TRANSGENIC APPROACHES TO DEFINE THE FUNCTIONAL-ROLE OF DUAL SITE PHOSPHOLAMBAN PHOSPHORYLATION

Phospholamban is a critical regulator of the sarcoplasmic reticulum Ca 2+-ATPase activity and myocardial contractility, Phosphorylation of ph ospholamban occurs on both Ser(16) and Thr(17) during isoproterenol st imulation, To determine the physiological significance of dual site ph ospholamban phosphorylation, we generated transgenic models expressing either wild-type or the Ser(16) --> Ala mutant phospholamban in the c ardiac compartment of the phospholamban knockout mice. Transgenic line s with similar levels of mutant or wild-type phospholamban were studie d in parallel, Langendorff perfusion indicated that the basal hyperdyn amic cardiac function of the knockout mouse was reversed to the same e xtent by reinsertion of either wild-type or mutant phospholamban, Howe ver, isoproterenol stimulation was associated with much lower response s in the contractile parameters of mutant phospholamban compared with wild-type hearts, These attenuated responses were due to lack of phosp horylation of mutant phospholamban, assessed in P-32 labelin, a perfus ion experiments, The lack of phospholamban phosphorylation in vivo was not due to conversion of Ser(16) to Ala, since the mutated phospholam ban form could serve as substrate for the calcium-calmodulin-dependent protein kinase in vitro. These findings indicate that phosphorylation of Ser(16) is a prerequisite for Thr(17) phosphorylation in phosphola mban, and prevention of phosphoserine formation results in attenuation of the beta-agonist stimulatory responses in the mammalian heart.