The full-length a and b variants of the rat plasma membrane calcium pump, isoform 2 (rPMCA2a and rPMCA2b), were constructed and expressed in COS-7 cells. To characterize these isoforms, calcium transport was determined in a microsomal fraction. Both rPMCA2a and rPMCA2b had a much higher affinity for calmodulin than the corresponding forms of hPMCA4, and rPMCA2b had the highest affinity among the isoforms that have been tested so far. When analyzed at a relatively high calmodulin concentration, rPMCA2b and, to a lesser extent, rPMCA2a showed higher apparent calcium affinity; i.e. they were more active at lower Ca 2؉ concentrations than hPMCA4b. This indicates that these two variants of rat isoform 2 will tend to maintain a lower free cytosolic Ca 2؉ level in cells where they are expressed. Both variants also showed a higher level of basal activity (in the complete absence of calmodulin) than hPMCA4b, a property which would reinforce their ability to maintain a low free cytosolic Ca 2؉ concentration. Experiments designed to determine the source of the higher apparent Ca 2؉ affinity of rPMCA2b showed that it came from the properties of the carboxyl terminus, rather than from any difference in the catalytic core.The plasma membrane Ca 2ϩ pump plays a key role in controlling the intracellular Ca 2ϩ concentration. This P-type ATPase is regulated by calmodulin and is responsible for the ATP powered removal of Ca 2ϩ from eukaryotic cells (1). The plasma membrane Ca 2ϩ pump (PMCA) 1 has a low level of activity in the absence of calmodulin. Calmodulin binds to an autoinhibitory domain (the C domain), and increases both the maximum velocity of the pump and the apparent Ca 2ϩ affinity.To date, at least four different genes have been found which encode for PMCA (2). Additional variability is obtained by alternate splices occurring at two sites in the pump (3-7). In each of the four genes, the alternative splice sites (8) are located in the middle of the cytosolic loop between transmembrane domains 2 and 3 (splice site A) (9) and downstream of the last transmembrane domain, in the middle of the calmodulinbinding domain (splice site C) (9 -11). The first 18 amino acids of the calmodulin-binding domain are conserved for all PMCA isoforms, but the presence of the alternative RNA splice site in the middle of this region (at splice site C) changes the remainder of the calmodulin-binding domain as well as the carboxyl terminus (10). The isoforms whose mRNA contains a spliced-in exon are called "a," while those isoforms lacking the additional exon are called "b." 2 The a variants of the isoforms have a less basic calmodulin-binding domain as well as a different carboxyl terminus than the b variants. When synthetic peptides corresponding to representative a and b forms of the calmodulinbinding domain were compared, the b form of the peptide showed a 10-fold higher affinity for calmodulin than the a form of the peptide (12). Additionally, full-length isoforms hPMCA4a and hPMCA4b were overexpressed in COS-1 cells and the calmodulin-res...

