Calcium-dependent levels of phospholamban pentamer in native heart membranes reflect interactions of monomers with calcium-free SERCA2a

Background

In sarcoplasmic reticulum (SR) membranes, phospholamban (PLB) exists in an equilibrium of non-inhibitory homopentamers (PLB₅) and inhibitory monomers (PLB₁) that bind to SERCA2a. A new approach is needed to determine the full scheme of interactions between PLB and SERCA2a in native cardiac SR membranes, which remains poorly understood.

Methods

Dog cardiac SR membranes (dSR) were switched between EGTA and Ca²⁺ buffers to convert SERCA2a between the Ca²⁺-free, E2 and the high Ca²⁺-affinity, E1 conformations. Reactions were stopped by SDS to preserve PLB₅ structures in dSR before immunoblotting.

Results

Converting SERCA2a from E2 to E1, Ca²⁺ addition significantly increased PLB₅/PLB₁ ratios, suggesting that PLB₁ is dissociated from E1, and assembled into PLB₅ in dSR. This Ca²⁺-induced increase in PLB₅/PLB₁ was reversed by the subsequent addition of EGTA, revealing the processes of PLB₁ binding to E2 and disassembly of PLB₅. In both cases, PLB₅/PLB₁ reached new steady states in <2 s. Furthermore, PLB antibody eliminated Ca²⁺-dependent shifts in PLB₅/PLB₁. PLB phosphorylation caused similar leftward shifts in the Ca²⁺-dependent curve for PLB₅/PLB₁ and Ca²⁺-ATPase activity.

Conclusions

We have developed a simple, effective method and revealed that the levels of SERCA2a inhibition are controlled by an equilibrium between PLB₁ association with E2 and its dissociation from E1, and the formation of PLB₅ in native cardiac SR membranes. With intact regulatory components in their natural phospholipid environment, Ca²⁺-dependent shifts in PLB₅/PLB₁ can expose PLB-SERCA2a protein-protein interactions in native membranes from normal and diseased hearts, in which proteomes and lipidomes are likely to vary.