I. INTRODUCTION

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Plasma membrane calcium pumps are now well recognized as a primary system for the specific expulsion of Ca²⁺ from eukaryotic cells. Together with Ca²⁺-specific ion channels and exchangers, these pumps are largely responsible for the regulated transport of Ca²⁺ between the intracellular and the extracellular milieu. Since their original identification in mammalian erythrocyte membranes in the mid 1960s (141), the calcium pumps have gained increasing attention as a ubiquitous mechanism for high-affinity calcium extrusion across the cell membrane. Due to the emergence of sophisticated protein biochemical and molecular techniques, tremendous progress has been made over the last two decades in elucidating their enzymatic properties, biochemical regulation, gross functional domain structure, and primary amino acid sequences. Accordingly, several reviews have been published on these transporters during the last few years, including contributions by Carafoli (24, 25), Carafoli and Stauffer (27), Monteith and Roufogalis (119), Lehotsky (111), Guerini (71), and Penniston and Enyedi (129). Several overviews comparing the calcium pumps of the plasma and the organellar membranes have also been published (128, 158), including one on the different calcium pumps in plants (54). In addition, the structural organization and mechanistic properties of P-type primary ion pumps (which include the plasma membrane calcium pumps) have received in-depth treatment in two excellent recent reviews by Andersen and Vilsen (5) and Møller et al. (118). Lastly, the molecular, functional, and physiological properties of Na⁺/Ca²⁺ exchangers, the major alternative to the calcium pumps for Ca²⁺ removal from a cell, have recently been reviewed in a comprehensive manner by Blaustein and Lederer in this journal (11).

The biochemical characteristics and overall domain structure of the plasma membrane calcium pumps have been comprehensively discussed in a review by Carafoli (24) published almost 10 years ago in this journal. Since then, rapid progress has been made in the identification of multiple isoforms of the pump and in the elucidation of their unique regulatory and functional properties. Of particular interest were findings showing that much of the isoform diversity among plasma membrane calcium pumps is due to complex alternative RNA splicing. This in turn has raised questions concerning the regulation of splicing, the cell and tissue specificity of isoform expression, and the functional significance of calcium pump isoforms differing only in a small and defined portion of the molecule. This review does not recapitulate the earlier findings on the characteristics of the calcium pump discussed extensively by Carafoli in 1991 (24) but, rather, continues where the previous review left off. The focus is on the unique aspect of the isoform complexity among plasma membrane calcium pumps, in particular as it relates to structural and functional differences among isoforms generated via alternative mRNA splicing. We begin with a brief overview of the general structural and regulatory properties of the plasma membrane calcium pumps and a section on the nomenclature of the different isoforms. We then compile the information concerning alternative splicing of the mammalian plasma membrane calcium pumps in a manner that will provide a comprehensive catalog of the splice variants, where and when they are expressed, and what consequences the alternate splices may have on the structural and functional properties of the encoded isoforms. This is followed by a discussion concerning the physiological significance of alternative splicing as it occurs in the plasma membrane calcium pump gene family. We conclude the review with a brief outlook into promising future developments, emphasizing the importance of transgenic animal models to study the physiological consequences of the selective ablation ("knockout") of specific plasma membrane calcium pump isoforms, as well as of investigations into naturally occurring diseases linked to mutations in specific calcium pump genes.

	II. OVERVIEW OF STRUCTURAL AND REGULATORY CHARACTERISTICS OF PLASMA MEMBRANE CALCIUM PUMPS

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The plasma membrane calcium pumps, also known as plasma membrane Ca²⁺-ATPases (PMCAs), belong to the P₂ (subtype 2B) subfamily of P-type primary ion transport ATPases (8, 115, 118), which are characterized by the formation of an aspartyl phosphate intermediate as part of their reaction cycle. The PMCAs appear to be ubiquitous in eukaryotic cells where they are thought to be the major high-affinity transporter for Ca²⁺ in the plasma membrane. A "generic" schematic model of a PMCA is shown in Figure 1. The PMCAs are predicted to contain 10 membrane-spanning segments, and the NH₂ and COOH termini are both located on the cytosolic side of the membrane. As shown in Figure 1B, the bulk of the protein mass is facing the cytosol and consists of three major parts: the intracellular loop between transmembrane segments 2 and 3, the large unit between membrane-spanning domains 4 and 5, and the extended "tail" following the last transmembrane domain (25, 71, 129). The first intracellular loop region between membrane-spanning domains 2 and 3 corresponds to the "transduction domain" thought to play an important role in the long-range transmission of conformational changes occurring during the transport cycle. The large cytosolic region of ~400 residues between membrane-spanning segments 4 and 5 contains the major catalytic domain including the ATP binding site and the invariate aspartate residue that forms the acyl phosphate intermediate during ATP hydrolysis. Finally, the extended COOH-terminal tail corresponds to the major regulatory domain of the PMCAs (25, 24, 129, 157). On the basis of computer modeling and sequence comparisons, the overall structure of the PMCAs closely resembles that of other P₂-type ATPases, notably that of the Ca²⁺ ATPases of the sarco/endoplasmic reticulum (SERCAs) (5, 118, 189). Indeed, the major "global" difference between the two types of calcium pumps is confined to the COOH-terminal tail, which is generally much smaller in the SERCAs (ranging from <20 to ~50 residues) than in the PMCAs (70 up to 200 residues). On the other hand, the global structural arrangement of the membrane-spanning domains and of the bulky intracellular loop regions appears to be surprisingly similar in different P₂-type ion pumps (140, 154), although differences in the size, structure, and relative spatial orientation of subdomains are obviously present and likely related to the specific cation(s) transported and the regulatory mechanisms operating on each transporter.

View larger version (31K): [in this window] [in a new window]   Fig. 1. Models of the plasma membrane Ca2+-ATPase (PMCA). A: linear representation of a generic PMCA showing the major domain substructure. The 10 putative transmembrane regions (TM) are numbered and indicated by shaded boxes. The NH2-terminal phospholipid-sensitive region (PL) preceding transmembrane domain 3 and the calmodulin binding domain (CaM-BD) are shown as black boxes. P is the site of the obligatory aspartyl-phosphate formed during pump function. The regions of highest sequence divergence among PMCA isoforms and splice variants are denoted by black bars above the model. The sites where alternative RNA splicing affects the protein are indicated below the model and are labeled A, B, and C. Site B may represent a splicing artifact and is shown in parentheses. B: two-dimensional model of the PMCA in its autoinhibited form (left) and upon stimulation by Ca2+-calmodulin (right). The domains are labeled as in A, and the location where the protein is affected by splicing at sites A and C is emphasized. At site A, the short peptide segment encoded by the alternatively spliced exon(s) is indicated by a hatched box. The PDZ domain binding/putative COOH-terminal targeting domain is shown as a shaded box at the extreme COOH terminus (C). N is the NH2 terminus. Note that the bulk of the protein mass is located on the cytosolic face of the membrane. Also note that the first cytosolic loop and the major catalytic loop are drawn differently in the inhibited (left) and stimulated (right) state to indicate the conformational changes that are likely to accompany the binding of Ca2+-calmodulin (Ca2+-CaM; hatched oval) to the autoinhibitory calmodulin binding site.

