Phospholamban inhibits Ca-ATPase conformational changes involving the E2 intermediate.
We have used steady-state fluorescence spectroscopy in combination with enzyme kinetic assays to test the hypothesis that phospholamban (PLB) stabilizes the Ca-ATPase in the E2 intermediate state. The cardiac muscle Ca-ATPase (SERCA2a) isoform was expressed either alone or coexpressed with PLB in High-Five insect cells and was isolated as insect cell microsomes. Fluorescence studies of the Ca-ATPase covalently labeled with the probe 5-(2-((iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid showed that PLB decreased the amplitude of the Ca-ATPase E2 --> E1 conformational transition by 45 +/- 3% and shifted the [Ca2+] dependence of the transition to higher Ca2+ levels (DeltaKCa = 230 nM), similar to the effect of PLB on Ca-ATPase activity. Similarly, PLB decreased the amplitude of Ca-ATPase phosphorylation by inorganic phosphate (Pi) by 55 +/- 2% and decreased slightly the affinity for Pi (DeltaK0.5 = 70 microM). However, PLB did not affect the Ca2+-dependent inhibition of Ca-ATPase phosphorylation by Pi. Finally, PLB decreased Ca-ATPase sensitivity to vanadate, increasing the IC50 value by 300 nM. The results suggest that PLB binding to Ca-ATPase stabilizes the enzyme in a conformation distinct from E2, decreasing the number of enzymes in the E2 state capable of undergoing ligand-dependent conformational changes involving the Ca-free E2 intermediate. The inability of conformation-specific ligands to fully convert this E2-like state into E1 or E2 implies that these states are not in a simple equilibrium relationship.
Waggoner
Jason R
JR
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA.
Huffman
Jamie
J
Froehlich
Jeffrey P
JP
Mahaney
James E
JE
D000818:Animals; D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004285:Dogs; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007313:Insecta; D008861:Microsomes / Q000378:metabolism; D009282:Naphthalenesulfonates; D010756:Phosphoric Acids; D010766:Phosphorylation; D011487:Protein Conformation; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D000099211:Phospholamban
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D010756:Phosphoric Acids; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban; C006421:1,5-I-AEDANS; C030242:phosphoric acid