Muscarinic Activation of a Novel Voltage-sensitive Inward Current in Rabbit Prevertebral Sympathetic Neurons

The muscarinic activation of rabbit prevertebral sympathetic neurons was studied in non-dissociated coeliac and superior mesenteric ganglia using whole-cell patch-clamp techniques. In the presence of nicotinic blockers, carbachol, muscarine and oxotremorine-M (1–50 μM) induced tonic firing by activating a persistent inward current. These effects were abolished by atropine. They persisted when the M-current was blocked with Ba²⁺ (1 mM) and intracellular Cs⁺. The muscarinic inward current was found to be time- and voltage-dependent. It peaked at -60 mV, decreased at large hyperpolarizations and was tonically activated between —110 and —20 mV, which gave steady-state I—V curves an N-shape between —96 and —54 mV. The negative slope accounted for the large hyperpolarizing responses generated by current pulses in carbachol-treated cells. The muscarinic current was abolished when Na⁺ was replaced by choline, Tris⁺, sucrose, N-methyl-D-glucamine and Cs⁺ but not Li⁺. It was resistant to tetrodotoxin (3 μM), amiloride (3 μM), benzamil (10 μM) and tetraethylammonium (5–20 mM). No involvement of K⁺ and Cl^- could be detected. We therefore styled it l_{Na, M}, in reference to its ionic selectivity and its coupling to muscarinic receptors. Low Ca²⁺-Mg²⁺ salines enhanced the Na, M-current. The current was blocked by Cd²⁺, Co²⁺, La³⁺ (1 mM) and Ba²⁺ (5 mM) but insensitive to methoxyverapamil hydrochloride, nicardipine, nifedipine and ω-conotoxin MVII A (2–20 μM). These effects were ascribed to the binding of di- and trivalent ions to the Na, M-channels. Spike bursts transiently blocked l_{Na, M}. With high intracellular ethylene glycol bis(b-aminoethyl ether)-N, N'-tetraacetic acid or 1, 2-bis(2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid (20–50 mM), this effect was reduced, whereas l_{Na, M} persisted in long-term recordings and its amplitude increased twofold, indicating that intracellular calcium negatively regulated the Na, M-channels. We conclude that we have described a novel muscarinic receptor-coupled channel which appears to play a major part in regulating the firing behaviour of sympathetic neurons.