A particularly distinctive feature of the PMCAs is the number of different regulatory mechanisms that alter their functionality (reviewed in Refs. 24, 119, 128, 129). Of interest to the topic of this review is how alternative splicing impacts the regulation of the PMCAs, primarily their activation by Ca²⁺-calmodulin, acidic phospholipids, serine/threonine phosphorylation, and the possibility that they are regulated by heterotrimeric G proteins. Ca²⁺-calmodulin binds to a region in the COOH-terminal portion of the PMCAs located ~40 residues downstream of the last transmembrane domain (90). In the absence of Ca²⁺-calmodulin, this sequence acts as an "autoinhibitory" domain; cross-linking studies using labeled peptides demonstrated that the calmodulin binding domain interacts intramolecularly with two separate regions of the pump, one located in the first cytosolic loop and the other in the major catalytic unit between the phosphorylation and the ATP binding site (53, 55, 56) (see Fig. 1B). In the absence of Ca²⁺-calmodulin, the autoinhibitory COOH-terminal domain is thought to prevent catalytic turnover, keeping the pump in an inactive state (Fig. 1B, left). An elevation in the cytoplasmic Ca²⁺ results in an increase in Ca²⁺-calmodulin, which then binds with high affinity to the autoinhibitory domain of the PMCA, thereby releasing the inhibition and stimulating pump activity to near-maximal potential (Fig. 1B, right). This regulatory mechanism is similar to that of other Ca²⁺-calmodulin-dependent enzymes such as smooth muscle myosin light-chain kinase or Ca²⁺/calmodulin-dependent protein kinase I (69, 97). In the latter case, a recent study of the crystal structure of the enzyme in its autoinhibited state showed convincingly that the COOH-terminal regulatory domain (which overlaps with the calmodulin binding domain) interacts with multiple sites in the catalytic core, preventing substrate access and obstructing ATP binding in the absence of Ca²⁺-calmodulin (69). Interestingly, alternative splicing affects the affinity of the PMCAs for Ca²⁺ and calmodulin as well as the maximum velocity (V_max) of the activated enzyme (47, 52, 129, 142). Because the Ca²⁺-calmodulin affinity of some isoforms of the PMCA is in the low nanomolar range (30, 47, 80), at least two to three orders of magnitude below calmodulin concentrations in tissues like the brain (23), it has been predicted that calmodulin may act as a pseudosubunit for some of the PMCAs (171).

In addition to mediating regulation by Ca²⁺-calmodulin, the COOH-terminal region of the calcium pumps has also been shown to be the target of phosphorylation by protein kinases A and C (reviewed in Refs. 25, 119, 129, 173, 181), to be affected by proteases such as calpain (25, 173), to contain structural as well as potentially regulatory Ca²⁺ binding sites (84), and to mediate interactions with PDZ domains (100). The PMCAs are also activated by self-association (104), and although they are still able to bind Ca²⁺-calmodulin in the oligomerized state (105), there is no further activation by calmodulin. These and additional studies using synthetic peptides and COOH-terminally truncated PMCAs have shown that the dimerization (oligomerization) of the PMCAs is mediated via the calmodulin binding domain itself (104, 105, 172). Finally, the calmodulin binding domain of the PMCAs also binds to phospholipids and may thereby participate in the known stimulation of the pumps by acidic phospholipids (20, 59). Taken together, the above data strongly suggest that alternative splicing affecting the COOH-terminal sequence of PMCA isoforms could have profound effects on the regulatory and protein interaction properties of the different splice variants. These are discussed in more detail in the appropriate sections below.

Acidic phospholipids, polyphosphoinositides in particular, are the most potent stimulators of the PMCA (117, 124, 125). They reduce the Michaelis constant for Ca²⁺ [K_m(Ca²⁺)] of the enzyme to ~0.3 µM compared with 0.4-0.7 µM in the case of calmodulin stimulation (117, reviewed in Refs. 111, 128, 173). Activation by acidic phospholipids renders the PMCA insensitive to calmodulin activation, i.e., Ca²⁺-calmodulin and phospholipids are not additive although acidic phospholipids can further reduce the K_m(Ca²⁺) of the Ca²⁺-calmodulin-stimulated pump (50, 173). Data showing that the fully Ca²⁺-calmodulin-stimulated pump (or a truncated pump lacking the COOH-terminal autoinhibitory, calmodulin binding domain) could be further stimulated by phospholipids indicated that a region(s) other than the COOH-terminal domain must also be involved in the lipid stimulation (50). Work with different proteolytic fragments of the PMCA and with synthetic peptides revealed that there are indeed two separate phospholipid binding regions in the pump: one corresponding to the previously mentioned COOH-terminal calmodulin binding domain and the other situated in the first cytosolic loop immediately preceding the third membrane-spanning domain (20, 59, 190) (Fig. 1). The mechanism by which different phospholipids activate the PMCAs is not known. However, given the location of the lipid-binding sequences in the pump, one may speculate that the interaction of acidic phospholipids (presumably via their charged headgroups) with the calmodulin binding domain leads to some structural rearrangement that weakens the autoinhibitory intramolecular interactions formed by the COOH-terminal tail (Fig. 1B). How phospholipid interactions with the first cytosolic loop further stimulate PMCA activity in a fully Ca²⁺-calmodulin-activated pump (corresponding to the model in Fig. 1B, right) is less obvious. Structural rearrangements of the transduction and catalytic domains could be invoked that may facilitate access of Ca²⁺ to its high-affinity transport sites and/or positively influence a rate-limiting step in the catalytic cycle. Regardless, it is intriguing that alternative splicing at the A site (Fig. 1) affects the first cytosolic loop in a region located between the phospholipid binding sequence and the sequence involved in autoinhibitory interactions (Fig. 1B). Alternative splicing may thus affect the phospholipid regulation of PMCA isoforms by altering the accessibility of the phospholipid binding domain to the acidic phospholipids. Similarly, alternative splice variants may show different levels of autoinhibition due to structural differences in their first cytosolic loop caused by the variable sequence insertions. In addition to protein-lipid and intramolecular protein interactions, the same alternate splice could also potentially affect interactions with external proteins. For example, the possibility that members of the heterotrimeric G protein family directly regulate the PMCA has been raised (94, 119). The amino acid sequence immediately preceding alternative splice site A displays sequence and structural similarity with G protein / binding regions in other effector proteins (33, 109), rendering this region of the PMCA well suited to participate in G protein interactions (see sect. VIIIA).

