Cholinergic regulation of PKA-dependent Ca2+ cycling in pacemaker cells

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Prior studies indicate that cholinergic receptor (ChR) activation is linked to beating rate reduction (BRR) in sinoatrial nodal cells (SANC) via: (1) a Gi coupled reduction in adenylyl cyclase (AC) activity, leading to a reduction of cAMP or protein kinase A (PKA) modulation of If, or ICa,L, respectively; and (2) direct Gi coupled activation of IKACh. More recent studies, however, have indicated that Ca2+ cycling by the sarcoplasmic reticulum within SANC (referred to as a Ca2+-clock) generates rhythmic, spontaneous local Ca2+ releases (LCRs) that are AC-PKA-dependent. LCRs activate Na+- Ca2+ exchange (NCX) current, which ignites the surface membrane ion channels to effect an AP. The purpose of the present study was to determine how ChR signaling initiated by a cholinergic agonist, carbachol (CCh) affects AC, cAMP and PKA or sarcolemmal ion channels and LCRs and how these effects become integrated to generate the net response to a given intensity of ChR stimulation in single, isolated rabbit SANC. The threshold CCh for BRR was 10 nM; half maximal inhibition (IC50) was achieved at 100 nM; and 1000 nM stopped spontaneous beating. Gi inhibition by pertussis toxin blocked all CCh effects on BRR. Using specific ion channel blockers, we established that If blockade did not affect BRR at any CCh, and that IKACh activation, evidenced by hyperpolarization, first became apparent at CCh>30 nM. At IC50, CCh reduced cAMP and reduced PKA-dependent phospholamban (PLB) phosphorylation by 50%. The dose response of BRR to CCh in the presence of IKACh blockade by a specific inhibitor, tertiapin Q (TQ) mirrored that of CCh to reduced PLB phosphorylation. At IC50, CCh caused a time dependent reduction in the number and size of LCRs and a time dependent increase in LCR period that paralleled coincident BRR. The phosphatase 3 inhibitor, calyculin A, reversed the effect of IC50 CCh on SANC LCRs and BRR. Numerical model simulations demonstrated that Ca2+ cycling is integrated into the cholinergic modulation of BRR via LCR-induced activation of NCX current, providing theoretical support for the experimental findings. Thus, ChR stimulation-induced BRR is entirely dependent on Gi activation, and the extent of Gi coupling to Ca2+ cycling via PKA signaling, or to IKACh: at low CCh, IKACh activation is not evident, and BRR is attributable to a suppression of cAMP-mediated, PKA-dependent Ca2+ signaling; as CCh increases beyond 30 nM, a tight coupling between suppression of PKA-dependent Ca2+ signaling and IKACh activation underlies a more pronounced BRR.