Mechanism of the Stimulation of Cardiac Sarcoplasmic Reticulum Calcium Pump by Calmodulin

Abstract

Calmodulin has been shown to stimulate the initial rates of Ca²⁺-uptake and Ca²⁺-ATPase in cardiac sarcoplasmic reticulum, when it is present in the reaction assay media for these activities. To determine whether the stimulatory effect of Calmodulin is mediated directly through its interaction with the Ca²⁺-ATPase, or indirectly through phosphorylation of phospholamban by an endogenous protein kinase, two approaches were taken in the present study. In the first approach, the effects of calmodulin were studied on a Ca²⁺-ATPase preparation, isolated from cardiac sarcoplasmic reticulum, which was essentially free of phospholamban. The enzyme was preincubated with various concentrations of calmodulin at 0±C and 37±C, but there was no effect on the Ca²⁺-ATPase activity assayed over a wide range of [Ca²⁺] (0.1–10 μM). In the second approach, cardiac sarcoplasmic reticulum vesicles were prephosphorylated by an endogenous protein kinase in the presence of calmodulin. Phosphorylation occurred predominantly on phospholamban, an oligomeric proteolipid. The sarcoplasmic reticulum vesicles were washed prior to assaying for Ca²⁺ uptake and Ca²⁺-ATPase activity in order to remove the added calmodulin. Phosphorylation of phospholamban enhanced the initial rates of Ca²⁺-uptake and Ca²⁺-ATPase, and this stimulation was associated with an increase in the affinity of the Ca²⁺-pump for calcium. The EC₅₀ values for calcium activation of Co²⁺-uptake and Ca²⁺-ATPase were 0.96±0.03 μM and 0.96±0.1 μM calcium by control vesicles, respectively. Phosphorylotion decreased these values to 0.64±0.12 μM calcium for Ca²⁺-uptake and 0.62±0.11 μM calcium for Ca²⁺-ATPase. The stimulatory effect was associated with increases in the apparent initial rates of formation and decomposition of the phosphorylated intermediate of the Ca²⁺-ATPase. These findings suggest that calmodulin regulates cardiac sarcoplasmic reticulum function by protein kinase-mediated phosphorylation of phospholamban.

Key Words:

• Cardiac muscle
• sarcoplasmic reticulum (SR)
• calmodulin
• protein kinase
• phospholamban
• calcium ATPase