The effects of activation of muscarinic receptors on the voltage-dependent calcium current, ICa, in parasympathetic neurones were examined. 2. Neurones were enzymatically isolated from the interatrial septum of bull-frog (Rana catesbeiana) heart, and were maintained in short-term (1-6 day) tissue culture. ICa was recorded from the cells using whole-cell patch-clamp methods (Clark, Tse & Giles, 1990). 3. External application of 2 nM to 10 microM acetylcholine (ACh) reduced the amplitude and slowed the time course of activation of ICa. These effects were dependent on membrane potential; they were most pronounced at potentials near the peak of the current-voltage relation for ICa (i.e. +10 to +15 mV), whereas at more-negative potentials (i.e. -15 to -25 mV) the effects on both amplitude and time course were relatively small. 4. Atropine (1 microM) completely blocked the action of 1 microM-ACh, indicating that the effects of ACh on ICa were mediated by activation of muscarinic receptors. 5. Other muscarinic agonists, such as carbamylcholine (0.1-10 microM), DL-muscarine (0.1-2.5 microM) and oxotremorine (5 microM), had similar effects on ICa to ACh. 6. A guanine nucleotide-binding protein (G-protein) is involved in this muscarinic inhibition of ICa. Inclusion of the non-hydrolysable guanosine triphosphate analogue guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S; 200 microM) in the intracellular solutions mimicked the effects of ACh, and application of external ACh in the presence of internal GTP-gamma-S produced smaller changes in ICa than in control conditions. Inclusion of another non-hydrolysable analogue, guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S; 0.5-5 mM), blocked the inhibitory effect of ACh on ICa. 7. The G-protein involved in the inhibition of ICa was sensitive to pertussis toxin (islet-activating protein; IAP). The inhibition of ICa by carbamylcholine (5 microM) was reduced by about 90% after incubating cells for 12-15 h in culture medium containing 200 ng/ml IAP. 8. The possible roles of cyclic AMP or cyclic GMP-dependent protein kinases, or protein kinase C, in the muscarinic inhibition of ICa were tested, but these enzymes appear not to be directly involved.
