A comparative functional analysis of plasma membrane Ca2+ pump isoforms in intact cells

The four basic isoforms of the plasma membrane Ca2+ pump and the two C-terminally truncated spliced variants PMCA4CII (4a) and 3CII (3a) were transiently overexpressed in Chinese hamster ovary cells together with aequorin targeted to the cytosol, the endoplasmic reticulum and the mitochondria. As PMCA3CII (3a) had not yet been cloned and studied, it was cloned for the study, partially purified, and characterized. At variance with the corresponding truncated variant of PMCA4, which had been studied previously PMCA3CII (3a) had very high calmodulin affinity. All four basic pump variants influenced the homeostasis of Ca2+ in the native intracellular environment. The level of Ca2+ refilling in the endoplasmic reticulum and the height of the [Ca2+] transients generated in the cytosol and in the mitochondria by the emptying of the endoplasmic reticulum store by inositol 1,4,5-trisphosphate were all reduced by the overexpression of the pumps. The effects were much greater with the neuron-specific PMCA2 and PMCA3 than with the ubiquitously expressed isoforms 1 and 4. Unexpectedly, the truncated PMCA3 and PMCA4 were as effective as the full length variants in influencing the homeostasis of Ca2+ in the cytosol and the organelles. In particular, PMCA4CII (4a) was as effective as PMCA4CI (4b), even if its affinity for calmodulin is much lower. The results indicate that the availability of calmodulin may not be critical for the modulation of PMCA pumps in vivo .


Introduction
The plasma membrane calcium pumps (PMCAs) belong to the family of P-type ATPases, which are characterised by the formation of an aspartyl phosphate intermediate during the reaction cycle (1). Four genes encode distinct isoforms in mammals. PMCA1 and 4 are ubiquitously expressed, whereas PMCA2 and PMCA3 are predominantly expressed in the central nervous system (2). Alternative RNA splicing, occurring mostly at two sites termed A and C, generate additional isoform variability. C-site splicing occurs in the C-terminal region containing the calmodulin-binding domain. In the resting state, the latter interacts with two sites in the PMCA molecule, maintaining it inhibited (3,4). Calmodulin removes the binding domain from these intramolecular receptors, freeing the pump from autoinhibition.
C site splicing occurs in the isoforms 1 and 3 through the piecemeal inclusion of portions of a performed on isolated pump domains or on membrane preparations from overexpressing mammalian or insect cells in which possible physiological pump regulators may have become lost.
It was thus decided to study the activity of PMCA isoforms under physiological conditions by transfecting CHO 1 cells with expression plasmids for the four basic isoforms and for the two truncated variants 4CII (4a) and 3CII (3a) together with the plasmids encoding the Ca 2+ -sensing probe aequorin targeted to the cytoplasm, to the mitochondria and to the endoplasmic reticulum (ER). At variance with fluorescent dyes, aequorin has very low Ca 2+ buffering capacity, and does not significantly perturb the resting cytosolic Ca 2+ level, which could influence the activity of the Ca 2+ transporters.
The work has shown that the isoforms typical of neurons were more effective in restoring the basal [Ca 2+ ] level after the transient induced by an inositol 1,4,5-trisphosphate (InsP 3 ) generating agonist than the ubiquitously expressed isoforms. The two neuronal isoforms reduced [Ca 2+ ] in the ER by about 30%, whereas PMCA4 only decreased it by about 15%.
Isoform 1 was even less effective, reducing ER [Ca 2+ ] only marginally. The effects on mitochondrial Ca 2+ uptake, which reflect the level of Ca 2+ filling of the ER (14), went along the same lines, i.e., the ubiquitous isoforms reduced it much less than PMCA2 and PMCA3.
Unexpectedly, under the conditions of the CHO cell environment, essentially no differences in the effects on cytosolic, ER and mitochondrial [Ca 2+ ] were observed between the full- The restriction sites are underlined.

Preparation of the construct for the hPMCA3CII (3a) isoform
The cDNA for the human PMCA3CII (3a) was assembled from fragments of the same. A short overview of the strategy used is outlined in Figure 1. Two partial clones containing the 5' end (800 bp) and the 3' end (1,000 bp) of the isoform were obtained by screening human brain libraries. The missing portion encompassing the central portion of the hPMCA3 cDNA was obtained by RT-PCR using human brain tissue and oligonucleotides hPMCA3-

Cell cultures and transfections
Spodoptera frugiperda (Sf9) cells were grown in TNM-FH supplemented with 10% fetal calf serum and 100 µg/ml gentamicin at 29 ± 1°C. All routine procedures involving these cells were performed according to (16

Calmodulin overlay
Proteins were transferred to nitrocellulose sheets (21). Nonspecific binding of calmodulin was blocked by 1%

Purification of the overexpressed pumps
Membranes of infected Sf9 cells were prepared as described above. All purification steps were carried out at 4°C. The solubilisation of the membrane proteins was performed by They were analysed on 7 or 10 % SDS-PAGE which were stained by a silver nitrate procedure.