A reconstitution system allowed us to measure the ATPase activity of specific isoforms of the plasma membrane Ca 2؉ pump continuously, and to measure the effects of adding or removing calmodulin. The rate of activation by calmodulin of isoform 4b was found to be very slow, with a half-time (at 235 nM calmodulin and 0.5 M free Ca 2؉ ) of about 1 min. The rate of inactivation of isoform 4b when calmodulin was removed was even slower, with a half-time of about 20 min. Isoform 4a has a lower apparent affinity for calmodulin than 4b, but its activation rate was surprisingly faster (half time about 20 s). This was coupled with a much faster inactivation rate, consistent with its low affinity. A truncated mutant of isoform 4b also had a more rapid activation rate, indicating that the downstream inhibitory region of fulllength 4b contributed to its slow activation. The results indicate that the slow activation is due to occlusion of the calmodulin-binding domain of 4b, caused by its strong interaction with the catalytic core. Since the activation of 4b occurs on a time scale comparable to that of many Ca 2؉ spikes, this phenomenon is important to the function of the pump in living cells. The slow response of 4b indicates that this isoform may be the appropriate one for cells which respond slowly to Ca 2؉ signals.Unlike other mechanisms for removing Ca 2ϩ from the cytosol, the plasma membrane Ca 2ϩ pump requires activation by another protein, calmodulin. The requirement for this extra step may have profound effects on the shape of Ca 2ϩ spikes, particularly if the binding of calmodulin to the pump is slow. The activation by calmodulin of several calmodulin-regulated enzymes is fast, but it has been observed that the activation of the plasma membrane Ca 2ϩ pump is slow in human erythrocytes (1). Since the binding of calmodulin to the plasma membrane Ca 2ϩ pump is very tight, this slowness in the activation was surprising. Erythrocytes contain a mixture of isoforms 1 and 4 of the plasma membrane Ca 2ϩ pump (2), so it was not clear which isoform was responsible for the slow activation. It was possible to study the rate of activation in erythrocytes because almost all of their Ca 2ϩ -stimulated ATPase activity is due to the plasma membrane Ca 2ϩ pump, but extension of such studies to other cell types has been difficult. The major difficulty is the presence in almost all kinds of cells of non-pump Ca 2ϩ ATPases whose activity swamps that of the pump. Some studies using Ca 2ϩ indicators in whole cells other than erythrocytes have given results consistent with slow activation of the pump. In human neutrophils (3) it was concluded that a Ca 2ϩ spike was caused by delayed activation of the plasma membrane Ca 2ϩ pump. In this case the arguments were based in part on the use of a calmodulin antagonist, which is rather nonspecific. In vascular endothelial cells (4) a similar conclusion was based on the use of La 3ϩ , VO 4 3Ϫ and Hg 2ϩ as inhibitors of the plasma membrane Ca 2ϩ pump. Each of these reagents also inhibits other pumps a...

The expression at the protein level of plasma membrane calcium pump (PMCA) isoforms in rat brain was detected by new antibodies that distinguished the four gene products and their alternatively spliced variants. All four gene products were distributed throughout hippocampus, cortex, and cerebellum, but the alternate splices showed more distinct distribution patterns. The b splice of isoform 1 was not detectable in any of the brain regions, which makes it unlikely that this isoform performs an essential housekeeping role as is frequently proposed. The b splices of isoforms 3 and 4, although expressed in all three regions, showed evidence of proteolysis, which removed a portion of the carboxyl terminus. In contrast, isoform 2b retained its full length, indicating that PMCA2b is more resistant to proteolysis than the other b forms. Whereas substantial amounts of isoforms 1a, 2a, and 3a were expressed in all regions, 4a was found only in frontal cortex. The distinct patterns of expression of the PMCA isoforms in brain suggest that some of them play a special role in intracellular Ca regulation.The plasma membrane calcium pump is one of the mechanisms responsible for maintaining the low cytosolic calcium concentration critical to cell function. Because of its high affinity for calcium, it is believed to have a unique role in the maintenance of calcium homeostasis in eukaryotic cells. Sequencing and molecular cloning work have revealed some 20 isoforms of the pump, generated by four different genes encoding the pump and by the alternative splicing of the primary gene transcripts in two sites identified as A and C in the pump molecule. Studies on the distribution of mRNA of the pump in rat and human tissues revealed that gene products 1 and 4 are transcribed in all tissues, whereas gene products 2 and 3 were more tissue-specific and were found enriched in excitable cells. The transcription of the splicing variants was also found to be specific. In tissues where there is wide cell-type diversity, the distribution of the spliced variants has been found to be cellspecific. For example, in the pancreas, where secretion of insulin is dependent on calcium concentration, the beta cells express only isoform 4b, whereas alpha and gamma cells express both 4a and 4b. The islets do not express the a but only the b form of isoforms 1 and 2 (1). In brain, where highly regulated signal transduction events occur, the mRNAs of the spliced variants were also localized to specific cells and regions (2-4). The specific presence of the proteins of the a and b variants of the different isoforms is particularly interesting, since these variants are products of the alternate splicing at site C in the region where most of the regulatory properties of the pump are located (5, 6). In fact, Enyedi et al. (7,8) show that these proteins have different affinities for calcium and calmodulin. The protein products of the four genes encoding the pump have been identified and have confirmed the reported distribution of the mRNA (9, 10). However, informat...