	III. NOMENCLATURE OF PLASMA MEMBRANE CALCIUM PUMP ISOFORMS

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In order that the reader understands the nomenclature used throughout this review, some history must be reviewed and some rules must be established. Shortly after the publication of the first full-length PMCA cDNAs (144, 169), others were cloned and a consensus on isoform naming was reached by comparing the sequences and by naming the isoforms numerically (1, 2, 3...) in the order of their discovery. A lowercase letter is added as a prefix to designate the species (e.g., h for human, r for rat, m for mouse, etc.). Currently, the cDNA sequences of four different PMCA isoforms are known in humans (hPMCA1, hPMCA2, hPMCA3, hPMCA4) and rats (rPMCA1, rPMCA2, rPMCA3, rPMCA4). The naming of the alternative splice variants has been a bit more controversial. A scheme for splice site designation was introduced shortly after the first description of alternative splicing in hPMCA1 (70, 162). In this scheme, the alternate splice variants are designated by lowercase letters following the isoform number (e.g., hPMCA1a, hPMCA1b, etc.) where the letters at the beginning of the alphabet (a, b, c...) are used to designate the historically earlier identified splices at the COOH terminus of the pumps, and those at the end of the alphabet (w, x, y, z) indicate the splices at the NH₂-terminal region (95, 128, 153, 156). This scheme of nomenclature has been used and extended in many subsequent publications concerning alternative splice variants, and although it is not without its problems, it is still widely used to maintain consistency with the earlier literature. An alternative system for the nomenclature of PMCA splice forms was later proposed by Carafoli (25). In this system, the lowercase letter designating the splice variant is replaced by a capital letter indicating the splice site (A, B, and C going from the NH₂-terminal to the COOH-terminal splices; see Fig. 1) followed by a Roman numeral (I, II, III...) denoting which exon(s) is inserted. An advantage of the newer system is that it explicitly specifies the splice site concerned. On the other hand, the addition of Roman numerals to indicate whether or not a specific exon is included at a given site and to designate variants with different combinations of multiple exons at the same site is still not intuitive. A comparison of the two systems as applied to the currently known PMCA splice variants is shown in Table 1. Given that the alternative system is not free of drawbacks, and to maintain consistency with the large number of earlier publications on the PMCA isoforms, we use the old system of nomenclature throughout this review.

	IV. FOUR GENES AND ALTERNATIVE RIBONUCLEIC ACID SPLICING GENERATE A MULTITUDE OF PLASMA MEMBRANE CALCIUM PUMP ISOFORMS

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The mammalian PMCAs are encoded by a gene family composed of at least four nonallelic members. Complete cDNA-derived coding sequences have been determined for all four PMCA isoforms in rats and humans, the two organisms in which the majority of the work on the genes and their products has been performed. The chromosomal loci for the human genes have been determined; they are 12q21-q23 for PMCA1, 3p25-p26 for PMCA2, Xq28 for PMCA3, and 1q25-q32 for PMCA4 (15, 110, 126, 175). Because of the large size of the genes, information on the gene structures of the different PMCA isoforms is still scarce. In most cases, only fragments of the genes have been characterized; however, these generally correspond to the areas of interest with respect to the alternative splicing options. Complete genomic structures have so far been reported for the rat PMCA3 (22) and the human PMCA1 gene (81), whereas partial information is available on the rat PMCA1, -2, and -4 genes (95, 96); the human PMCA2, -3, and -4 genes (17, 21, 77, 108); and the mouse PMCA genes (43, 106; Zacharias and Strehler, unpublished results). With the rapid progress of the Human Genome Project, complete sequence and structural information for all human genes should become available within a few years, and the corresponding information from other species will likely follow in rapid succession from many ongoing and future whole genome sequencing efforts. The mammalian PMCA genes appear to be very closely related in their exon-intron structure, as demonstrated by the almost perfect conservation of intron locations in all currently known human, rat, and mouse PMCA genes and gene fragments. The currently known genes are large, ranging from ~70 kb for the rat PMCA3 gene (22) to well over 100 kb for the human PMCA1 and -2 genes (81, 108). The rat PMCA3 and the human PMCA1 genes contain 24 and at least 22 exons, respectively, and several of these exons specify only 5'-untranslated sequences or are subject to alternative splicing (22, 81).

Each of the genes' primary transcripts is subject to alternative splicing. The occurrence of alternative splicing in PMCA transcripts was first suggested by Shull and Greeb (144) and first demonstrated for hPMCA1 by Strehler et al. (162). The sites where alternative RNA splicing may occur in the protein-coding sequence of the PMCA transcripts are indicated in Figure 1 and are termed A, B, and C (157, 160). Because of the combinatorial potential of the various splice options at each site, a large number of splice variants are theoretically possible (see also Table 1), of which over 20 have been detected at the RNA (cDNA) level. In the following section, we systematically survey the different splice options that have so far been observed for each PMCA isoform at each of the three splice "hot spots" A, B, and C.

	V. ALTERNATIVE SPLICING OPTIONS OF MAMMALIAN PLASMA MEMBRANE CALCIUM PUMPS

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The alternative splice options at site A (Fig. 2) have been extensively characterized in all four human and rat PMCA isoforms (22, 96, 153, 183). The splicing affects a small exon of 36-42 nucleotides (nt) present in all PMCA genes and coding for a short segment of the first intracellular loop of the pump molecule. This exon can be optionally inserted or excluded in the mature transcripts from all PMCA genes, with the apparent exception of PMCA1. Despite intense efforts to detect alternative usage of the corresponding 39-nt exon in PMCA1, this exon has always been found to be included in every PMCA1 transcript. Thus all PMCA1 variants are of the x splice type (see Table 1 and Fig. 2).

View larger version (27K): [in this window] [in a new window]   Fig. 2. Observed alternative splicing options at site A of the PMCAs. The exon structure of the region affected by alternative splicing at site A is shown for each of the 4 PMCA genes. The constitutively spliced exons flanking site A are represented by hatched boxes; the alternatively spliced exons are displayed as open boxes with their size indicated in nucleotides (nt). Introns are shown by solid lines connecting the exons; exon and intron sizes are not to scale. The different splice options are indicated for each PMCA, and the resulting splice variants are labeled by their lowercase symbol. Note that the 39-nt exon in PMCA1 transcripts corresponds to alternatively spliced exons of 42, 42, and 36 nt, respectively, in transcripts of the genes for PMCAs 2, 3, and 4. This exon has never been found to be excluded from any PMCA1 mRNA; hence, all PMCA1 variants are of the "x" splice type. y*, splicing of this type has so far only been observed in the rat.

