Role of Phospholamban in Regulating Cardiac Sarcoplasmic Reticulum Calcium Pump

Abstract

Cardiac sarcoplasmic reticulum plays a critical role in the excitation-contraction cycle and hormonal regulation of heart cells. Catecholamines exert their ionotropic action through the regulation of Ca transport into the sarcoplasmic reticulum. cAMP causes the cAMP-dependent protein kinase to phosphorylate the regulatory protein phospholamban, which results in the stimulation of Ca transport. Calmodulin also phosphorylates phospholamban by a Ca-dependent mechanism. The isolation and purification of phospholamban with low deoxycholate (DOC) concentrations (5 .times. 10-6 M) are reported on. The isolation and purification of Ca2+ + Mg2+-ATPase with a similar procedure is also reported. Both phospholamban and Ca2+ + Mg2+-ATPase retained their native properties associated with sarcoplasmic reticulum vesicles. The removal of phospholamban from membranes of sarcoplasmic reticulum vesicles evidently uncouples Ca2+-uptake from ATPase without any effect on Ca2+ + Mg2+-ATPase activity or Ca2+ efflux. Phospholamban appears to be the substrate for both the Ca2+-calmodulin system and the cAMP-dependent protein kinase system. The phosphorylation of phospholamban by the Ca2+-calmodulin system apparently is reuired for the normal basal level of Ca2+ transport, and that the phosphorylation of phospholamban at another site by the cAMP-dependent protein kinase system causes the stimulation of Ca2+-transport above the basal level. The functional effects of the phosphorylation of phospholamban by cAMP-dependent protein kinase system are expressed only after the phosphorylation of phospholamban with Ca2+-calmodulin system. A model for the cardiac Ca2+ + Mg2+-ATPase, whereby the enzyme is normally uncoupled from Ca2+ uptake is proposed. The enzyme becomes coupled to Ca2+ transport after the first site of phospholamban is phosphorylated with the Ca2+-calmodulin system. When the 2nd site of phospholamban is phosphorylated with cAMP-dependent protein kinase both Ca2+ transport and ATPase are stimulated and phospholamban becomes inaccessible to DOC solubilization and trypsin.