Phosphorylation by protein kinase C of the "a" and "b" variants of plasma membrane Ca 2؉ pump isoforms 2 and 3 was studied. Full-length versions of these isoforms were assembled and expressed in COS cells. Whereas the "a" forms were phosphorylated easily with PKC, isoform 2b was phosphorylated only a little, and isoform 3b was not phosphorylated at all. Phosphorylation of isoforms 2a and 3a did not affect their basal activity, but prevented the stimulation of their activity by calmodulin and their binding to calmodulin-Sepharose. This indicated that phosphorylation prevented activation of these isoforms by preventing calmodulin binding. Based on these results, phosphorylation of the pump with PKC would be expected to increase free intracellular Ca 2؉ levels in those cells where isoforms 2a and 3a are expressed.The plasma membrane Ca 2ϩ pump is an important element in removing Ca 2ϩ from the cell during intracellular signaling and in maintaining the very low resting level of cytosolic Ca 2ϩ of the unstimulated cell. The pump is known to be activated in many different ways: by calmodulin, acidic phospholipids, phosphorylation with protein kinases, proteolysis, and dimerization. Although the mechanisms of all of these regulations have not been determined, the way calmodulin stimulates the pump is fairly well understood. Calmodulin binds tightly to a specific domain that is 92 amino acids upstream of the carboxyl terminus of hPMCA4b 1 (1, 2). This domain is about 28 residues long in hPMCA4b and constitutes part of an autoinhibitory region (3, 4). Binding of calmodulin to the autoinhibitor releases the inhibition and activates the pump. Although phosphorylation and/or stimulation of the pump with PKC has been described in various cells and tissues (5-11), details of the molecular basis of PKC action have been difficult to obtain. Until very recently the subjects available for study have been intact cells or biological membranes of unknown isoform composition; only in the case of the erythrocyte membrane had the isoform composition of the pump been determined, consisting primarily of hPMCA4b.Cloning of the pump has revealed the existence of at least four different genes coding for the plasma membrane Ca 2ϩ pump. PMCA1 and PMCA4 are widely expressed, whereas PMCA2 and PMCA3 are more specialized forms which are expressed primarily in brain, skeletal muscle, and heart. Alternative splices at two different sites raise the number of possible pump isoforms to more than 20. One of the alternative splices occurs at the C hot spot near the middle of the calmodulin-binding domain; this splice changes the carboxyl-terminal third of the calmodulin-binding domain and the rest of the regulatory region (12). We have shown that this alternate splice in hPMCA4 changed the structure of the calmodulinbinding domain and the autoinhibitory region, and as a result, hPMCA4a had a higher basal activity and a much lower calmodulin affinity than hPMCA4b (13,14).Studies on the erythrocyte Ca 2ϩ pump (which is mainly hPMCA4b) indicated that phos...

We have determined the DNA sequence of the gene encoding the protein of the plasma membrane Ca(2+)-ATPase in Paramecium tetraurelia. The predicted amino acid sequence of the plasma membrane Ca(2+)-ATPase shows homology to conserved regions of known plasma membrane Ca(2+)-ATPases and contains the known binding sites for ATP (FITC), acylphosphate formation, and calmodulin, as well as the "hinge" region: all characteristics common to plasma membrane Ca(2+)-ATPases. The deduced molecular weight for this sequence is 131 kDa. The elucidation of this gene will assist in the studies of the mechanisms by which this excitable cell removes calcium entering through voltage gated calcium channels and the pump functions in chemosensory signal transduction.