Regulation of the skeletal sarcoplasmic reticulum Ca(2+)-ATPase by phospholamban and negatively charged phospholipids in reconstituted phospholipid vesicles.
The Ca(2+)-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in proteoliposomes containing phosphatidylcholine (PC). When reconstitution occurred in the presence of PC and the acidic phospholipids, phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP), the Ca(2+)-uptake and Ca(2+)-ATPase activities were significantly increased (2-3 fold). The highest activation was obtained at a 50:50 molar ratio of PS:PC and at a 10:90 molar ratio of PIP:PC. The skeletal SR Ca(2+)-ATPase, reconstituted into either PC or PC:PS proteoliposomes, was also found to be regulated by exogenous phospholamban (PLB), which is a regulatory protein specific for cardiac, slow-twitch skeletal, and smooth muscles. Inclusion of PLB into the proteoliposomes was associated with significant inhibition of the initial rates of Ca(2+)-uptake, while phosphorylation of PLB by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects. The effects of PLB on the reconstituted Ca(2+)-ATPase were similar in either PC or PC:PS proteoliposomes, indicating that inclusion of negatively charged phospholipid may not affect the interaction of PLB with the skeletal SR Ca(2+)-ATPase. Regulation of the Ca(2+)-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by crosslinking experiments, using a synthetic peptide which corresponded to amino acids 1-25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca(2+)-ATPase may be also regulated by phospholamban although this regulator is not expressed in fast-twitch skeletal muscles.
Szymańska
G
G
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0575.
Kim
H W
HW
Cuppoletti
J
J
Kranias
E G
EG
D000345:Affinity Labels; D000818:Animals; D002135:Calcium-Binding Proteins / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004789:Enzyme Activation; D066298:In Vitro Techniques; D008563:Membrane Lipids / Q000502:physiology; D009132:Muscles / Q000201:enzymology*; D010716:Phosphatidylinositols / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry; D010743:Phospholipids / Q000502:physiology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D000099211:Phospholamban
D000345:Affinity Labels; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D008563:Membrane Lipids; D010716:Phosphatidylinositols; D010718:Phosphatidylserines; D010743:Phospholipids; D000099211:Phospholamban