title: Alpha-cobratoxin inhibits T-type calcium currents through muscarinic M4 receptor and Gο-protein βγ subunits-dependent protein kinase A pathway in dorsal root ganglion neurons.
abstract: 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(o)α-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. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.
authors.affiliation: Department of Neurobiology and Psychology, Key Laboratory of Pain Research & Therapy, Medical College of Soochow University, Suzhou 215123, PR China.
authors.forename: Ling
authors.initials: L
authors.lastname: Zhang
authors.forename: Yiming
authors.initials: Y
authors.lastname: Zhang
authors.forename: Dongsheng
authors.initials: D
authors.lastname: Jiang
authors.forename: Paul F
authors.initials: PF
authors.lastname: Reid
authors.forename: Xinghong
authors.initials: X
authors.lastname: Jiang
authors.forename: Zhenghong
authors.initials: Z
authors.lastname: Qin
authors.forename: Jin
authors.initials: J
authors.lastname: Tao
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D020747:Calcium Channels, T-Type / Q000378:metabolism*; D002478:Cells, Cultured; D003039:Cobra Neurotoxin Proteins / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D043588:Receptor, Muscarinic M4 / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
chemical_list: D020747:Calcium Channels, T-Type; D003039:Cobra Neurotoxin Proteins; D043588:Receptor, Muscarinic M4; C025154:alpha-cobratoxin; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D002118:Calcium