M2 muscarinic receptor-mediated inhibition of the Ca2+ current in rat magnocellular cholinergic basal forebrain neurones

1. The actions of muscarinic agonists and antagonists upon the Ca2+ current (ICa) in acutely dissociated magnocellular cholinergic basal forebrain neurones from 11 to 14-day-old postnatal rats were studied using the whole-cell patch-clamp technique. 2. In all cells studied, muscarinic agonists inhibited a transient component of high-voltage-activated (HVA) current, but had no effect upon the low-voltage-activated (LVA) current. The mean IC50 values for ACh and oxotremorine methiodide (oxo-M), obtained from non-cumulative dose-response curves, were 204 and 363 nM respectively. Superfusion with the K+ channel blocker, tetraethylammonium chloride (TEA; 30 mM) shifted the ACh dose-response curve to the right giving an IC50 value of 22:9 microM. 3. Pirenzepine (0.1-1 microM) and methoctramine (0.03-0.3 microM) produced parallel shifts to the right in the agonist dose-response curves. Schild analysis of the agonist dose ratios yielded pA2 (negative log of the apparent dissociation constant KB) values for pirenzepine and methoctramine of 6.8 and 8.2 respectively, indicating the involvement of an M2 receptor subtype. 4. In the presence of GTP-gamma-S (10-500 microM) in the patch pipette, the agonist-induced inhibition of ICa became irreversible. Dialysis with GDP-beta-S (1 mM) abolished all agonist-induced inhibition of the Ca2+ current. The agonist-induced inhibition of ICa was totally blocked by pretreatment with pertussis toxin (500 ng ml-1) but unaffected by preincubation with cholera toxin (500 ng ml-1). Superfusion with 8-bromo cAMP (0.5-1 mM) did not mimic or prevent the effect of agonist application. 5. Inhibition of the Ca2+ current by muscarinic agonists was only partially blocked by omega-conotoxin GVIA (omega-CgTX GVIA), with approximately 46% of the agonist-sensitive current being resistant to omega-CgTX GVIA. Both the agonist- and omega-CgTX GVIA-sensitive components of the current were abolished following maximal inhibition of ICa by GTP-gamma-S. 6. These results indicate that inhibition of the Ca2+ current by muscarinic agonists is mediated via an M2 muscarinic receptor coupled to a pertussis toxin-sensitive G-protein. The possible modulation of multiple HVA Ca2+ channels by muscarinic agonists and the role that these receptors may play in presynaptic modulation of neurotransmitter release are discussed.