title: Three distinct muscarinic signalling pathways for cationic channel activation in mouse gut smooth muscle cells.
abstract: Using mutant mice genetically lacking certain subtypes of muscarinic receptor, we have studied muscarinic signal pathways mediating cationic channel activation in intestinal smooth muscle cells. In cells from M2 subtype-knockout (M2-KO) or M3-KO mice, carbachol (100 microM) evoked a muscarinic cationic current (mI(Cat)) as small as approximately 10% of mI(Cat) in wild-type (WT) cells. No appreciable current was evoked in M2/M3 double-KO cells. All mutant type cells preserved normal G-protein-cationic channel coupling. The M3-KO and WT mI(Cat) each showed a U-shaped current-voltage (I-V) relationship, whereas the M2-KO mI(Cat) displayed a linear I-V relationship. Channel analysis in outside-out patches recognized 70-pS and 120-pS channels as the major muscarinic cationic channels. Active patches of M2-KO cells exhibited both 70-pS and 120-pS channel activity usually together, either of which consisted of brief openings (the respective mean open times O(tau) = 0.55 and 0.23 ms). In contrast, active M3-KO patches showed only 70-pS channel activity, which had three open states (O(tau) = 0.55, 3.1 and 17.4 ms). In WT patches, besides the M2-KO and M3-KO types, another type of channel activity was also observed that consisted of 70-pS channel openings with four open states (O(tau) = 0.62, 2.7, 16.9 and 121.1 ms), and patch current of this channel activity showed a U-shaped I-V curve similar to the WT mI(Cat). The present results demonstrate that intestinal myocytes are endowed with three distinct muscarinic pathways mediating cationic channel activation and that the M2/M3 pathway targeting 70-pS channels, serves as the major contributor to mI(Cat) generation. The delineation of this pathway is consistent with the formation of a functional unit by the M2-Go protein and the M3-PLC systems predicted to control cationic channels.
authors.affiliation: Department of Pathogenetic Veterinary Science, United Graduate School of Veterinary Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Sakamoto
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Unno
authors.forename: Takio
authors.initials: T
authors.lastname: Kitazawa
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Taneike
authors.forename: Masahisa
authors.initials: M
authors.lastname: Yamada
authors.forename: Jürgen
authors.initials: J
authors.lastname: Wess
authors.forename: Masakazu
authors.initials: M
authors.lastname: Nishimura
authors.forename: Seiichi
authors.initials: S
authors.lastname: Komori
mesh_terms: D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002412:Cations / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007082:Ileum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry / Q000378:metabolism*; D007583:Jejunum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008564:Membrane Potentials; D051379:Mice; D018345:Mice, Knockout; D008958:Models, Molecular; D018721:Muscarinic Agonists / Q000494:pharmacology; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D043585:Receptor, Muscarinic M2 / Q000819:agonists / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D043587:Receptor, Muscarinic M3 / Q000819:agonists / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D002412:Cations; D007473:Ion Channels; D018721:Muscarinic Agonists; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002217:Carbachol; D010738:Type C Phospholipases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go