Mechanism of cAMP-dependent modulation of cardiac sodium channel current kinetics

beta-Adrenergic modulation is one of the most important regulatory mechanisms of ion channel function. Only recently, however, have beta-adrenergic effects on cardiac Na+ channel activity been recognized, and some diversity of effects has been reported in different preparations. We report studies of protein kinase A-dependent phosphorylation effects on cardiac Na+ current using the macropatch on-cell mode voltage-clamp technique to maintain cytoplasmic composition intact. During the first 5 minutes after addition of 8-(4-chlorophenylthio)cAMP to the bath, the midpoints of both voltage-dependent availability and conductance shifted in the hyperpolarizing direction an average of -7.5 +/- 2.8 mV (n = 31). Moreover, these effects were not species specific; similar results were obtained in canine, rabbit, and guinea pig myocytes, and a similar shift occurred after exposure to 5 microM isoproterenol. Maximum conductance did not change, nor did single-channel conductance. The shifts of conductance and voltage-dependent availability that were induced by protein phosphorylation were distinct from and independent of the slow background shift in kinetics. We measured the background shift to be less than 0.3 mV/min and to be restricted to the channels within the patch. Pretreatment of cells with a blocker of protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H-89), prevented the effect of 8-(4-chlorophenylthio)cAMP while not affecting the background shift in kinetics. Although clearly not the result of addition of a negatively charged phosphate to the inside face of the channel, cAMP-dependent phosphorylation affects the voltage-dependent kinetics, as expected, by an electrostatic interaction with the voltage sensor.