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Abstract Acetylcholine, the main neurotransmitter of the parasympathetic nervous system, depolarizes various smooth muscles and initiates their contraction via activating muscarinic cholinergic receptors. In most visceral smooth muscle tissues, such as the gastrointestinal tract, airways, and the urinary system, muscarinic receptors are comprised of predominant M2 (about 80%)and minor M3 (about 20%) subtypes. Cholinergic excitation is generally mediated by the opening of ion channels selective for monovalent cations (under physiological conditions, Na+ and K+); among them the cationic channel of an about 60 pS unitary conductance has been recently identified as the main target for acetylcholine action. The signal transduction leading to channel opening is very complex and involves activation of Gao protein (an M2 effect), activation of phospholipase C (an M3 effect), and [Ca2+]i and voltage dependence of channel opening. These multiple signaling pathways were difficult to reconcile with the channel gating mechanisms since only a simplified two-state channel mechanism (e.g., one open and one shut state) was until recently available. However, our recent studies of channel gating in isolated outside-out membrane patches revealed a greater complexity. Thus, this cationic channel shows transitions between at least eight states, four open and four shut, with strong connections between adjacent shut and open states. Therefore, four pairs of connected states have been identified, which showed voltage-dependent transitions in each pair of shut/open states. Since the membrane potential did not affect the relative proportions between the pairs, we have assumed that these effects are controlled by ligands that bind to the channel and, thus, stabilize its various open conformations. In this work, direct tests of the above hypothesis have been performed, and their results showed that spontaneous brief channel gating exists in the absence of receptor or G-protein activation, which is strongly voltage-dependent (increasing at depolarized potentials). Furthermore, this activity was potentiated at a low agonist concentration, while channel openings generally remained brief. An increasing receptor occupancy by the agonist produced long channel openings, indicating a shift of gating towards a long open/brief shut pair of the channel states. These findings are interpreted in the context of the established signal transduction pathways;certain predictions for the whole-cell current are also examined. [ABSTRACT FROM AUTHOR]

Abstract The properties of cationic channels of an average unitary conductance were studied in guinea-pig ileum smooth muscle cells using a patch-clamp technique in the outside-out configuration. Cationic channels were activated by addition of 200 M GTP?S to an intrapipette solution, which resulted in stable activation of G proteins. The replacement of external cesium-containing (in the absence of Ca2+ and Mg2+ ions) solution with a more physiological sodium solution containing 2.5 mM Ca2+ and 2.0 mM Mg2+ led to smaller values of both the amplitude of currents through the unitary cationic channels under study and the probability of the stay of the channel in the open state (Po). The drop in current amplitude was related mostly to the blocking effect of bivalent cations, while a decrease in the Po resulted from the replacement of Cs+ with Na+. Just a drop in the Po, which was responsible for approximately 85% of the inhibitory effect, played a crucial role in the suppression of the integral transmembrane current. [ABSTRACT FROM AUTHOR]