Regulation of Ca 2+ transport by cyclic 3′,5′-AMP-dependent and calcium-calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum

Abstract

Canine cardiac sarcoplasmic reticulum is phosphorylated by cyclic AMP-dependent and by Ca 2+-calmodulin-dependent protein kinases on a 22 kDa protein, called phospholamban. Both types of phosphorylation have been shown to stimulate the initial rates of Ca 2+ transport. To establish the interrelationship of the cAMP-dependent and Ca 2+-calmodulin-dependent phosphorylation on Ca 2+ transport, cardiac sarcoplasmic reticulum vesicles were preincubated under optimum conditions for: (a) cAMP-dependent phosphorylation, (b) Ca 2+-calmodulin-dependent phosphorylation, and (c) combined cAMP-dependent and Ca 2+-calmodulin-dependent phosphorylation. Control vesicles were treated under identical conditions, but in the absence of ATP, to avoid phosphorylation. Control and phosphorylated sarcoplasmic reticulum vesicles were subsequently centrifuged and assayed for Ca 2+ transport in the presence of 2.5 mM Tris-oxalate. Our results indicate that cAMP-dependent and Ca 2+-calmodulin-dependent phosphorylation can each stimulate calcium transport in an independent manner and when both are operating, they appear to have an additive effect. Stimulation of Ca 2+ transport was associated with a statistically significant increase in the apparent affinity for calcium by each type of phosphorylation. The degree of stimulation of the calcium affinity was relatively proportional to the degree of phospholamban phosphorylation. These findings suggest the presence of a dual control system which may operate in independent and combined manners for regulating cardiac sarcoplasmic reticulum function.