Cuevas, J. and Adams, D. J. M4muscarinic receptor activation modulates calcium channel currents in rat intracardiac neurons. J. Neurophysiol. 78: 1903–1912, 1997. Modulation of high-voltage–activated Ca2+channels by muscarinic receptor agonists was investigated in isolated parasympathetic neurons of neonatal rat intracardiac ganglia using the amphotericin B perforated-patch whole cell recording configuration of the patch-clamp technique. Focal application of the muscarinic agonists acetylcholine (ACh), muscarine, and oxotremorine-M to the voltage-clamped soma membrane reversibly depressed peak Ca2+channel current amplitude. The dose-reponse relationship obtained for ACh-induced inhibition of Ba2+current ( IBa) exhibited a half-maximal inhibition at 6 nM. Maximal inhibition of IBaamplitude obtained with 100 μM ACh was ∼75% compared with control at +10 mV. Muscarinic agonist-induced attenuation of Ca2+channel currents was inhibited by the muscarinic receptor antagonists pirenzepine (≤300 nM) and m4-toxin (≤100 nM), but not by AF-DX 116 (300 nM) or m1-toxin (60 nM). The dose-response relationship obtained for antagonism of muscarine-induced inhibition of IBaby m4-toxin gave an IC50of 11 nM. These results suggest that muscarinic agonist-induced inhibition of high-voltage–activated Ca2+channels in rat intracardiac neurons is mediated by the M4muscarinic receptor. M4receptor activation shifted the voltage dependence and depressed maximal activation of Ca2+channels but had no effect on the steady-state inactivation of Ca2+channels. Peak Ca2+channel tail current amplitude was reduced ≥30% at +90 mV in the presence of ACh, indicating a voltage-independent component to the muscarinicreceptor-mediated inhibition. Both dihydropyridine- and ω-conotoxin GVIA–sensitive and -insensitive Ca2+channels were inhibited by ACh, suggesting that the M4muscarinic receptor is coupled to multiple Ca2+channel subtypes in these neurons. Inhibition of IBaamplitude by muscarinic agonists was also observed after cell dialysis using the conventional whole cell recording configuration. However, internal perfusion of the cell with 100 μM guanosine 5′-O-(2-thiodiphosphate) trilithium salt (GDP-β-S) or incubation of the neurons in Pertussis toxin (PTX) abolished the modulation of IBaby muscarinic receptor agonists, suggesting the involvement of a PTX-sensitive G-protein in the signal transduction pathway. Given that ACh is the principal neurotransmitter mediating vagal innervation of the heart, the presence of this inhibitory mechanism in postganglionic intracardiac neurons suggests that it may serve for negative feedback regulation.