Acetylcholine modulates I(f) and IK(ACh) via different pathways in rabbit sino-atrial node cells

The aim of the present study was to examine whether a direct G-protein pathway was necessary to explain the depression of the cardiac pacemaker current (I(f)) by acetylcholine (ACh). Pacemaker current and spontaneous fluctuations of muscarinic K+ current (IK(ACh)) were simultaneously measured in sino-atrial (SA) node cells isolated from rabbit hearts, using cell-attached and outside-out macro-patches. The effects of 1 microM ACh, added to the bathing solution, were compared on I(f) and on IK(ACh), known to be activated via a direct G-protein mechanism. In a cell-attached configuration where cytoplasmic substrates were present, ACh depressed I(f) by a negatively directed shift of the open probability curve of 7.6 +/- 0.3 mV. ACh never induced modifications in spontaneous openings of muscarinic K+ channels. These results indicate that ACh added to the external solution is unable to modulate IK(ACh) recorded in the membrane-delimited mode, via a G-protein pathway. The ACh depressing effect on I(f) is related to changes in second messenger activity. In outside-out conditions, with guanosine triphosphate (GTP) added to the pipette solution. ACh increased the variance of IK(ACh) fluctuations from 1.66 +/- 0.50 pA2 to 6.60 +/- 2.05 pA2 (at -120 mV). indicating direct G-protein action. ACh had no effect on I(f). It is concluded that in SA node cells, the regulation of I(f) by muscarinic receptors does not involve a direct G-protein pathway. ACh depresses I(f) by a mechanism that probably implicates reduction of intracellular cAMP production.