J Physiol Volume 582, Number 1, 41-61, July 1, 2007 DOI: 10.1113/jphysiol.2007.133165
Three distinct muscarinic signalling pathways for cationic channel activation in mouse gut smooth muscle cells
Takashi Sakamoto1,
Toshihiro Unno2,
Takio Kitazawa3,
Tetsuro Taneike3,
Masahisa Yamada4,
Jürgen Wess5,
Masakazu Nishimura1 and
Seiichi Komori2
1 Department of Pathogenetic Veterinary Science, United Graduate School of Veterinary Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
2 Laboratory of Pharmacology, Department of Veterinary Medicine, Faculty of Applied Biological Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
3 Department of Pharmacology, Faculty of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501, Japan
4 Yamada Research Unit, RIKEN Brain Science Institute, Saitama 351-0198, Japan
5 Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892, USA
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 µM) evoked a muscarinic cationic current (mICat) as small as 
10% of mICat 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 mICat each showed a U-shaped current–voltage (I–V) relationship, whereas the M2-KO mICat 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
= 0.55 and 0.23 ms). In contrast, active M3-KO patches showed only 70-pS channel activity, which had three open states (O
= 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
= 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 mICat. 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 mICat 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.
(Received 22 March 2007;
accepted after revision 23 April 2007;
first published online 26 April 2007)
Corresponding author S. Komori: Laboratory of Pharmacology, Department of Veterinary Medicine, Faculty of Applied Biological Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan. Email: skomori{at}gifu-u.ac.jp
T. Sakamoto and T. Unno contributed equally to this work.
Copyright © 2007 The Physiological Society.