The situation for PMCA2 at splice site A is more complex. There are three exons of 33, 60, and 42 nt (see Fig. 2) whose alternative usage theoretically gives rise to eight splice variants at this site (77). This is possible because all exons contain integral multiples of three nucleotides, and any combination of their insertion or exclusion will maintain an open protein reading frame. However, only four of the eight possible combinations have so far been detected in mRNAs from a variety of tissues. These are the variants w (with all 3 exons included), z (all 3 exons excluded), x (only the 42-nt exon included), and y (inclusion of the 33- and 60-nt exons). In humans, only variants w, x, and z have been detected, whereas in rat, variant y also has been found (2). Compared with the z and x variants, PMCA2 splice variants of the "w" type contain an additional 45 and 31 amino acid residues, respectively, in their first cytosolic loop (see Fig. 1).

In PMCAs 3 and 4, there is a single exon (exon 8 in the rat PMCA3 gene) that may be included or excluded in the mature mRNA (yielding the x and z splice variants, respectively; see Fig. 2). In both human and rat, the size of this exon is 42 bp in the PMCA3 and 36 bp in the PMCA4 gene.

The occurrence in vivo of alternative splicing at site B has been a point of some controversy. The isolation of a hPMCA4 cDNA that lacked a 108-nt segment in the COOH-terminal coding region led Strehler et al. (156, 159) to postulate that alternative splicing may occur at this site. This was supported by the finding that the 108-nt sequence is encoded in a single exon in the hPMCA1 and rPMCA3 genes (22, 81). Furthermore, as in the case of the alternatively spliced exons at site A, the 108-nt exon encodes an integral multiple of codons, and its exclusion does not alter the reading frame of the remainder of the protein. Independent cDNA clones and PCR products corresponding to PMCA1 and PMCA4 transcripts alternatively spliced at this site were subsequently isolated. However, they appear to represent only an extremely minor proportion of the total message and have only been detected in the intestine and liver (85, 87).

Several studies by other laboratories have found no indication that the 108-nt exon at splice site B is ever removed in any of the tissues analyzed (95, 153). The failure by most researchers to positively identify this splice variant, and/or its very low abundance in those cases where it has been detected, support the view that mRNAs lacking the 108-nt exon are possibly the products of aberrant splicing (87, 95, 153).

If translated into protein, the physiological significance of such splice site B variants would also be questionable. Exclusion of the 108-nt exon sequence leads to a pump protein lacking the tenth putative transmembrane domain (see Fig. 1A). Numerous studies have shown that the long COOH-terminal tail of the PMCA is cytosolic (reviewed in Ref. 25). Given the proposed 10-transmembrane domain model, splicing at site B would alter the topology of the PMCA such that the COOH terminus would protrude into the extracellular space. It should be noted, however, that the precise number of membrane-spanning domains of the PMCAs has not yet been experimentally determined. In particular, the number and arrangement of membrane-spanning domains in the COOH-terminal half of the molecule are still a matter of debate, although the recent determination of the closely related SERCA Ca²⁺ pump (189) and Neurospora plasma membrane H⁺ pump structures (7) at a resolution of 8 Å leaves little doubt that the PMCAs will also contain 10 transmembrane domains. Interestingly, when a recombinant hPMCA4 splice variant lacking the exon at site B was expressed in insect Sf9 cells, it showed no Ca²⁺-dependent ATPase activity but was still able to form the phosphoenzyme intermediate from inorganic phosphate. When it was expressed in COS cells, it was retained in the endoplasmic reticulum with the COOH terminus apparently in the cytosol, indicating that an even number of transmembrane domains had been eliminated (142). Considering the activity data reported by Seiz-Preiano et al. (142), however, the physiological significance of such splice site B variants remains doubtful regardless of their COOH-terminal transmembrane topology.

Alternative splicing at site C occurs in all isoforms, albeit in varying degrees of complexity. Generally, however, similar structural options have been found in all PMCA isoforms at this site (Fig. 3). The variations, as they occur in rat, have all been examined in a single comprehensive work by Keeton et al. (95), whereas those for the human PMCAs were most thoroughly analyzed by Stauffer et al. (153).

View larger version (28K): [in this window] [in a new window]   Fig. 3. Alternative splicing options at site C of the PMCAs. The exon structure of the region affected by alternative splicing at site C is shown for each of the 4 PMCA genes. The constitutively spliced exons are represented by hatched boxes, and the alternatively spliced exons are displayed as open boxes. The sizes of the alternatively spliced exons are indicated in nucleotides (nt) and are those for the human genes. Introns are shown by solid lines connecting the exons; exon and intron sizes are not to scale. The different splice options are indicated for each PMCA gene, and the resulting splice variants are labeled by their lowercase symbol. Note that the "b" splice variants exclude the alternatively spliced exon(s), whereas the "a" variants include an entire large exon of 154, 172, 154, and 175 nt, respectively, in PMCAs 1, 2, 3, and 4. In PMCA2, splice variant a was originally proposed to arise from insertion of both the 172- and 55-nt exons; however, based on exon utilization and amino acid comparisons, this variant should be renamed "c," and the former c should henceforth be called variant a. This change is indicated by asterisks in the splicing diagram for PMCA2. In PMCA3, splice variant "e" results from a read-through of the 154-nt exon into the following intron (indicated as thin open box). The position of the translation stop codon for each splice form is indicated by a corresponding capital letter.

For PMCA1, five different variants (a, b, c, d, e) have been shown to arise by inclusion or exclusion of a single 154-nt exon, and/or by utilization of multiple internal donor sites within that exon (95, 153, 162).

In PMCA2, an exon of 172 nt corresponds to the 154-nt alternatively spliced exon found in PMCA1. However, the most frequent insertion at this site in PMCA2 is a sequence of 227 nt consisting of the 172-nt exon and an additional 55-nt exon (95, 153). In the rat PMCA2 gene, these two exons are separated by an intronic sequence of ~3.5 kb (95). There have been several reports of the insertion of the 172-nt exon independently of the smaller 55-nt exon, resulting in a variant designated "2c" (74, 95). In contrast, the splice variant produced from mRNA containing both exons is known as "2a." However, based on COOH-terminal amino acid sequence similarities, we propose that the 2c variant should be more appropriately named 2a (see Figs. 3 and 6). This would also be consistent with the mechanism for the generation of the 1a, 3a, and 4a splice variants that all arise from the insertion of a single large exon (see also below).

