We have recently shown that RyR2 (cardiac ryanodine receptor) is phosphorylated by PKA (protein kinase A/cAMP-dependent protein kinase) at two major sites, Ser-2030 and Ser-2808. In the present study, we examined the properties and physiological relevance of phosphorylation of these two sites. Using site- and phospho-specific antibodies, we demonstrated that Ser-2030 of both recombinant and native RyR2 from a number of species was phosphorylated by PKA, indicating that Ser-2030 is a highly conserved PKA site. Furthermore, we found that the phosphorylation of Ser-2030 responded to isoproterenol (isoprenaline) stimulation in rat cardiac myocytes in a concentration- and time-dependent manner, whereas Ser-2808 was already substantially phosphorylated before beta-adrenergic stimulation, and the extent of the increase in Ser-2808 phosphorylation after beta-adrenergic stimulation was much less than that for Ser-2030. Interestingly, the isoproterenol-induced phosphorylation of Ser-2030, but not of Ser-2808, was markedly inhibited by PKI, a specific inhibitor of PKA. The basal phosphorylation of Ser-2808 was also insensitive to PKA inhibition. Moreover, Ser-2808, but not Ser-2030, was stoichiometrically phosphorylated by PKG (protein kinase G). In addition, we found no significant phosphorylation of RyR2 at the Ser-2030 PKA site in failing rat hearts. Importantly, isoproterenol stimulation markedly increased the phosphorylation of Ser-2030, but not of Ser-2808, in failing rat hearts. Taken together, these observations indicate that Ser-2030, but not Ser-2808, is the major PKA phosphorylation site in RyR2 responding to PKA activation upon beta-adrenergic stimulation in both normal and failing hearts, and that RyR2 is not hyperphosphorylated by PKA in heart failure. Our results also suggest that phosphorylation of RyR2 at Ser-2030 may be an important event associated with altered Ca2+ handling and cardiac arrhythmia that is commonly observed in heart failure upon beta-adrenergic stimulation.

Cholinergic modulation of heart rate in isolated spontaneously beating single cells from the rabbit sino‐atrial node was investigated by measuring transmembrane ionic currents using the nystatin‐perforated patch whole‐cell voltage‐clamp technique. Carbamylcholine (CCh), a stable analogue of acetylcholine (ACh), significantly inhibited L‐type calcium currents (Ica(L) which had been augmented by beta‐adrenergic stimulation. In addition, CCh activated a potassium outward current (IK(ACh)). Both effects were blocked by atropine. The possible involvement of nitric oxide (NO) in these responses was evaluated by inhibiting NO synthesis. In the presence of NG‐monomethyl‐L‐arginine (L‐NMMA, 100 microM) or nitro‐L‐arginine methyl ester (L‐NAME, 1 mM), two specific inhibitors of nitric oxide synthase (NOS), CCh no longer inhibited ICa(L). IK(ACh) could still be activated. Co‐incubation of cells in L‐NAME or in L‐NMMA with arginine (the endogenous substrate of NOS) restored the CCh‐induced attenuation of ICa(L), indicating that L‐NAME or L‐NMMA did not interfere directly with the muscarinic action of CCh on ICa(L). Effects of the NO‐releasing agent molsidomine (SIN‐1) on CCh‐induced changes in ICa(L) were also investigated. After ICa(L) had been augmented by beta‐adrenergic stimulation, SIN‐1 (0.1 mM) inhibited ICa(L); however, SIN‐1 had no further inhibitory effect after a maximal CCh concentration had been applied. These findings suggest that NO generation is an obligatory process in cholinergic inhibition of ICa(L) in mammalian cardiac pacemaker tissue.

