Phospholamban Inhibits Ca-ATPase Conformational Changes Involving the E2 Intermediate

• Published in: Biochemistry (Easton) Vol. 46; no. 7; pp. 1999 - 2009
• Main Authors: Waggoner, Jason R, Huffman, Jamie, Froehlich, Jeffrey P, Mahaney, James E
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 20.02.2007
• Subjects: Animals; Antibodies, Monoclonal; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - metabolism; Cells, Cultured; Dogs; Fluorescent Dyes; In Vitro Techniques; Insecta; Microsomes - metabolism; Naphthalenesulfonates; Phosphoric Acids; Phosphorylation; Protein Conformation; Sarcoplasmic Reticulum Calcium-Transporting ATPases - genetics; Sarcoplasmic Reticulum Calcium-Transporting ATPases - metabolism; Spectrometry, Fluorescence
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi061365k

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 (ΔK Ca = 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 (ΔK 0.5 = 70 μM). 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.