 6-22, blocked cobratoxin -induced T-current inhibition, whereas inhibition of PKC or PLC elicited no such effects. Intrathecal administration of cobratoxin (5μg/kg) produced antinociceptive effects in mechanical, thermal, and inflammatory pain models. Moreover, these cobratoxin -induced antinociceptions in mice could be abrogated by tropicamide.Conclusions: These results together showed that cobratoxin inhibited small DRG neuron T-currents via a M4 mAChR-dependent PTX-sensitive PKA pathway, which could contribute to its analgesic effects in mice.