PROTEIN-KINASE INHIBITORS REDUCE SR CA TRANSPORT IN PERMEABILIZED CARDIAC MYOCYTES

Phosphorylation of the sarcoplasmic reticulum (SR) protein phospholamban by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) and Ca-calmodulin-dependent protein kinase (CaM-KII) stimulates Ca-adenosinetriphosphatase (ATPase) activity and SR Ca transport, but the role of CaM-KII-dependent phosphorylation is not well defined. We studied the PKA- and CaM-KII-dependent regulation of SR Ca transport in digitonin-permeabilized rabbit ventricular myocytes. SR Ca uptake and free Ca concentration were measured on line with indo 1 and Ca electrodes in the presence of 20 mu M ruthenium red and 10 mM oxalate. Neither N-6,2'-O-dibutyryl-cAMP (up to 500 mu M) nor the nonhydrolyzable cAMP agonist adenosine 3'5'-cyclic monophosphorothioate sodium salt (Sp-cAMP[S]; up to 275 mu M) affected the maximum uptake rate (V-max) or the dissociation constant (K-d) for Ca uptake. However, the PKA inhibitor H-89 significantly increased K-d (e.g., from 307 +/- 67 to 826 +/- 62 nM Ca at 40-65 mu M H-89) without significantly affecting V-max. Both CaM-KII inhibitors, KN-62 (60 mu M) and a CaM-KII inhibitory peptide (10 mu M), significantly decreased V-max from 11.95 +/- 0.5 to 9.48 +/- 0.6 nmol.mg(-1).min(-1) and from 10.95 +/- 1.72 to 7.37 +/- 0.94 nmol.mg(-1).min(-1), respectively, without consistently changing K-d The effects of H-89 on K-d and of KN-62 on V-max were prevented by a monoclonal antibody to phospholamban 2D12 (consistent with the antibody removing the inhibitory effect of phospholamban on the SR Ca-ATPase). Clear effects of protein kinase inhibitors and lack of stimulatory effects lead us to conclude that the Ca pump may be close to maximal activation in our system (such that only inhibitory effects are apparent). The results suggest that phospholamban phosphorylation by PKA and CaM-KII, respectively, may produce functionally distinct effects on Ca transport by the SR. That is, phosphorylation of phospholamban by endogenous CaM-KII appears to increase the V-max of the SR Ca-ATPase, whereas PKA increases the Ca affinity of the pump.