M4 MUSCARINIC RECEPTOR ACTIVATION MODULATES CALCIUM CHANNEL CURRENTS IN RAT
INTRACARDIAC NEURONS
J. CUEVAS and D.J. ADAMS
Department of Molecular and Cellular Pharmacology, University of Miami
School of Medicine, Miami, FL 33101 USA and Department of Physiology and
Pharmacology, University of Queensland, Brisbane, QLD 4072 Australia. Present
address: Department of Biology, University of California, San Diego, La Jolla,
CA 92093 USA
APStracts 4:111N, 1997.
ABSTRACT
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 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 IBa
amplitude obtained with 100 (M ACh was ÿ7E75% compared to 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 IBa by
m4-toxin gave an IC50 of 11 nM. These results suggest that muscarinic agonist-
induced inhibition of high voltage-activated Ca2+ channels in rat intracardiac
neurons is mediated by the M4 muscarinic receptor. M4 receptor 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 ³ 30% at +90 mV in the
presence of ACh, indicating a voltage-independent component to the muscarinic
receptor-mediated inhibition. Both dihydropyridine- and w-conotoxin GVIA-
sensitive and -insensitive Ca2+ channels were inhibited by ACh, suggesting
that the M4 muscarinic receptor is coupled to multiple Ca2+ channel subtypes
in these neurons. Inhibition of IBa amplitude by muscarinic agonists was also
observed following cell dialysis using the conventional whole-cell recording
configuration. However, internal perfusion of the cell with 100 (M GDP-b-S or
incubation of the neurons in Pertussis toxin (PTX) abolished the modulation of
IBa by 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.
Received 29 January 1997; accepted in final form 20 June 1997.
APS Manuscript Number J092-7.
Article publication pending J. Neurophysiol.
ISSN 1080-4757 Copyright 1997 The American Physiological Society.
Published in APStracts on 15 July 1997