title: Activation of M3 muscarinic receptors inhibits T-type Ca(2+) channel currents via pertussis toxin-sensitive novel protein kinase C pathway in small dorsal root ganglion neurons.
abstract: Cobrotoxin (CbT), a short-chain postsynaptic α-neurotoxin, has been reported to play a role in analgesia. However, to date, the detailed mechanisms still remain unknown. In the present study, we identify a novel functional role of CbT in modulating T-type Ca(2+) channel currents (T-currents) in small dorsal root ganglia (DRG) neurons as well as pain behaviors in mice. We found that CbT inhibited T-currents in a dose-dependent manner. CbT at 1μM reversibly inhibited T-currents by ~26.3%. This inhibitory effect was abolished by the non-selective muscarinic acetylcholine receptor (mAChR) antagonist atropine, or the selective M3 mAChR antagonist 4-DAMP, 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 CbT. Using depolarizing prepulse, we found the absence of direct binding between G-protein βγ subunits and T-type Ca(2+) channels in CbT-induced T-current inhibition. CbT responses were abolished by the phospholipase C inhibitor U73122 (but not the inactive analog U73343). The classical and novel protein kinase C (nPKC) antagonist chelerythrine chlorid or GF109203X abolished CbT responses, whereas the classical PKC antagonist Ro31-8820 or inhibition of PKA elicited no such effects. Intrathecal administration of CbT (5μg/kg) produced antinociceptive effects in mechanical, thermal, and inflammatory pain models. Moreover, CbT-induced antinociception could be abrogated by 4-DAMP. Taken together, these results suggest that CbT acting through M3 mAChR inhibits T-currents via a PTX-sensitive nPKC pathway in small DRG neurons, which could contribute to its analgesic effects in mice.
authors.affiliation: Key Laboratory of Pain Research & Therapy, Department of Neurobiology and Medical Psychology, Medical College of Soochow University, Suzhou 215123, China.
authors.forename: Yiming
authors.initials: Y
authors.lastname: Zhang
authors.forename: Ling
authors.initials: L
authors.lastname: Zhang
authors.forename: Fen
authors.initials: F
authors.lastname: Wang
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Jiangong
authors.initials: J
authors.lastname: Wang
authors.forename: Zhenghong
authors.initials: Z
authors.lastname: Qin
authors.forename: Xinghong
authors.initials: X
authors.lastname: Jiang
authors.forename: Jin
authors.initials: J
authors.lastname: Tao
mesh_terms: D000818:Animals; D020747:Calcium Channels, T-Type / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002772:Cholera Toxin / Q000494:pharmacology; D003039:Cobra Neurotoxin Proteins / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D005727:Ganglia, Spinal / Q000166:cytology* / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D008813:Mice, Inbred ICR; D018727:Muscarinic Antagonists / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D010147:Pain Measurement; D037342:Pertussis Toxin / Q000494:pharmacology*; D010880:Piperidines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011760:Pyrrolidinones / Q000494:pharmacology; D043587:Receptor, Muscarinic M3 / Q000819:agonists*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
chemical_list: D020747:Calcium Channels, T-Type; D003039:Cobra Neurotoxin Proteins; D004963:Estrenes; D007211:Indoles; D008301:Maleimides; D018727:Muscarinic Antagonists; D010880:Piperidines; D011760:Pyrrolidinones; D043587:Receptor, Muscarinic M3; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C042375:4-diphenylacetoxy-1,1-dimethylpiperidinium; D002772:Cholera Toxin; D037342:Pertussis Toxin; D011493:Protein Kinase C; D010738:Type C Phospholipases; C070515:bisindolylmaleimide I