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MOLECULAR PHARMACOLOGY 52:725-734 (1997).

Effects of Protein Phosphatase and Kinase Inhibitors on Ca²⁺ and Cl Currents in Guinea Pig Ventricular Myocytes

Yoshiyuki Hirayama and H. Criss Hartzell

Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322-3030

It is well-established that in heart, both the L-type Ca²⁺ channel and the cystic fibrosis transmembrane conductance regulator Cl channel are regulated by cAMP-dependent phosphorylation. However, it is not clear whether both of these channels are regulated in concert by protein kinase A (PKA) or whether there are mechanisms that independently control the phosphorylation of these two PKA targets. The purpose of this study was to compare the effects of various protein phosphatase and protein kinase inhibitors on these two ionic currents (I_Ca and I_Cl) in guinea pig ventricular myocytes to gain insight into these questions. We found that both the stimulation and washout of the effects of isoproterenol on I_Cl are about twice as fast as the effects on I_Ca, probably because the dephosphorylation reaction for I_Cl is faster than that for I_Ca. In contrast, inhibition of protein phosphatases with 10 µM microcystin stimulated both I_Ca and I_Cl, but the stimulation of I_Cl was much slower and smaller than the stimulation of I_Ca. The effect of microcystin was inhibited by staurosporine (K_i = 171.5 and 161 nM for I_Ca and I_Cl, respectively), suggesting that the stimulation was due to a kinase. The kinase was not protein kinase C (PKC) because it was not inhibited by the specific pseudosubstrate inhibitor of PKC, PKC_(19-31), and it was not PKA because it was not inhibited by adenosine 3,5-cyclic phosphorothioate. These results suggest that although both the Ca²⁺ and Cl channels are regulated by cAMP-dependent phosphorylation, another protein kinase may also regulate these channels, and the kinetics of the response of the channels to phosphorylation can be modulated independently by protein phosphatases.