Phospholamban is a phosphoprotein regulator of cardiac sarcoplasmic reticulum which is phosphorylated in response to ␤-adrenergic stimulation. Previous results have shown that phospholamban forms Ca 2؉ -selective channels in lipid bilayers. The channel-forming domain has been localized to amino acid residues 26 -52, which form a stable pentameric, helical structure. The specific residues responsible for stabilizing the pentameric membrane domain of phospholamban have been identified by mutational analysis.

Phospholamban (PLB), a 52-residue protein integral to the cardiac sarcoplasmic reticulum, is a key regulator of the Ca pump. PLB has been shown to form pentamers in the denaturing detergent sodium dodecyl sulfate (SDS), but its oligomeric state in the natural environment of the lipid membrane remains unknown. In order to address this issue, we performed electron paramagnetic resonance (EPR) experiments on two types of lipid-reconstituted, recombinant PLB: wild type (WT PLB) and a mutant substituted with alanine at leucine 37 (L37A PLB), whose propensity to oligomerize in SDS is greatly diminished. The lipid used in reconstitution was dioleoylphosphatidylcholine (DOPC) doped with a phospholipid spin-label that detects protein contact. EPR spectroscopy was used to determine the fraction of the total lipid molecules in contact with PLB. Our results show that, in phospholipid bilayers, WT PLB is oligomeric (effective oligomeric size of 3.52 +/- 0.71), while L37A PLB is monomeric (effective oligomeric size of 1.15 +/- 0.15). Thus, the oligomeric states of these proteins in the lipid membrane are remarkably similar to those in SDS solution. In particular, the point mutation in L37A PLB greatly destabilizes the PLB oligomer. Phosphorylation of PLB by protein kinase A, which has been shown to relieve inhibition of the cardiac Ca pump, changes the lipid-PLB interactions, decreasing the number of lipids restricted by contact with protein. The results are consistent with a phosphorylation-dependent increase of the effective oligomer size of WT PLB from 3.52 to 5.34 and of L37A PLB from 1.15 to 1.91. These phosphorylation effects were abolished in a medium with a high ionic strength. We conclude that the oligomeric states of PLB in lipid membranes are in a dynamic equilibrium that is perturbed by phosphorylation due to reduced electrostatic repulsion among PLB protomers.

The utility of the baculovirus cell expression system for investigating Ca 2؉ -ATPase and phospholamban regulatory interactions was examined. cDNA encoding the canine cardiac sarco(endo)plasmic Ca 2؉ -ATPase pump (SERCA2a) was cloned for the first time and expressed in the presence and absence of phospholamban in Spodoptera frugiperda (Sf21) insect cells. The recombinant Ca 2؉ pump was produced in high yield, contributing 20% of the total membrane protein in Sf21 microsomes. At least 70% of the expressed pumps were active. Coexpression of wild-type, pentameric phospholamban with the Ca 2؉ -ATPase decreased the apparent affinity of the ATPase for Ca 2؉ , but had no effect on the maximum velocity of the enzyme, similar to phospholamban's action in cardiac sarcoplasmic reticulum vesicles. To investigate the importance of the oligomeric structure of phospholamban in ATPase regulation, SERCA2a was coexpressed with a monomeric mutant of phospholamban, in which leucine residue 37 was changed to alanine. Surprisingly, monomeric phospholamban suppressed SERCA2a Ca 2؉ affinity more strongly than did wild-type phospholamban, demonstrating that the pentamer is not essential for Ca 2؉ pump inhibition and that the monomer is the more active species. To test if phospholamban functions as a Ca 2؉ channel, Sf21 microsomes expressing either SERCA2a or SERCA2a plus phospholamban were actively loaded with Ca 2؉ and then assayed for unidirectional 45 Ca 2؉ efflux. No evidence for a Ca 2؉ channel activity of phospholamban was obtained. We conclude that the phospholamban monomer is an important regulatory component inhibiting SERCA2a in cardiac sarcoplasmic reticulum membranes, and that the channel activity of phospholamban previously observed in planar bilayers is not involved in the mechanism of ATPase regulation.

Phospholamban is a small phosphoprotein inhibitor of the Ca 2؉ -pump in cardiac sarcoplasmic reticulum, which shows a distinct oligomeric distribution between monomers and homopentamers that are stabilized through Leu/Ile zipper interactions. A two-faced model of phospholamban inhibition of the Ca 2؉ -pump was proposed, in which the Leu/Ile zipper residues located on one face of the transmembrane ␣-helix regulate the pentamer to monomer equilibrium, whereas residues on the other face of the helix bind to and inhibit the pump. Here we tested this two-faced model of phospholamban action by analyzing the functional effects of a new series of Leu/Ile zipper mutants. Pentameric stabilities of the mutants were quantified at different SDS concentrations. We show that several phospholamban mutants with hydrophobic amino acid substitutions at the Leu/ Ile zipper region retain the ability to form pentamers but at the same time give the same or even stronger (i.e. L37I-PLB) inhibition of the Ca 2؉ -pump than do mutants that are more completely monomeric. Steric constraints prevent the Leu/Ile zipper residues sequestered in the interior of the phospholamban pentamer from binding to the Ca 2؉ -pump, leading to the conclusion that the zipper residues access the pump from the phospholamban monomer, which is the active inhibitory species. A modified model of phospholamban transmembrane domain action is proposed, in which the membrane span of the phospholamban monomer maintains contacts with the Ca 2؉ -pump around most of its circumference, including residues located in the Leu/Ile zipper region. PLB1 is a 52-amino acid phosphoprotein integral to the cardiac sarcoplasmic reticulum membrane, which is an inhibitor of the Ca 2ϩ -pump in its dephosphorylated state (1, 2). The small phosphoprotein is composed of a cytosolic N-terminal domain (residues 1-30) and a highly hydrophobic, C-terminal transmembrane domain (residues 31-52). In cardiac sarcoplasmic reticulum, PLB mediates the hormonally controlled regulation of the Ca 2ϩ -pump by modulating the apparent affinity of the ATPase for calcium (3, 4). Dephospho-PLB inhibits the Ca 2ϩ -pump by lowering its apparent affinity for ionized calcium. Phosphorylation of PLB at Ser-16 and/or at Thr-17 in the cytosolic domain restores the high apparent Ca 2ϩ affinity, causing an activation of ATP hydrolysis and Ca 2ϩ -transport at submicromolar ionized calcium while leaving the V max of the enzyme measured at saturating ionized calcium concentration largely unchanged (2).On SDS-PAGE, PLB exhibits a remarkable pattern of oligomerization, being distributed mainly between populations of monomers (20%) and homopentamers (80%) (5). Alanine-and phenylalanine-scanning mutagenesis has been utilized to identify key amino acid residues in the transmembrane domain of PLB that are critical for pentamer formation. The pentamerstabilizing amino acids are Leu residues 37, 44, and 51 and Ile residues 40 and 47, all of which are located in the transmembrane ␣-helix (6). When plotted on a heptad repeat, these ...