FUNCTIONAL COEXPRESSION OF THE CANINE CARDIAC CA2-FRUGIPERDA (SF21) CELLS REVEALS NEW INSIGHTS ON ATPASE REGULATION( PUMP AND PHOSPHOLAMBANIN SPODOPTERA)
Jm. Autry et Lr. Jones, FUNCTIONAL COEXPRESSION OF THE CANINE CARDIAC CA2-FRUGIPERDA (SF21) CELLS REVEALS NEW INSIGHTS ON ATPASE REGULATION( PUMP AND PHOSPHOLAMBANIN SPODOPTERA), The Journal of biological chemistry, 272(25), 1997, pp. 15872-15880
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.