title: Auto-positive feedback regulation for nicotinic acetylcholine receptors by protein kinase C activation.
abstract: OBJECTIVE
  Protein kinase C (PKC) is well-recognized to modify ligand-gated ion channels such as nicotinic ACh (nACh) receptors by phosphorylating the receptors. The aim of the present study was to obtain direct evidence for PKC activation through nACh receptors.
  
  
  METHODS
  Two-electrode voltage-clamp was made to Xenopus oocytes expressing wild-type and mutant Torpedo nACh receptors. Western blotting using antibodies against phospho-serine and phospho-threonine was carried out in oocytes expressing Torpedo nACh receptors. In situ PKC activation was monitored in cultured rat skeletal muscle cells expressing nACh receptors.
  
  
  RESULTS
  In the Xenopus oocyte expression systems, ACh-evoked whole-cell membrane currents through wild-type Torpedo nACh receptors were depressed by GF109203X, a PKC inhibitor, while currents through mutant receptors lacking PKC phosphorylation sites was not affected. In the Western blot analysis, ACh produced immunoreactive bands against an anti-phospho-serine or an anti-phospho-threonine antibody in oocytes expressing wild-type Torpedo nACh receptors, and those signals were attenuated by alpha-bungarotoxin, an inhibitor of nACh receptors, or GF109203X. In the in situ PKC assay using cultured rat muscle cells that expressed all the mRNAs for muscle nACh receptor subunits such as the alpha, beta, gamma, delta, and e subunit, ACh activated PKC in the presence of atropine, an inhibitor of muscarinic ACh receptors, and the activation was abolished by alpha-bungarotoxin or GF109203X.
  
  
  CONCLUSIONS
  The results of the present study show that ACh activates PKC through nACh receptors and that in turn, activated PKC constantly enhances ACh receptor responses by phosphorylating the receptors. This may represent a new auto-positive feedback regulation for nACh receptors by PKC activation.
authors.affiliation: Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, Nishinomiya, Japan.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Yamada
authors.forename: Takahiro
authors.initials: T
authors.lastname: Yaguchi
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kanno
authors.forename: Takeshi
authors.initials: T
authors.lastname: Mukasa
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Nishizaki
mesh_terms: D000818:Animals; D001285:Atropine / Q000494:pharmacology; D002038:Bungarotoxins / Q000494:pharmacology; D025461:Feedback, Physiological; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000494:pharmacology; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D011976:Receptors, Muscarinic / Q000737:chemistry / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000378:metabolism*; D014981:Xenopus / Q000378:metabolism
chemical_list: D002038:Bungarotoxins; D007211:Indoles; D008301:Maleimides; D018727:Muscarinic Antagonists; D011976:Receptors, Muscarinic; D011978:Receptors, Nicotinic; D001285:Atropine; D011493:Protein Kinase C; C070515:bisindolylmaleimide I