Muscarinic Cholinergic Excitation of Smooth Muscle: Signal Transduction and Single Cationic Channel Properties

Acetylcholine, the principal neurotransmitter of the parasympathetic nervous system, evokes smooth muscle excitation and contraction by acting at the muscarinic receptors which, in many tissues, including the gastrointestinal tract, are comprised of the M₂ and M₃ subtypes. The opening of ion channels selective for monovalent cations (e.g., Na⁺ and K⁺) is the major mechanism of cholinergic excitation. We have studied signal transduction pathways and single cationic channel properties using patch-clamp recording and Ca²⁺ imaging techniques in guinea-pig single ileal myocytes. Cationic channels were found to couple to both M₂ and M₃ receptors via the GTP-bound G_oα and phospholipase C activation, respectively. When these primarily signaling links are established, cationic channel opening can be further potentiated by membrane depolarization and an increase in the intracellular Ca²⁺ concentration. A strong synergism exists between the receptor occupancy by the agonist and intrinsic voltage dependence of the current as the former can effectively modulate the voltage range of cationic channel activation, while membrane depolarization produces a strong sensitizing effect. However, at potentials close to 0 mV ion flux is terminated by channel flickery block, while further depolarization induces long-lasting channel inactivation. Channel flicker is not caused by intracellular Mg²⁺, polyamines, or any other freely diffusible molecule and is confined to potentials around 0 mV irrespective of the driving force. Thus, it appears to be an intrinsic channel property of physiological importance as it improves conditions for the action potential discharge and propagation. Similarly, intracellular Ca²⁺-dependent facilitation of channel opening is counteracted by a slower desensitization. Further, the most intriguing negative control was discovered in the experiments whereby all cellular G proteins were non-selectively and persistently activated by GTPγS infusion, in which case, over time, carbachol instead of activation caused strong and almost irreversible inhibition of the cationic current. In cell-attached and outside-out membrane patches exposed to 50 μM carbachol or 200 μM internal GTPγS, the activity of three types of cationic channels was observed. They had dissimilar conductances (10, 50, and 130 pS), voltage dependence, and kinetics. The properties of the 50 pS channel are consistent with the whole-cell current behavior, at least when [Ca²⁺] _i is “clamped” at 100 nM. The voltage-independent component of the cationic conductance, which appears at higher levels of [Ca²⁺] _i , is likely mediated by the 130 pS channel, while the role of the 10 pS channel at present is unclear. Thus, smooth muscle cationic channels can uniquely detect and integrate many of the most important physiological signals such as the active conformation of two different muscarinic receptors, their associated G proteins and enzymes, as well as membrane potential and [Ca²⁺] _i levels. Moreover, some signals act in synergy, while most of them, depending on the intensity, can be either stimulatory or inhibitory.