Cholinergic excitation of the gastrointestinal smooth muscles is mediated by cation channels via a synergistic action of the predominant M₂ (~80%) and minor M₃ (~20%) muscarinic receptors. Cation channel of 57±7 pS conductance (means±S.E.M., n=55) carries most of the muscarinic inward current, mI_CAT (Zholos et al. 2004a). The gating signals apart from [Ca²⁺]_i elevation and membrane depolarization include Pertussis toxin sensitive M₂-mediated activation of Gαo protein (Yan et al. 2003) and concurrent M₃-dependent activation of phospholipase C, PLC. mI_CAT appears to be directly activated by the M₂ receptor since adenylyl cyclase is not involved while the nature of the M₃ receptor action remains enigmatic. Apart from [Ca²⁺]_i-dependent potentiation of mI_CAT due to InsP₃-induced Ca²⁺ release it also includes another important but yet unidentified process, in which PLC products, diacylglycerols and InsP₃, as well as Ca²⁺ store depletion are not involved (Zholos et al. 2004b). Experiments were performed on single collagenase-dispersed myocytes isolated from the small intestine of humanely killed adult guinea-pigs using a combination of patch-clamp recordings, confocal [Ca²⁺]_i imaging (0.1 mM fluo-3 signal) and flash photolysis of ‘caged’ InsP₃ (30 μM in the pipette) or Ca²⁺ (5 mM NP-EGTA/3.8 mM Ca²⁺). We aimed to relate the multiple signalling pathways to the channel mechanism. The 60 pS channel in outside-out patches exposed to 50 μM carbachol externally or 200 μM GTPγS internally showed transitions between 8 states with strong correlations between the dwell times of adjacent shut and open states. Thus, four pairs of connected states have been identified which showed voltage-dependent vertical transitions in each pair (Fig. 1, note that mean dwell times and fractional contribution of each open state to the channel open probability, P_O, are indicated). Prominent regular cycles of P_O occurred because of a variable number of long openings between consecutive long shuttings, that is due to the horizontal transitions between the ‘low-P_O mode’ in C1-O1 and the ‘high-P_O mode’ in C4-O4 states. These probably occurred in a ligand-dependent manner since horizontal redistribution between the channel states was voltage independent. In Cs⁺-rich (125 mM) divalent cation-free solution which maximizes mI_CAT spontaneous cation current, sI_CAT, was revealed with voltage dependence similar to mI_CAT (e.g. U-shaped I-V curve at negative potentials). Its amplitude was 192±9 pA at −40 mV increasing to 1590±209 pA at 80 mV (n=8). sI_CAT was insensitive to atropine (50 nM) suggesting that mAChR constitutive activity was not involved. sI_CAT relaxations during steps from −40 to −120 mV were fast consistent with the O1 mean open time; in 3 out of 8 cells a slower component with τ=4.7±1.2 ms was also detected. Photorelease of Ca²⁺ significantly potentiated this current in a voltage-dependent manner (760±120 and 4860±137 pA at −40 and 80 mV, respectively), while slower relaxations with the time constant in the range 2.8-9.5 ms (7.5±0.4 ms; n=18) became apparent in the macroscopic kinetics — the values typical for the O2 microstate. Carbachol application (10 μM) initiated Ca²⁺ waves and mI_CAT, the latter occurred with a latency of 229±55 ms (n=7) and peak lagging by 1.22±0.11 s (n=17) compared to the fluo-3 signal. However, at later times mI_CAT mirrored changes in [Ca²⁺]_i induced by an additional photorelease of InsP₃ (time-to-peak 279±25 and 298±23 ms, respectively; n=7). Moreover, 5 mM caffeine-induced Ca²⁺ release was totally inefficient in modulating mI_CAT while photorelease of Ca²⁺ was poorly efficient compared to the InsP₃-mediated modulation of mI_CAT. This suggested an important synergy between InsP₃ and Ca²⁺-dependent activation of the channel although InsP₃ alone was inefficient when [Ca²⁺]_i was clamped at 100 nM using 10 mM BAPTA. Our results suggest that in the absence of ligands intrinsic voltage-dependent channel gating occurs in the ‘low-P_O‘ C1-O1 mode. The ‘high P_O‘ C4-O4 mode is probably dominated by the activated Gαo interaction with the channel since Pertussis toxin nearly abolishes mI_CAT. Intracellular Ca²⁺ and the M₃/PLC/InsP₃ system may therefore shift the gating through the intermediate states (Fig. 1).