In PMCA3, which appears to be expressed at high levels only in skeletal muscle and brain, two exons are subject to alternative splicing at site C (Fig. 3). These are a 68-nt exon (exon 22 in the rat gene) followed by a 154-nt exon (exon 23 in the rat gene) that contains multiple internal donor sites (22) and is analogous to the 154- and 172-nt exons found at splice site C in PMCA1 and -2, respectively, and the 175/178-nt exon in isoform 4 (see below). As in all other PMCA isoforms, the splice variant lacking any of the alternatively inserted exons is the "b" variant (Fig. 3). The splice variant including the 154-nt exon is named 3a. Variants 3c and 3d utilize internal splice donor sites at positions 87 and 114, respectively, within the 154-nt exon. Splice variant 3e is generated when a "read through" of the 154-nt exon occurs, adding 88 nt of the following intron and a poly(A) tail (22). This read through leads to a short alternate COOH-terminal sequence in this variant compared with variant 3a because the open reading frame terminates just three codons into the intron. Finally, the inclusion of the 68-nt exon located 5' to the 154-nt exon results in variant 3f. Insertion of this exon introduces an in-frame translation-termination codon after 45 nt, thereby generating a PMCA3 variant with the shortest COOH-terminal tail of all known PMCAs (22, 95). Insertion of the 68-nt exon generates variant 3f irrespective of the splice options utilized further downstream (e.g., whether or not the following 154-nt exon is inserted in the mRNA).

In PMCA4, alternative splicing at site C is relatively simple. In humans, cows, and rats, there is a single alternatively spliced exon of 178, 181, or 175 nt, respectively, first described by Brandt et al. (17) and Keeton et al. (95). As in the case of variants 1a, 2a, and 3a, the inclusion of this exon (generating variant 4a) causes a truncation of the open reading frame that results in a protein with a shorter regulatory COOH-terminal domain that differs significantly from that of the b variant.

	VI. TISSUE DISTRIBUTION OF PLASMA MEMBRANE CALCIUM PUMP ISOFORMS AND SPLICE VARIANTS

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Many studies have documented the tissue- and developmental-specific pattern of expression of several PMCAs. These studies have also shown that the brain expresses by far the greatest abundance and diversity of isoforms and splice variants (17, 95, 149, 153, 183; reviewed in Refs. 27, 111). PMCA isoforms 1 and 4 are, in general, expressed throughout most tissues, whereas PMCA isoforms 2 and 3 are expressed in a much more restricted manner and, in the adult, are found predominantly in the brain and striated muscle. Within the brain, PMCA2 is primarily expressed within specialized cell types such as cerebellar Purkinje cells (152) and cochlear hair cells (64, 155). However, significant amounts of PMCA2 are also present in tissues such as uterus, liver, and kidney, and very high local levels of PMCA2 are apparently found in lactating mammary glands (132). Expression of PMCA3 appears to be even more restricted, and in the brain is particularly high in the choroid plexus (149, 152). In rats, high levels of PMCA3 expression are also found in skeletal muscle (70), whereas in humans, PMCA3 expression in skeletal muscle appears to be transient and is essentially undetectable in neonatal and adult skeletal muscle (153). Recently, isoform-specific, mono- and polyclonal antibodies have been produced against peptide epitopes corresponding to small, nonconserved portions of the NH₂ termini of each PMCA isoform. Using these as well as other independently generated antibodies to detect PMCA expression at the protein level, several studies have shown that there is generally a good correlation between the proteins and the previously demonstrated mRNAs in terms of their localization and relative abundance (31, 44, 58, 151, 152).

Very few studies have appeared in the literature on the developmental pattern of expression of the different PMCA isoforms. Such information is crucial, however, if we are to understand the potential involvement of the different PMCAs in specific processes of organ and tissue development. It will also be essential for proper interpretation of the phenotypic consequences of PMCA knockout mice that are certain to be generated in the very near future or, as for PMCA2, have already been reported (106).

The most comprehensive analysis of the expression of the four PMCA genes during development was recently published in a study using in situ hybridization on tissue sections of mouse embryos between days 9.5 and 18.5 in utero (186) (see Fig. 4). In this study, PMCA1 expression was detected throughout the embryo from the earliest time point analyzed, and although there appear to be some fluctuations in the level of expression in different tissues during development, the data confirm that PMCA1 is ubiquitously expressed and may be considered a "house-keeping" isoform of the pump. All other PMCA isoforms were first detectable by in situ hybridization around day 12.5, and all showed pronounced changes in the level and/or tissue distribution during further development (186). PMCA2 expression was essentially confined to the developing nervous system and reached high levels in the dorsal root ganglia, the retinoblasts of the developing eye, and the central nervous system, where the external granular layer of the developing cerebellum showed the most intense labeling. Interestingly, PMCA3, which shows a very restricted tissue distribution in the adult, was widely expressed at the transcript level early in development (between 12.5 and 15.5 days). Only at later time points (starting at around 16.5 days) did the expression of this isoform become more restricted and was high in the nervous system, the developing limb skeletal muscles, and the lung. This finding is of interest as it suggests that "knocking out" the PMCA3 gene may not merely affect specific areas of the brain (such as the choroid plexus and habenula) but could have severe, potentially lethal consequences during early embryonic development due to the possible importance of this pump isoform in the development of vital organs such as the lung. Finally, PMCA4 was generally found to be expressed at much lower levels than the other isoforms throughout mouse development, which is in contrast to the situation in adult human tissues where PMCA4 is almost as abundant and ubiquitous as PMCA1. An exception appears to be the liver, where PMCA4 gene expression was relatively high during early time points (12.5-16.5 days) but then decreased at 18.5 days. Significant levels of PMCA4 expression were detected at the later time points in the brain, dorsal root ganglia, heart, and intestine (186).

View larger version (72K): [in this window] [in a new window]   Fig. 4. Temporal and spatial expression of the 4 PMCA genes during mouse development. Contact autoradiograms are shown of in situ hybridizations to sagittal/parasagittal sections of embryos isolated from 9.5 to 18.5 days postcoitum (dpc). Sections on the left were stained with Alcian blue (AB) and Nuclear Fast Red (NFR) to illustrate overall anatomy and morphology for each stage (scale bar, 0.5 cm). PMCA1 is expressed from at least 9.5 dpc on. Signal is distributed throughout the embryo but is greatest in the nervous system, heart, skeletal muscle, and intestine. PMCA2 is first detected at 12.5 dpc. Its expression is confined to the nervous system (central nervous system, dorsal root ganglia, and pituitary) throughout development. PMCA3 is first detected at 12.5 dpc. At the early stages, expression is greatest in the nervous system but is also evident transiently in skeletal muscle. Onset of PMCA4 expression is also at ~12.5 dpc, although expression levels seem to remain low throughout development. PMCA4 expression is highest in the liver but decreases by 18.5 dpc. [From Zacharias and Kappen (186) with permission from Elsevier Science.]

