Co-reconstitution of phospholamban mutants with the Ca-ATPase reveals dependence of inhibitory function on phospholamban structure.

Phospholamban (PLB), a 52-amino acid integral membrane protein, regulates the Ca-ATPase (calcium pump) in cardiac sarcoplasmic reticulum through PLB phosphorylation mediated by beta-adrenergic stimulation. Based on site-directed mutagenesis and coexpression with Ca-ATPase (SERCA2a) in Sf21 insect cells or in HEK 293 cells, and on spin label detection of PLB oligomeric state in lipid bilayers, it has been proposed that the monomeric form of PLB is the inhibitory species, and depolymerization of PLB is essential for its regulatory function. Here we have studied the relationship between PLB oligomeric state and function by in vitro co-reconstitution of PLB and its mutants with purified Ca-ATPase. We compared wild type-PLB (wt-PLB), which is primarily a pentamer on SDS-polyacrylamide gel electrophoresis (PAGE) at 25 degrees C, with two of its mutants, C41L-PLB and L37A-PLB, that are primarily tetramer and monomer, respectively. We found that the monomeric mutant L37A-PLB is a more potent inhibitor than wt-PLB, supporting the previous proposal that PLB monomer is the inhibitory species. On the other hand, C41L-PLB, which has a monomeric fraction comparable to that of wt-PLB on SDS-PAGE at 25 degrees C, has no inhibitory activity when assayed at 25 degrees C. However, at 37 degrees C, a 3-fold increase in the monomeric fraction of C41L-PLB on SDS-PAGE resulted in inhibitory activity comparable to that of wt-PLB. Upon increasing the temperature from 25 to 37 degrees C, no change in fraction monomer or inhibitory activity for wt-PLB and L37A-PLB was observed. Based on these results, the extent of inhibition of Ca-ATPase by PLB or its mutants appears to depend not only on the propensity of PLB to dissociate into monomers but also on the relative potency of the particular PLB monomer when interacting with the Ca-ATPase.