PHYSIOLOGICAL REVIEWS
Vol. 81, No. 1, January 2001
Printed in U.S.A.
Role of Alternative Splicing in Generating Isoform
Diversity Among Plasma Membrane Calcium Pumps
EMANUEL E. STREHLER AND DAVID A. ZACHARIAS
Department of Biochemistry and Molecular Biology, Mayo Graduate School, Mayo Clinic/Foundation,
Rochester, Minnesota; and Howard Hughes Medical Institute, Cell and Molecular Medicine,
University of California San Diego, La Jolla, California
V.
VI.
VII.
VIII.
IX.
Introduction
Overview of Structural and Regulatory Characteristics of Plasma Membrane Calcium Pumps
Nomenclature of Plasma Membrane Calcium Pump Isoforms
Four Genes and Alternative Ribonucleic Acid Splicing Generate a Multitude of Plasma Membrane
Calcium Pump Isoforms
Alternative Splicing Options of Mammalian Plasma Membrane Calcium Pumps
A. Splice site A: highly variable complexity among different PMCA genes
B. Splice site B: a splicing artifact?
C. Splice site C: a multitude of options with some conserved principles
Tissue Distribution of Plasma Membrane Calcium Pump Isoforms and Splice Variants
A. Differential expression of the four PMCA genes in adult tissues
B. Differential expression of the four PMCA genes during development
C. Differential expression of alternative splice variants
Regulation of Alternative Splicing in the Plasma Membrane Calcium Pump Family
Physiological Significance of Alternative Splicing in the Plasma Membrane Calcium Pump Family
A. Experimental challenges in determining the function of individual PMCA isoforms
B. Splicing at site A: differential phospholipid sensitivity and/or differential interaction with
regulatory proteins?
C. Splicing at site C: multiple effects on the complex and modular COOH-terminal regulatory
domain
Conclusions and Future Perspectives
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IV.
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Strehler, Emanuel E., and David A. Zacharias. Role of Alternative Splicing in Generating Isoform Diversity
Among Plasma Membrane Calcium Pumps. Physiol Rev 81: 21–50, 2001.—Calcium pumps of the plasma membrane
(also known as plasma membrane Ca2⫹-ATPases or PMCAs) are responsible for the expulsion of Ca2⫹ from the
cytosol of all eukaryotic cells. Together with Na⫹/Ca2⫹ exchangers, they are the major plasma membrane transport
system responsible for the long-term regulation of the resting intracellular Ca2⫹ concentration. Like the Ca2⫹ pumps
of the sarco/endoplasmic reticulum (SERCAs), which pump Ca2⫹ from the cytosol into the endoplasmic reticulum,
the PMCAs belong to the family of P-type primary ion transport ATPases characterized by the formation of an
aspartyl phosphate intermediate during the reaction cycle. Mammalian PMCAs are encoded by four separate genes,
and additional isoform variants are generated via alternative RNA splicing of the primary gene transcripts. The
expression of different PMCA isoforms and splice variants is regulated in a developmental, tissue- and cell
type-specific manner, suggesting that these pumps are functionally adapted to the physiological needs of particular
cells and tissues. PMCAs 1 and 4 are found in virtually all tissues in the adult, whereas PMCAs 2 and 3 are primarily
expressed in excitable cells of the nervous system and muscles. During mouse embryonic development, PMCA1 is
ubiquitously detected from the earliest time points, and all isoforms show spatially overlapping but distinct
expression patterns with dynamic temporal changes occurring during late fetal development. Alternative splicing
affects two major locations in the plasma membrane Ca2⫹ pump protein: the first intracellular loop and the
COOH-terminal tail. These two regions correspond to major regulatory domains of the pumps. In the first cytosolic
loop, the affected region is embedded between a putative G protein binding sequence and the site of phospholipid
sensitivity, and in the COOH-terminal tail, splicing affects pump regulation by calmodulin, phosphorylation, and
differential interaction with PDZ domain-containing anchoring and signaling proteins. Recent evidence demonstrating differential distribution, dynamic regulation of expression, and major functional differences between alternative
splice variants suggests that these transporters play a more dynamic role than hitherto assumed in the spatial and
http://physrev.physiology.org
0031-9333/01 $15.00 Copyright © 2001 the American Physiological Society
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EMANUEL E. STREHLER AND DAVID A. ZACHARIAS
Volume 81
temporal control of Ca2⫹ signaling. The identification of mice carrying PMCA mutations that lead to diseases such
as hearing loss and ataxia, as well as the corresponding phenotypes of genetically engineered PMCA “knockout”
mice further support the concept of specific, nonredundant roles for each Ca2⫹ pump isoform in cellular Ca2⫹
regulation.