The developmental regulation of PMCA isoform and COOH-terminal splice variant expression has also been studied by PCR amplification of reverse-transcribed mRNA in the developing rat brain (16). As expected, PMCA1 is expressed at all time points from embryonic day 10 to postnatal day 30. PMCA1b is the predominant splice form early on but slowly decreases as expression of the 1a variant gradually increases until it reaches a steady-state level around embryonic day 18. Clearly, a splice shift from variant 1b to the "brain-specific" 1a variant occurs during maturation of the neuronal system. In the study by Brandt and Neve (16), PMCA2 mRNA expression in the brain was first detected around day 18, and both PMCA2a/c and 2b appeared to be about equally abundant at all time points analyzed. During postnatal development, significant changes in the relative expression of PMCA2a/c were apparent, however, in several subregions of the brain. For example, expression of PMCA2a/c appears to be upregulated in the developing cerebellum, whereas this splice variant decreases in septum, caudate, and hypothalamus during the same time period (postnatal days 2-30) (16). PMCA3 transcripts showed a similar pattern of expression in the developing rat brain as PMCA1: PMCA3b was detected from embryonic day 10, whereas expression of PMCA3a did not become apparent until embryonic day 18. As in the case of PMCA1, the emergence of the PMCA3a splice form thus appears to accompany neuronal maturation. In accordance with the in situ hybridization study on mouse embryos (186), and previous indications of relatively low expression of PMCA4 in rat brain (95, 96), PMCA4 mRNAs were not detected in the developing rat brain by the methods used in the study by Brandt and Neve (16).

Northern blots have been extensively used to determine the overall size, tissue distribution, and relative abundance of PMCA gene transcripts. The most detailed studies have been performed in the rat by Shull and co-workers (22, 70, 96). The major disadvantage of this method is its relative insensitivity, requiring large amounts of high-quality RNA as starting material. The problem is exacerbated in the case of the PMCAs because of their apparently low abundance in most tissues. Therefore, it has been generally necessary to analyze the poly(A) RNA fraction to detect the PMCA transcripts with the required sensitivity. This fact, combined with the often limited amount of a specific tissue source, severely limits the usefulness of the Northern technique to determine the expression pattern of the various PMCA isoforms and subtle changes in alternative splice forms. However, an advantage of this method is that it allows the determination of the full-length transcript size and that relative steady-state transcript levels of specific PMCAs can be directly compared. Northern blots have been used to show, for example, that several PMCA3 transcripts of widely divergent size (7.5 and 4.5 kb) are expressed in brain and skeletal muscle and that they are generated by alternative splicing (22, 70).

Once a site for alternative splicing has been characterized, RT-PCR is the easiest method to examine the expression of differently spliced variants. However, its major advantage, exquisite sensitivity, may also be one of its main problems. PCR artifacts such as the creation of heteroduplex molecules and spurious amplification of very minor transcripts (including aberrant splice products) may lead to erroneous conclusions concerning the presence of "novel" splice variants (95, 183, 185). This problem may have arisen in a number of studies on the expression of (rare) PMCA alternative splice variants in different cell types and tissues. For example, the sensitivity of the RT-PCR approach may be responsible for the detection in some tissues of alternative splice products of PMCAs 1 and 4 at splice site B (87) and may have led to an overestimate of the diversity of alternative splice options utilized in some cell types (74). The importance of a careful analysis of all RT-PCR products by nucleotide sequencing and the inadequacy of agarose gel electrophoresis and Southern blotting alone to determine alternative splice variants have been pointed out by Zacharias et al. (185) and White et al. (180). Information concerning the exact cellular distribution of the various splice variants is generally not obtainable by RT-PCR because all tissue structure and cellular identity is destroyed in the process of extracting the RNA. There have been some attempts at microdissection of various subregions from tissues such as the kidney, but the potential for obtaining a mixed population of cell types is still very high (28, 29, 116). This problem is exemplified in the human brain; a study in which 14 regions of the brain were carefully dissected and assayed for the expression of the various splice variants (183) was not able to define which of the different cell types expressed the different spliced variants, although it was still possible to detect gross differences among the brain regions in overall splice variant expression. Single-cell RT-PCR approaches are able to solve this problem, but they are technically demanding and not always feasible in highly complex tissues.

Although this method is labor intensive, technically demanding, and more expensive than most of the other methods, it also has several advantages. 1) RPA is usually sensitive enough to detect even very rare messages. 2) The data produced are not prone to the same artifacts that occur in RT-PCR analyses (see sect. VIC2). 3) It can be quantitative. If one uses known quantities of radiolabeled antisense probes for an mRNA of interest and an internal standard such as glyceraldehyde-3-phosphate dehydrogenase, each with a known specific activity, then the signal obtained by detection of the protected fragments by an instrument such as the PhosphorImager (Molecular Dynamics) theoretically provides a highly quantitative measure of a given mRNA species in a mixed population. 4) RPA can detect alternatively spliced mRNA species. RPA methods were first used to detect the alternative splice options at site C in human PMCA1 transcripts from skeletal muscle (162) and have also been successfully employed to determine changes in the expression of PMCA2 splice site A variants in human IMR32 neuroblastoma cells (187).

The major advantage of this method is that it allows the direct visualization of the transcripts of interest in specific cell types. However, for low-abundance transcripts, sensitivity issues and problems with background signals may arise. The method is also technically challenging and time consuming, and the interpretation of results is not always straightforward, particularly when complex tissues with multiple cell types are being studied. The detection of alternatively spliced products by in situ hybridization can also be difficult, if not impossible, if the alternatively spliced sequence is too short to generate sufficiently specific probes. To date, there are no in situ hybridization studies that have specifically examined the location of alternatively spliced variants of the PMCAs, only those in which probes common to most or all splice variants of an isoform were utilized. The most comprehensive studies examining the cellular distribution of the PMCA transcripts are those by Stahl and co-workers (44, 149, 150) in which the gene products for all four isoforms were localized in the rat brain, and the recent publication by Zacharias and Kappen (186) where the four PMCA gene transcripts were localized in the developing mouse embryo. These studies have shown, for example, that PMCA mRNAs are primarily expressed in neurons and at much lower levels in glial cells and astrocytes (however, see Ref. 61) and that dramatic differences exist in the distribution of the different isoform transcripts in various brain areas and cell layers. In situ hybridization has also revealed species differences in the distribution of some PMCA transcripts. PMCA4 was found to be highly expressed in granule cells of the dentate gyrus and in the CA2 region of the human hippocampus (184), whereas studies in the rat brain showed little, if any, PMCA4 in the hippocampus. In the rat, PMCA4 transcripts were detected at high levels in the piriform cortex, the amygdaloid nucleus, and several cortical layers (150). In situ hybridization has also been used to detect temporal changes in PMCA transcript expression in the rat hippocampus following kainic acid-induced seizure activity (65). Lastly, in situ hybridization has recently been used in an elegant study to detect the cellular distribution of the four PMCA isoform transcripts in the developing and adult rat cochlea (64) as well as to study their appearance and tissue localization during mouse embryonic development (186) (see sect. VIB).

