Phospholamban-Dependent Effects of C12E8 on Calcium Transport and Molecular Dynamics in Cardiac Sarcoplasmic Reticulum

Abstract

We have studied the effects of the nonionic detergent C₁₂E₈ on Ca-ATPase enzymatic activity and oligomeric state (detected by time-resolved phosphorescence anisotropy, TPA) in skeletal and cardiac sarcoplasmic reticulum (SR). In skeletal SR, C₁₂E₈ inhibits the Ca-ATPase, both at high (micromolar and above) and low (submicromolar) Ca. In cardiac SR, C₁₂E₈ inhibits at high Ca but activates at low Ca. Thus C₁₂E₈ activates enzymatic activity only in cardiac SR and only under conditions (submicromolar Ca) where phospholamban (PLB) regulates (inhibits) the enzyme [Lu, Y.-Z., & Kirchberger, M. A. (1994) Biochemistry 33, 5056−5062]. TPA of skeletal SR at low and high Ca demonstrates that C₁₂E₈ induces aggregation of ATPase monomers and small oligomers. C₁₂E₈ also aggregates the Ca-ATPase in cardiac SR at high Ca. In cardiac SR at low Ca, the Ca-ATPase is already highly aggregated, and C₁₂E₈ partially dissociates these aggregates. Thus the TPA results provide a simple physical explanation for the functional effects: C₁₂E₈ inhibits the ATPase when it aggregates the enzyme (skeletal SR at high and low Ca; cardiac SR at high Ca), and the detergent activates when it dissociates ATPase oligomers (cardiac SR at low Ca). C₁₂E₈ stabilizes the E2P conformation of the Ca-ATPase with respect to the E2 conformation, and this stabilization is PLB-dependent. Both the physical and the functional effects of C₁₂E₈ on the Ca-ATPase are PLB-dependent, with C₁₂E₈ reversing the effects of PLB. The results provide insight into the mechanism by which PLB regulates the Ca-ATPase in cardiac SR.