FUNCTIONAL COEXPRESSION OF THE CANINE CARDIAC CA2-FRUGIPERDA (SF21) CELLS REVEALS NEW INSIGHTS ON ATPASE REGULATION( PUMP AND PHOSPHOLAMBANIN SPODOPTERA)

The utility of the baculovirus cell expression system for investigatin g Ca2+-ATPase and phospholamban regulatory interactions was examined, cDNA encoding the canine cardiac sarco(endo)plasmic Ca2+-ATPase primp (SERCA2a) was cloned for the first time and expressed in the presence and absence of phospholamban in Spodoptera frugiperda (Sf21) insect ce lls, The recombinant Ca2+ pump was produced in high yield, contributin g 20% of the total membrane protein in Sf21 microsomes. At least 70% o f the expressed pumps were active, Coexpression of wild-type, pentamer ic phospholamban with the Ca2+-ATPase decreased the apparent affinity of the ATPase for Ca2+, but had no effect on the maximum velocity of t he enzyme, similar to phospholamban's action in cardiac sarcoplasmic r eticulum vesicles, To investigate the importance of the oligomeric str ucture 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 suppress ed SERCA2a Ca2+ affinity more strongly than did wild-type phospholamba n, demonstrating that the pentamer is not essential for Ca2+ pump inhi bition and that the monomer is the more active species, To test if pho spholamban functions as a Ca2+ channel, Sf21 microsomes expressing eit her SEBCA2a or SERCA2a plus phospholamban were actively loaded with Ca 2+ and then assayed for unidirectional Ca-45(2+) efflux. No evidence f or a Ca2+ channel activity of phospholamban was obtained, We conclude that the phospholamban monomer is an important regulatory component in hibiting SERCA2a in cardiac sarcoplasmic reticulum membranes, and that the channel activity of phospholamban previously observed in planar b ilayers is not involved in the mechanism of ATPase regulation.