Taken together, the data from the above approaches provide a fairly detailed picture of the identity, expression level, and tissue (in some cases even cell type) distribution of the various PMCA transcripts and their splice variants. The studies at the mRNA level are being complemented at the protein level by a growing number of studies using PMCA isoform- and splice variant-specific antibodies. Tables 2-6 summarize the data on the currently known PMCA splice variants with respect to their tissue distribution and abundance. Because the vast majority of these studies were performed in the rat and human systems, our current knowledge of PMCA isoforms and splice variants in other mammals (and definitely in nonmammalian species) is more limited. Where possible, however, data from other mammalian species have been included in Tables 2-6.

	VII. REGULATION OF ALTERNATIVE SPLICING IN THE PLASMA MEMBRANE CALCIUM PUMP FAMILY

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There are numerous examples of specific expression of the PMCA splice variants in different cells or tissues and at different time points during development. In addition, there are an increasing number of reports showing the plasticity of PMCA splice variant expression, i.e., the shift from the expression of a particular variant to the expression of additional or different splice forms. Although the mechanism(s) affecting such splice shifts are virtually unknown, it is becoming evident that rapid signaling events, for example, in response to growth factors, can induce these shifts. Throughout a body, whether rat or human, both the PMCA isoforms as well as their individual splice variants have a relatively unique distribution. Isoforms 1 and 4 are expressed in virtually all tissues, but not all the known splice variants of these isoforms are expressed in all tissues. PMCA isoforms 2 and 3 are expressed almost exclusively in excitable tissues, although certain cell types in other tissues such as kidney, liver, or mammary gland may also express significant amounts of either or both of these isoforms (see Tables 3 and 4). It should be noted, however, that most of these studies rely on data obtained by RT-PCR and that isoform-specific antibodies have so far failed to detect either isoform outside of the brain by Western blotting (151).

The data accumulated over the last few years clearly demonstrate that the process of alternative splicing in the PMCA family is a regulated event. For example, some of the variants of PMCA1 appear to be specific to excitable tissue (1a, 1c, 1d, 1e) (17, 95, 153), and their expression is regulated during embryonic development and during differentiation in vivo and in vitro (16, 18, 37, 74). In the case of muscle, the alternative splicing events that occur as a result of myogenic differentiation may be mimicked by the application of the muscle differentiation factor myogenin (74). Although this does not establish a causative link between the splicing event and the differentiation agent, it is an interesting finding and certainly fits with in vitro data showing the induction of the 1c splice form upon myotube formation (18, 37). Nerve growth factor treatment of PC12 pheochromocytoma cells also leads to the appearance of the "differentiation-specific" splice variants of PMCAs 1, 2, and 4 (i.e., 1c, 2a, 4a) (74), suggesting a link between nerve growth factor signaling and PMCA splice variant expression.

Recent reports have shown that the mRNAs (and the corresponding proteins) of several PMCA isoforms and splice variants are regulated by changes in Ca²⁺ itself (26, 72, 107, 187). For example, rat cerebellar granule cells kept under depolarizing conditions for several days (leading to increased Ca²⁺ influx) showed a marked upregulation of PMCA1a as well as of PMCA2 and PMCA3 at the mRNA and protein level (72). In contrast, elevation of intracellular Ca²⁺ resulted in a rapid (within hours) and specific downregulation of the PMCA4a splice variant. This downregulation likely occurs at the transcriptional level and has very recently been shown to be mediated by the Ca²⁺/calmodulin-sensitive phosphatase calcineurin (73). In a study in the human neuroblastoma cell line IMR32, we showed that splicing at site A of hPMCA2 can be regulated by a second messenger signaling pathway elicited by a rise in intracellular Ca²⁺ (187). These data also illustrated that at least some changes in the expression of mRNA alternative splice variants of the PMCA occur rapidly (within minutes) and independently of new protein synthesis. In the IMR32 cells, differentiation is accompanied by a marked upregulation of PMCA isoforms 2 and 4 (and to a lesser extent, of PMCA1) which in turn leads to an improved Ca²⁺ extrusion efficiency of these cells (Y. M. Usachev, S. L. Toutenhoofd, E. E. Strehler, and S. A. Thayer, unpublished data). Very recent studies also indicate that PMCA1 expression can be altered (repressed) on a relatively rapid time scale by glucocorticoids in the rat hippocampus (10) or by c-myb-mediated transcriptional repression during the cell cycle in vascular smooth muscle cells (4). These findings are of interest not only because of their implications for PMCA expression but more generally as a potential mechanism of inducing rapid changes in protein (isoform) components in response to specific signaling events. Such signals are likely part of the molecular events leading to specific biological outcomes such as synaptic plasticity in neurons.

	VIII. PHYSIOLOGICAL SIGNIFICANCE OF ALTERNATIVE SPLICING IN THE PLASMA MEMBRANE CALCIUM PUMP FAMILY

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For a number of reasons, determining the physiological role of each PMCA isoform and splice variant in the context of an intact cell has proven to be extremely difficult. One significant reason is that there are no specific pharmacological inhibitors for any of the PMCA isoforms or splice variants. This barrier has arguably been the greatest impediment to a complete understanding of the function of these pumps in a living cell or tissue. The PMCAs are believed to have turnover rates on the order of only 10-50 Ca²⁺ translocated/s (138). This rate is considerably below that which is necessary to make successful measurements of pump activity by standard methodologies like patch clamping, which have proven so useful in the ion channel field. Another confounding factor is that in most cell lines suitable for calcium imaging, more than one PMCA isoform is expressed (most cells express at least PMCA1 and PMCA4), and multiple splice variants may be present simultaneously. Therefore, assignment of calcium extrusion characteristics to a particular isoform or splice variant is not readily possible in vivo. Because of these technical limitations, our knowledge of the specific physiological role of each PMCA isoform, and of each of the multiple alternative splice variants, in a specific cell type and under dynamic Ca²⁺ loads is still in its infancy.

The best strategies currently available for determining the in vivo participation of the PMCAs in calcium regulation attempt to isolate their contribution from that of other calcium regulatory processes by selectively inhibiting major alternative Ca²⁺ transporting pathways. Generally, this involves blocking the SERCA pumps of the endoplasmic reticulum with thapsigargin, "poisoning" mitochondrial calcium transport by reagents such as the protonophore carbonyl cyanide m-chlorophenylhydrazone, and blocking Na⁺/Ca²⁺ exchange by replacing extracellular Na⁺ with N-methyl-D-glucamine or tetraethylammonium ions (9, 79, 177, 178). Combined with genetic manipulations, i.e., selective "knock down" of specific PMCA isoforms via antisense strategies or overexpression of specific PMCA isoforms and splice variants from recombinant expression vectors, the contributions of PMCA isoforms in shaping intracellular Ca²⁺ signals and maintaining resting Ca²⁺ levels are beginning to be unraveled (19, 67, 75, 76, 112, 166).

