Autoinhibition mechanism of the plasma membrane calcium pump isoforms 2 and 4 studied through lipid-protein interaction

• Published in: Biochemical journal Vol. 443; no. 1; p. 125
• Main Authors: Mangialavori, Irene C, Corradi, Gerardo, Rinaldi, Débora E, de la Fuente, María Candelaria, Adamo, Hugo P, Rossi, Juan Pablo F C
• Format: Journal Article
• Language: English
• Published: England 01.04.2012
• Subjects: Animals; Calmodulin - chemistry; Chromatography, Affinity; Enzyme Activation; Erythrocytes - enzymology; Humans; Isoenzymes - biosynthesis; Isoenzymes - chemistry; Isoenzymes - isolation & purification; Phospholipids - chemistry; Plasma Membrane Calcium-Transporting ATPases - biosynthesis; Plasma Membrane Calcium-Transporting ATPases - chemistry; Plasma Membrane Calcium-Transporting ATPases - isolation & purification; Protein Binding; Protein Conformation; Rats; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Saccharomyces cerevisiae; Staining and Labeling; Titrimetry
• ISSN: 1470-8728
• DOI: 10.1042/BJ20111035

The autoinhibition/activation of the PMCA (plasma membrane Ca2+-ATPase) involves conformational changes in the membrane region of the protein that affect the amount of lipids directly associated with the transmembrane domain. The lipid-protein-dependence of PMCA isoforms 2 and 4 expressed and obtained in purified form from Saccharomyces cerevisiae was investigated using the phosphatidylcholine analogue [125I]TID-PC/16 {l-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromemyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine}, which was incorporated into mixtures of dimyristoylphosphatidylcholine and the non-ionic detergent C12E10 [deca(ethylene glycol) dodecyl ether]. We found no differences between the recombinant PMCA4 and PMCA purified from erythrocytes (ePMCA). However, titration of the half-maximal activation by Ca2+/calmodulin of PMCA2 showed 30-fold higher affinity than PMCA4. PMCA2 exhibited a lower level of labelling in the autoinhibited conformation relative to PMCA4, indicating that the lower autoinhibition was correlated with a lower exposure to lipids in the autoinhibited state. Analysis of the lipid-protein stoichiometry showed that the lipid annulus of PMCA varies: (i) in accordance to the conformational state of the enzyme; and (ii) depending on the different isoforms of PMCA. PMCA2 during Ca2+ transport changes its conformation to a lesser extent than PMCA4, an isoform more sensitive to modulation by calmodulin and acidic phospholipids. This is the first demonstration of a dynamic behaviour of annular lipids and PMCA.