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 calcium transport into the sarcoplasmic reticulum. Cyclic 3′-5′-adenosine monophosphate (cAMP) causes the cAMP-dependent protein kinase to phosphorylate the regulatory protein phospholamban, which results in the stimulation of calcium transport. Calmodulin also phosphorylates phospholamban by a calcium-dependent mechanism.

We have reported the isolation and purification of phospholamban with low deoxycholate (DOC) concentrations (5 × 10⁻⁶ M). We have also reported the isolation and purification of Ca²⁺ + Mg²⁺-ATPase with a similar procedure. Both phospholamban and Ca²⁺ + Mg²⁺-ATPase retained their native properties associated with sarcoplasmic reticulum vesicles. Further, we have shown that the removal of phospholamban from membranes of sarcoplasmic reticulum vesicles 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. It is found that the phosphorylation of phospholamban by the Ca²⁺-calmodulin system is required 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.

We propose a model for the cardiac Ca²⁺ + Mg²⁺-ATPase, whereby the enzyme is normally uncoupled from Ca²⁺ uptake. The enzyme becomes coupled to Ca²⁺ transport after the first site of phospholamban is phosphorylated with the Ca²⁺-calmodulin system. When the second 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.