Ca2+ -stimulated adenylyl cyclases (AC) are known to play important roles in neurons but have not previously been reported in the heart. Here we present the first evidence for selective expression of Ca 2+ -stimulated AC in the sino-atrial node (SAN) but not in ventricular muscle of the guinea-pig heart. The AC1 isoform of Ca 2+ -stimulated AC was shown to be present in SAN, both as mRNA using RT-PCR and as protein using immuno-blotting with a specific antibody. Confocal immuno-fluorescence studies detected membrane localization of AC1 in SAN cells, but no AC1 in ventricular muscle. Ca 2+ -stimulated AC8 may also be present in SAN. The functional importance of AC activity was investigated by monitoring activation of I f (gated by hyperpolarization and regulated by cAMP, which shifts activation to more depolarized voltages). Basal activity of AC in isolated SAN myocytes was demonstrated by the observations that an inhibitor of AC activity (MDL 12330A, 10 μM) shifted activation in the hyperpolarizing direction, while inhibition of phosphodiesterases (IBMX, 100 μM) shifted I f activation in the depolarizing direction. Buffering cytosolic Ca 2+ with the Ca 2+ chelator BAPTA (by exposure to BAPTA-AM) shifted activation of I f in the hyperpolarizing direction, and under these conditions the AC inhibitor MDL had little or no further effect. The actions of BAPTA were overcome by exposure to forskolin (10 μM), a direct stimulator of all AC isoforms, to restore cAMP levels. These effects are consistent with the functional importance of Ca 2+ -stimulated AC, which is expected to be fundamental to initiation and regulation of the heartbeat.

Na + -Ca 2+ exchange (NCX) current has been suggested to play a role in cardiac pacemaking, particularly in association with Ca 2+ release from the sarcoplasmic reticulum (SR) that occurs just before the action potential upstroke. The present experiments explore in more detail the contribution of NCX to pacemaking. Na + -Ca 2+ exchange current was inhibited by rapid switch to low-Na + solution (with Li + replacing Na + ) within the time course of a single cardiac cycle to avoid slow secondary effects. Rapid switch to low-Na + solution caused immediate cessation of spontaneous action potentials. ZD7288 (3 μM), to block I f (funny current) channels, slowed but did not stop the spontaneous activity, and tetrodotoxin (10 μM), to block Na + channels, had little effect, but in the presence of either of these agents, rapid switch to low-Na + solution again caused immediate cessation of spontaneous action potentials. Spontaneous electrical activity was also stopped following loading of the cells with the Ca 2+ chelators BAPTA and EGTA, and by exposure to the NCX inhibitor KB-R7943 (5 μM). When rapid switch to low-Na + solution caused cessation of spontaneous activity, this was found (using confocal microscopy, with fluo-4 as the

Abstract-Phosphorylation of ion channels plays an important role in the regulation of cardiac function, but signaling mechanisms controlling dephosphorylation are not well understood. We have tested the hypothesis that p 21 -activated kinase-1 (Pak1), a serine-threonine protein kinase regulated by Ras-related small G proteins, regulates sinoatrial node (SAN) ion channel activity through a mechanism involving protein phosphatase 2A. We report a novel role of Pak1-mediated signaling in attenuating isoproterenol-induced enhancement of L-type Ca 2ϩ current (I CaL ) and delayed rectifier potassium current (I K ) in guinea pig SAN pacemaker cells. We demonstrate that in guinea pig SAN: (1) ignaling pathways activated by ␤-adrenoceptors that increase heart rate have been well studied (reviewed elsewhere 1-4 ), but parasympathetic pathways that attenuate this signaling and cause a slowing of heart rate are less well understood. Although there is evidence that the antiadrenergic effect of acetylcholine is achieved at least in part by a depression in adenylate cyclase activity and reduced cAMP levels mediated via inhibitory G proteins, 5 emerging evidence suggests an important role for a signaling pathway involving protein phosphatase 2A (PP2A). 6,7 This idea fits with evidence indicating a dynamic balance between kinase and phosphatase activity in the control of cardiac ion channel phosphorylation and dephosphorylation even in the absence of autonomic stimulation. 8 -11 Pathways regulating the phosphorylation of ion channels in cardiac cells are relatively well understood, 12 but possible signaling pathways controlling dephosphorylation remain unclear.We have tested the hypothesis that signaling through p 21 -activated kinase-1 (Pak1), a serine-threonine protein kinase regulated by Ras-related small G proteins, regulates sinoatrial node (SAN) ion channel activity. Our hypothesis is based on our demonstration that Pak1 binds to PP2A and induces dephosphorylation of cardiac myofilament proteins. 7 Results of our approach, which involved infection of SAN pacemaker cells with an adenovirus expressing constitutively active Pak1 (Ad-Pak1), support this hypothesis. Materials and Methods AnimalsAge-matched (12-week-old) female guinea pigs were purchased from Charles River (Wilmington, Mass; Margate Kent, UK); procedures were performed in accordance with national and institutional guidelines (US and UK). Viral InfectionFreshly isolated guinea pig SAN pacemaker cells were prepared by procedures based on previous methods 13 and were allowed to settle onto laminin-coated coverslips in 6-well tissue culture plates. These cells were cultured for 2 hours in RPMI medium 1640ϩ4% FBS Original