SUMMARY1. WVhole-cell voltage-clamp measurements were made of the time-and voltagedependenit properties of the inwardly rectifying background potassium current K'1, in single myocytes from rabbit ventricle. The main goal of these experiments was to define the role of IKI in the plateau and repolarization phases of the action potential (AP).2. Action potentials from single ventricular myocytes were used as the command signals for voltage-clamp measurements. In these 'action potential voltage-clamp' experiments, IKi was isolated from other membrane currents by taking the difference between control currents and currents in Ks-free bathing solution. The results show that IK1 is small during the plateau, but then rapidly increases during repolarization and declines in early diastole. 4. Rectangular voltage-clamp steps were used to study time-and voltagedependent changes in IKi at membrane potentials corresponding to the repolarization phase of the AP. 'Slow' relaxations or tail currents, lasting 100-300 ms, were consistently recorded when the cell was repolarized to potentials in the range -30 to -70 mV, following depolarizations between + 10 and -10 mV.5. The close correlation between the magnitude of the steady-state IK1 (in an external K+ concentration of 5 4 mM), which was outward for membrane poterntials in the range -30 to -70 mV, and the magnitude of the tail currents, suggests that they resulted from a slow increase, or reactivation, of 'K1V 6. The component of the slow tails due to reactivation Of 'Kl can be separated from a previously described component due to Na+-Ca2" exchange since the IK1 also depends on the rate of change of membrane potential during the ramp; 'faster' ramps produced less outward IK1.8. These results show that IK1 is 'inactivated' during the upstroke and plateau phases of an AP and consequently the amount of IK. available for repolarization is less than the maximal IKl present in the cell. Thus, heart rate-induced changes in IK.may contribute to alterations in the plateau and/or the duration of the action potential, as well as the threshold for firing Ca2+-dependent action potentials, or slow responses.

The electrophysiological properties of single ventricular myocytes from control rats and from rats made diabetic by streptozotocin (STZ) injection (100 mg/kg body weight) have been investigated using whole-cell voltage-clamp measurements. Our major goal was to define the effects of diabetes on rate-dependent changes in action potential duration and the underlying outward K' currents. As early as 4 to 6 days after STZ treatment, significant elevation of plasma glucose levels occurs, and the action potential duration increases. In both control and diabetic rats, when the stimulation rate is increased, the action potential is prolonged, but this lengthening is considerably more pronounced in myocytes from diabetic rats. In ventricular myocytes from diabetic rats, the Ca2`-independent transient outward K' current (It) is reduced in amplitude, and its reactivation kinetics are slowed. These changes result in a smaller It at physiological heart rates. The steady-state outward K' current (IK) also exhibits rate-T he cardiovascular complications of diabetes mellitus are well known.1,2 In addition to coronary vessel disease, there are significant derangements in the myocardium itself, including alterations in both mechanical and electrical activity.34 Changes in both the action potential configuration5,6 and the ECG of diabetic patients have been described and may be responsible for the increased propensity for cardiac arrhythmias.7Recent findings have provided important biochemical/electrophysiological evidence concerning the cellular mechanism(s) for the functional changes that occur in the heart in the diabetic state. Changes in the ionic currents that generate the action potential8 9 have been described, and the amounts of GTP-binding proteins that mediate hormonal and neurotransmitter transduction mechanisms10 are known to be altered significantly. In addition to well-known changes in membrane phospholipid composition," ion pump and exchange systems,12 and overall cellular metabolic activity,13.'4 there are alterations in the levels of various hormones that, in turn, may also affect cardiac function. For example, a hypothyroid state develops in diabetic animals and patients.5,15In most previous studies, an animal model of chemically induced diabetes has been used, and the effects of diabetes have been investigated after a period of no less Received April 30, 1993; accepted December 8, 1993 dependent attenuation, and this phenomenon is more pronounced in cells from diabetic rats. These STZ-induced changes in It and IK also develop when a lower dose (55 mg/kg) of STZ is used and measurements are made after 7 weeks of treatment. These electrophysiological effects are not related to the hypothyroid conditions that accompany the diabetic state, since they cannot be reversed by replacement of the hormone L-triiodothyronine to physiological levels. Direct effects of STZ could be ruled out, since preceding the STZ injection with a bolus injection of 3-O-methylglucose, which prevents development of hyperglycemia, pr...

The biochemical signaling pathways involved in nitric oxide (NO)-mediated cholinergic inhibition of L-type Ca 2+ current (Ic, tL1) were investigated in isolated primary pacemaker cells from the rabbit sinoatrial node (SAN) using the nystatin-perforated whole-cell voltage clamp technique. Carbamylcholine (CCh; 1 v.M), a stable analogue of acetylcholine, significantly inhibited/C~