Presently, the best estimations of functional differences among isoforms and splice variants stem from in vitro assays measuring the uptake of ⁴⁵Ca²⁺ into reconstituted microsomal vesicles (prepared from cells overexpressing recombinant pump isoforms) and/or from the ATPase activity of specific PMCA isoforms purified from eukaryotic overexpression systems such as recombinant baculovirus-infected insect Sf9 cells (3, 30, 52, 78, 80). These methods are valid to determine the isolated functional characteristics of the isoforms and their splice variants with respect to enzyme kinetics [V_max, K_m(Ca²⁺)] and regulation by calmodulin, phosphorylation, and phospholipids. However, they provide only limited information concerning the true physiological properties of the isoforms and splice variants because in these systems, the PMCAs are separated from the entire network of endogenous regulatory factors of which many are likely to exist. Despite these caveats, a number of distinct differences among the major splice variants of several PMCA isoforms have been established. In the following sections, we discuss these findings as well as speculate on additional or alternative roles of alternative splicing in providing specific functionalities to the various pumps. We first consider potential consequences of alternative splicing at site A, followed by those at site C. We do not discuss any further the potential consequences of alternative splicing at site B because all available evidence suggests that such splicing events happen only in a small proportion of the PMCA transcripts and can only be detected by sensitive PCR methods in a few tissues (see Tables 2 and 5).

B.  Splicing at Site A: Differential Phospholipid Sensitivity and/or Differential Interaction With Regulatory Proteins?

1.  Differential interactions with phospholipids and with the intramolecular autoinhibitory domain may contribute to functional differences among splice variants

The functional consequence of alternative splicing at site A is still unknown, but a few speculations on its possible impact on pump regulation and/or localization may appropriately be made here. The splicing event that occurs at site A affects the sequence immediately 5' to the region that encodes a phospholipid-sensitive portion of the PMCA (see Fig. 1). Acidic phospholipids, in particular polyphosphoinositides, are potent activators of the PCMA (reviewed in Refs. 24, 128, 131, 173). Splice site A is also situated between the phospholipid binding region and a sequence further upstream that appears to be involved in an intramolecular inhibitory interaction with the COOH-terminal calmodulin binding domain (56) (see also sect. II). Insertions of different length and sequence at site A are thus likely to alter the overall conformation of the second cytosolic loop of the pump and change the spatial connectivities between the phospholipid binding and upstream autoinhibitory domain.

To date, only one study has attempted to measure functional differences between site A alternative splice variants of the PMCA (80). In this study, the three site A splice variants of human PMCA2b (2z/b, 2x/b, and 2w/b) were overexpressed in recombinant baculovirus-infected Sf9 cells, and the ATP-dependent production of the phosphorylated intermediate as well as the Ca²⁺-dependent ATPase activity were determined directly on membrane preparations from these cells. The data were compared with those obtained with the hPMCA4x/b isoform that has become the standard comparator for these studies because it was the first PMCA to be overexpressed in eukaryotic cell systems (3, 78). All PMCA2 splice variants had a higher affinity for ATP than hPMCA4x/b and showed a much higher basal ATPase activity in native membranes than hPMCA4x/b. This activity could be stimulated only marginally by the addition of calmodulin. Calmodulin stimulation of all PMCA2b variants was observed, however, in EDTA-washed membranes, suggesting that calmodulin had not been completely removed from the native membranes. The study also showed that PMCA2b (all site A variants) has a 5- to 10-fold higher affinity for calmodulin than PMCA4b (80). This has been confirmed more recently in an independent study that also indicated that the high basal activity may be an intrinsic, calmodulin-independent property of all PMCA2 splice variants (47). In a comparison of the three site A splice variants, a decreased maximal activity was found for 2z compared with 2x and 2w in both native and EDTA-washed membranes (80). This difference was particularly obvious in the EDTA-washed membranes where both the basal and the calmodulin-stimulated activity were reduced to ~50% of the activity of the other two splice variants. As pointed out by the authors (80), the potential difference in activity between PMCA2z/b and the other splice forms must be interpreted with caution, however, because of potential problems with calmodulin release from the EDTA-washed membranes and the limited amounts of material available for these studies. The latter may also be responsible for the fact that no reliable data are so far available on the phospholipid sensitivity of the splice forms. Phosphatidylserine was found to be a more potent activator than phosphatidylinositol for both hPMCA2w/b and hPMCA4x/b when added exogenously to the PMCA preparations (80).

Unfortunately, however, no comprehensive comparison of the effects of different phospholipids on the different site A splice variants has yet been performed, leaving open the possibility that they show major functional differences as a consequence of differential lipid regulation. The first two alternatively spliced exons of PMCA2 at site A encode a relatively hydrophobic stretch of amino acids that is positioned amidst a highly charged region. This could have significant consequences for the interaction of the first intracellular loop with the membrane environment (2). Alternative splice variants may well differ in their ability to interact optimally with different phospholipids containing different head groups (with respect to both size and charge). With the development of improved expression systems for individual PMCAs (e.g., the baculovirus system), these issues will hopefully soon be addressed in more detail.

The region of the protein around and including alternative splice site A is predicted to form an amphipathic, -helical structure. This particular feature may indicate that this region of the PMCA is involved in intra- or intermolecular protein-protein interactions that may be responsible for some of its observed regulatory properties. A notable feature of the sequence encoded by the site A exons is the occurrence of a KxxDG motif at the beginning of each insert. In PMCA2, this motif is thus found only once in the z splice variant, duplicated in the x variant, and repeated three times in the w variant (2). Repeated elements of defined structure are often involved in specific molecular interactions.

There is evidence that the PMCAs are regulated by both the - and the -subunits of heterotrimeric G proteins (93, 94, 113). However, it is not yet clear if these regulatory phenomena reflect a membrane-delimited event, meaning that the effects observed on the PMCA molecules could be occurring circuitously via stimulation of other signaling or regulatory pathways that also interact on the PMCA. Direct regulation of several membrane-bound effector proteins (e.g., ion channels) by the - or the -G protein subunits occurs via interactions with specific cytosolic loops in these proteins (e.g., the linker loop between membrane domains I and II in the ₁-subunit of voltage-gated Ca²⁺ channels, Refs. 40, 188). The alternatively spliced region at site A of the PMCAs would seem to be an ideal candidate domain for the interaction with regulatory proteins such as G protein subunits. Indeed, the sequence immediately preceding splice site A shows significant similarity to a consensus motif for G protein binding identified earlier in a variety of membrane receptors and channels (33). Figure 5 shows that the putative -interacting consensus sequence is conserved in all PMCA isoforms and not directly affected by alternative splicing. However, alternative splicing will change the immediate COOH-terminal extension of this sequence. Because the extent and overall structure of the complete -interacting sequence is not known, alternative splicing would be expected to have significant consequences for a possible G protein-PMCA interaction in this region. Although purely speculative at this time, the above hypothesis for a direct G protein interaction of the PMCAs should be fairly easy to address experimentally.

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