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Expression and functional characterization of isoforms 4 of the plasma membrane calcium pump
B S Preianò1, D Guerini, E Carafoli
1Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), Zürich, Switzerland.
Abstract:
PMCA isoforms 4CII (generated by splicing at the C-terminus) and 4BICI (a pump version lacking the 10th transmembrane domain) were expressed in Sf9 cells using the baculovirus system. The purified PMCA4CII had a 20-fold lower affinity for calmodulin than the PMCA4CI, the PMCA4 isoform of the erythrocytes' membranes, but had a higher activity in the absence of calmodulin. The amount of phosphoenzyme intermediate formed by PMCA4CII in the presence of Ca2+ alone was almost 3 times higher than in PMCA4CI and was increased by La3+ less than in the PMCA4CI. The isoform lacking the 10th transmembrane domain (PMCA4BICI) had no Ca2+-dependent ATPase activity, but was still able to form the phosphoenzyme intermediate starting from phosphate. When expressed in COS cells, this isoform was retained in the endoplasmic reticulum; changes in membrane architecture apparently occurred during its expression; the C-terminal portion of the isoform was located in the cytosol, indicating that the deletion of the 10th transmembrane domain resulted in the loss of at least another transmembrane domain.
Insights
Investigating plasma membrane calcium ATPase (PMCA) isoforms revealed distinct functional properties. PMCA4CII exhibits altered calmodulin affinity and activity, while PMCA4BICI, lacking a transmembrane domain, loses Ca2+-dependent ATPase function.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Plasma membrane calcium ATPase (PMCA) pumps are crucial for maintaining cellular calcium homeostasis.
- Alternative splicing and protein modifications generate diverse PMCA isoforms with potentially distinct functions.
- Understanding PMCA isoform variations is key to elucidating calcium signaling regulation.
Purpose of the Study:
- To characterize the functional properties of two novel PMCA isoforms: PMCA4CII and PMCA4BICI.
- To investigate the impact of C-terminal splicing and transmembrane domain deletion on PMCA activity and calmodulin interaction.
- To determine the cellular localization and membrane integration of the PMCA4BICI isoform.
Main Methods:
- Expression of PMCA isoforms in Sf9 and COS cells using the baculovirus system.
- Purification of PMCA4CII for biochemical assays.
- Enzyme kinetics studies to assess Ca2+-dependent ATPase activity and calmodulin affinity.
- Analysis of phosphoenzyme intermediate formation.
- Cellular expression and localization studies using COS cells.
Main Results:
- PMCA4CII displayed a 20-fold lower calmodulin affinity but higher activity without calmodulin compared to PMCA4CI.
- PMCA4CII showed increased phosphoenzyme formation with Ca2+ alone and reduced La3+ stimulation.
- PMCA4BICI lacked Ca2+-dependent ATPase activity but retained phosphate-dependent phosphoenzyme formation.
- PMCA4BICI was retained in the endoplasmic reticulum and appeared to disrupt membrane architecture, suggesting loss of additional transmembrane domains.
Conclusions:
- The C-terminal splicing in PMCA4CII significantly alters its calmodulin sensitivity and catalytic activity.
- Deletion of the 10th transmembrane domain in PMCA4BICI abolishes its pump function and affects its membrane integration.
- These findings highlight the functional diversity of PMCA isoforms and the critical role of transmembrane domains in pump activity and localization.