Abstract
The utility of the baculovirus cell expression system for investigating Ca2+-ATPase and phospholamban regulatory interactions was examined. cDNA encoding the canine cardiac sarco(endo)plasmic Ca2+-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 Ca2+ 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. Co-expression of wild-type, pentameric phospholamban with the Ca2+-ATPase decreased the apparent affinity of the ATPase for Ca2+, 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 co-expressed with a monomeric mutant of phospholamban, in which leucine residue 37 was changed to alanine. Surprisingly, monomeric phospholamban suppressed SERCA2a Ca2+ affinity more strongly than did wild-type phospholamban, demonstrating that the pentamer is not essential for Ca2+ pump inhibition and that the monomer is the more active species. To test if phospholamban functions as a Ca2+ channel, Sf21 microsomes expressing either SERCA2a or SERCA2a plus phospholamban were actively loaded with Ca2+ and then assayed for unidirectional 45Ca2+ efflux. No evidence for a Ca2+ 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.
Highlights
Phospholamban is a pentameric transmembrane phosphoprotein regulator of the Ca2ϩ-transport ATPase of cardiac sarcoplasmic reticulum [1, 2]
The SERCA2a ATPase contributes 20% of the total microsomal protein, a protein content comparable to that found in cardiac sarcoplasmic reticulum vesicles, and an expression level far greater than that previously reported
Phospholamban co-expressed with SERCA2a is functional, producing regulatory effects identical to those observed with use of cardiac sarcoplasmic reticulum vesicles
Summary
Phospholamban is a pentameric transmembrane phosphoprotein regulator of the Ca2ϩ-transport ATPase of cardiac sarcoplasmic reticulum [1, 2]. To understand the molecular mechanism of phospholamban regulation, several mammalian cell expression systems have recently been developed in which phospholamban and the Ca2ϩ-ATPase are co-expressed after transient transfection of cells with plasmid expression vectors [11,12,13]. These studies have provided useful insights into the mechanism of phospholamban inhibition, including identification of some of the amino acid residues of phospholamban required for Ca2ϩ pump regulation [14]. We report on the further development of this system for
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