Alpha-cobratoxin inhibits T-type calcium currents through muscarinic M4 receptor and G o-protein βγ subunits-dependent protein kinase A pathway in dorsal root ganglion neurons

摘要

The long-chain neurotoxic protein, alpha-cobratoxin (α-CTx), has been shown to have analgesic effects. However, the underlying mechanisms still remain unclear. In this study, we examined the effects of α-CTx on T-type calcium channel currents (T-currents) and elucidated the relevant mechanisms in mouse dorsal root ganglion (DRG) neurons. Our results showed that α-CTx reversibly inhibited T-currents in a dose-dependent manner. This inhibitory effect was blocked by the selective muscarinic M4 receptor antagonist tropicamide, while methyllycaconitine, a specific antagonist for the α7 subtype of nicotinic receptor had no effect. siRNA targeting the M4 receptor in small DRG neurons abolished α-CTx-induced T-current inhibition. Intracellular application of GDP-β-S or a selective antibody against the G oα-protein, as well as pretreatment of the cells with pertussis toxin, abolished the inhibitory effects of α-CTx. The M4 receptor-mediated response was blocked by dialyzing cells with QEHA peptide or anti-G β antibody. Pretreatment of the cells with protein kinase A (PKA) inhibitor H89 or intracellular application of PKI 6-22 abolished α-CTx-induced T-current inhibition in small DRG neurons, whereas inhibition of phosphatidylinositol 3-kinase or PKC elicited no such effects. In addition, α-CTx significantly increased PKA activity in DRG neurons, whereas pretreatment of the cells with tropicamide abolished this effect. In summary, our results suggest that activation of muscarinic M4 receptor by α-CTx inhibits T-currents via the G βγ of G o-protein and PKA-dependent pathway.