Three distinct muscarinic signalling pathways for cationic channel activation in mouse gut smooth muscle cells

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 M 2 subtype‐knockout (M 2 ‐KO) or M 3 ‐KO mice, carbachol (100 μ m ) evoked a muscarinic cationic current (m I Cat ) as small as ∼10% of m I Cat in wild‐type (WT) cells. No appreciable current was evoked in M 2 /M 3 double‐KO cells. All mutant type cells preserved normal G‐protein–cationic channel coupling. The M 3 ‐KO and WT m I Cat each showed a U‐shaped current–voltage ( I–V ) relationship, whereas the M 2 ‐KO m I 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 M 2 ‐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 τ = 0.55 and 0.23 ms). In contrast, active M 3 ‐KO patches showed only 70‐pS channel activity, which had three open states (O τ = 0.55, 3.1 and 17.4 ms). In WT patches, besides the M 2 ‐KO and M 3 ‐KO types, another type of channel activity was also observed that consisted of 70‐pS channel openings with four open states (O τ = 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 m I Cat . The present results demonstrate that intestinal myocytes are endowed with three distinct muscarinic pathways mediating cationic channel activation and that the M 2 /M 3 pathway targeting 70‐pS channels, serves as the major contributor to m I Cat generation. The delineation of this pathway is consistent with the formation of a functional unit by the M 2 ‐G o protein and the M 3 ‐PLC systems predicted to control cationic channels.