Functional specificity of PMCA isoforms?

In mammals, four different genes encode four PMCA isoforms. PMCA1 and PMCA4 are expressed ubiquitously. PMCA2 and PMCA3 are expressed prevalently in the central nervous systems. More than 30 variants are generated by mechanisms of alternative splicing. The physiological meaning of the existence of such elevated number of isoforms is not clear, but it would be plausible to relate it to the cell-specific demands of Ca2+ homeostasis. To characterize functional specificity of PMCA variants we have investigated two aspects: the effects of the overexpression of the different PMCA variants on cellular Ca2+ handling and the existence of possible isoform-specific interactions with partner proteins using a yeast two-hybrid technique. The four basic PMCA isoforms were coexpressed in CHO cells together with the Ca2+-sensitive recombinant photoprotein aequorin. The effects of their overexpression on Ca2+ homeostasis were monitored in the living cells. They had revealed that the ubiquitous isoforms 1 and 4 are less effective in reducing the Ca2+ peaks generated by cell stimulation as compared to the neuron-specific isoforms 2 and 3. To establish whether these differences were related to different and new physiological regulators of the pump, the 90 N-terminal residues of PMCA2 and PMCA4 have been used as baits for the search of molecular partners. Screening of a human brain cDNA library with the PMCA4 bait specified the epsilon-isoform of protein 14-3-3, whereas no 14-3-3 epsilon clone was obtained with the PMCA2 bait. Overexpression of PMCA4/14-3-3 epsilon (but not of PMCA2/14-3-3 epsilon) in HeLa cells together with targeted aequorins showed that the ability of the cells to export Ca2+ was impaired. Thus, the interaction with 14-3-3 epsilon inhibited PMCA4 but not PMCA2. The role of PMCA2 has been further characterized by Ca2+ measurements in cells overexpressing different splicing variants. The results indicated that the combination of alternative splicing at two different sites in the pump structure was responsible for different functional characteristics of the pumps.