Calmodulin-Sepharose 4B
Recombinant calmodulin was immobilised on CNBr-activated-Sepharose 4B according to the procedure supplied by the manufacturer (Amersham-Pharmacia, Buckinghamshire, UK).
The yield of the coupling reaction was controlled by SDS-PAGE.

Measurement of Ca 2+ -ATPase activity
The Ca 2+ -ATPase activity was measured by the colorimetric method described by (24). The

Preparation of membranes from CHO cells and Western blotting analysis
Thirty-six hours after transfection, CHO cells were harvested in 10 mM Tris-HCl, pH 8.0, 2 mM EDTA, 2 mM PMSF, 1 mM DTT. They were disrupted by three cycles of freeze and thaw at -80°C/37°C and the insoluble proteins were sedimented at 11,000 x g for 30 min (4°C). The supernatant was discarded and the pellet resuspended in 5 mM Tris-HCl, pH 8.0, and 10% sucrose. Proteins were separated by 7.5% SDS-PAGE and transferred to nitrocellulose membranes. 100 µg of membrane proteins were loaded onto each lane. The sheets were probed with antibody 5F10 (diluted 1:1,000

Cloning, partial purification and characterisation of PMCA3CII (3a)
To construct a full-length expression vector for PMCA3CII (3a), nucleotide mutations were inserted in the coding sequence, generating a NsiI site at position 2061 had been already expressed in previous studies (8,9) and was thus used as a control. Figure 2 shows that both isoforms were expressed at similar levels. The PMCA3CII (3a) protein migrated in the gel faster than PMCA4CI (4b), since its molecular mass is 129 kDa as  and PMCA4 (traces a and b), were less effective than the neuronal isoforms, PMCA2 and PMCA3 (trace d and c).

ER Ca 2+
As shown in previous work (30) (Figure 9 and Table II Unfortunately, the information presently available on functional differences among isoforms is fragmentary at best. The problem is further exacerbated by the existence, next to the four basic gene products of the pump, of numerous alternatively spliced variants, which also display tissue-specific expression and which in some cases may be even more abundantly expressed than the un-spliced versions (12,13). Although the most extensively studied splice variants involve splice site C in the C-terminal cytosolic unit, splice variants involving site which has the shortest C-terminal cytosolic unit so far described for PMCA pumps. This variant, which is the major isoform in rat skeletal muscles, was only negligibly stimulated by calmodulin in membrane preparations of overexpressing COS cells, even if it still bound to calmodulin columns (13).
The finding that isoform 3CVI (3f) had lost almost completely calmodulin regulation has been the main motivation for the decision to clone, express, and characterise the 3CII (3a) version of this isoform which is homologous to 4CII (4a), which interacts with calmodulin, with very lower affinity. As all other CII (a) variants, the 3CII (3a) variant becomes truncated at the 1117 th residue, within the calmodulin-binding domain (32). PMCA3CII (3a) was thus predicted to retain, at best, the some modest degree of calmodulin regulation of PMCA4CII (4a). It was thus surprising to find that it instead reacted with calmodulin with an affinity which was even higher than that of the full-length PMCA4 (K m = 5-10 nM) and was in fact in the same range of that of PMCA2, which has the highest calmodulin affinity of all PMCAs. It is thus clear that no generalisation can be made on the effects of C-terminal truncation on the affinity for calmodulin, since the process may lead to a pump with markedly decreased affinity (PMCA4CII, 4a), to one that has all but lost calmodulin regulation (PMCA3CVI, 3f), but also to pump variants that retain optimal calmodulin affinity, as in the case of the PMCA3CII (3a) variant studied here. The finding that some isoforms may interact with calmodulin with very high affinity, whereas others may have poor to extremely poor affinity for it, is interesting in principle, but cannot be placed in a logical physiological framework in the absence of reliable information on the amount of calmodulin available to the pumps within cells.
The experiments reported here have thus evaluated the activity of the isoforms in the native cellular environment. They lead to some conclusions of general significance. The first is that the two ubiquitous isoforms PMCA1 and PMCA4 are far less effective in controlling the homeostasis of Ca 2+ than the two neuron-specific isoforms PMCA2 and PMCA3. The second is that the CII (a) truncated versions of the two pumps studied (one ubiquitous, one tissue-specific) are just as effective in controlling cellular Ca 2+ than the corresponding fulllength variants. Even if truncated isoforms differ widely in calmodulin affinity they all function very efficiently in the intracellular ambient. Therefore, in this ambient the availability of calmodulin may be but one of the factors that regulate the activity of the pumps. Other factors/processes could take primacy, perhaps depending on particular demands of cell physiology. Interestingly, one of the first studies of PMCA regulation (33) had calculated that in the membrane environment the pump would be permanently activated to about 50% of maximum by acidic phospholipids. The finding that one of the truncated versions (PMCA4CII, 4a) had significantly higher activity in the absence of calmodulin than the full-length counterpart (9), and the demonstration that two C-terminally truncated pumps         4CII (4a), respectively.