I. INTRODUCTION
II. OVERVIEW OF STRUCTURAL AND
REGULATORY CHARACTERISTICS OF
PLASMA MEMBRANE CALCIUM PUMPS
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
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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.
January 2001
ISOFORMS OF THE PLASMA MEMBRANE CALCIUM PUMP
23
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.
A particularly distinctive feature of the PMCAs is the
number of different regulatory mechanisms that alter
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2⫹
FIG. 1. Models of the plasma membrane Ca -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.
24
EMANUEL E. STREHLER AND DAVID A. ZACHARIAS
ization (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⫹ [Km(Ca2⫹)] of the enzyme to ⬃0.3 ␮M compared
with 0.4 – 0.7 ␮M 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 Km(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
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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 COOHterminal 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 (Vmax) 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 COOHterminally truncated PMCAs have shown that the dimer-
Volume 81
January 2001
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ISOFORMS OF THE PLASMA MEMBRANE CALCIUM PUMP
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.
III. NOMENCLATURE OF PLASMA MEMBRANE
CALCIUM PUMP ISOFORMS
TABLE
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
1. Nomenclature for known alternative splice
variants of the PMCAs
Isoform
Splice Site
Original
Nomenclature
Alternative
Nomenclature
PMCA1
A
B
B
C
C
C
C
C
A
A
A
A
C
C
C
A
A
C
C
C
C
C
C
A
A
B
B
C
C
x*
⫺†
⫺†
a
b
c
d
e
w
x
y
z
a
b
c
x
z
a
b
c
d
e
f
x
z
(g)†
⫺†
a
b
AII*
(BI)†
(BII)†
CII
CI
CIII
CIV
CV
AIII
AII
AIV
AI
CII
CI
CIII
AII
AI
CII
CI
CIII
CIV
CV
CVI
AII
AI
(BI)†
(BII)†
CII
CI
PMCA2
PMCA3
PMCA4
For a schematic overview of the exon configuration for each splice
variant, see Figures 2 and 3.
For original nomenclature, see References 95, 153.
For alternative nomenclature, see Reference
25.
* This site in plasma membrane Ca2⫹-ATPase (PMCA) 1 does not
appear to be alternatively spliced; on the basis of the exon structure, all
currently known PMCA1 splice variants correspond to the “x” or “AII”
type.
† Alternative splicing at site B has only been observed in a few
intestinal tissues (see Ref. 86) and may represent aberrant splicing. In
the alternative nomenclature, the “normal” variant would be called BII
because it includes the alternatively spliced exon.
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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).
26
EMANUEL E. STREHLER AND DAVID A. ZACHARIAS
IV. FOUR GENES AND ALTERNATIVE
RIBONUCLEIC ACID SPLICING GENERATE
A MULTITUDE OF PLASMA MEMBRANE
CALCIUM PUMP ISOFORMS
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
A. Splice Site A: Highly Variable Complexity
Among Different PMCA Genes
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).
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.
B. Splice Site B: A Splicing Artifact?
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
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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
Volume 81
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ISOFORMS OF THE PLASMA MEMBRANE CALCIUM PUMP
27
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 membranespanning 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.
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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.
28
EMANUEL E. STREHLER AND DAVID A. ZACHARIAS
C. Splice Site C: A Multitude of Options With
Some Conserved Principles
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).
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.
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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).
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
Volume 81
January 2001
ISOFORMS OF THE PLASMA MEMBRANE CALCIUM PUMP
VI. TISSUE DISTRIBUTION OF PLASMA
MEMBRANE CALCIUM PUMP ISOFORMS
AND SPLICE VARIANTS
A. Differential Expression of the Four PMCA
Genes in Adult Tissues
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 predomi-
nantly 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, monoand 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).
B. Differential Expression of the Four PMCA
Genes During Development
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
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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.
29
30
EMANUEL E. STREHLER AND DAVID A. ZACHARIAS
Volume 81
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).
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 steadystate 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
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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.]
January 2001
ISOFORMS OF THE PLASMA MEMBRANE CALCIUM PUMP
C. Differential Expression of Alternative
Splice Variants
The problem of the tissue distribution and relative
abundance of the various alternative splice variants has
been more difficult to address. At the mRNA level, this
issue has been examined extensively by Northern blots,
reverse transcriptase (RT)-PCR, ribonuclease protection
assays (RPA), and in situ hybridization, with the bulk of
these studies being performed on rat and human tissues.
In the following sections, we review each of these techniques with respect to their relative merit and potential
problems as they relate to the characterization of different PMCA isoforms and alternative splice variants. The
collected set of data on the expression of PMCA splice
variants is then summarized in tabular form at the end.
1. Northern blots
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).
2. RT-PCR
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.
3. RPA
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
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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).
31
32
EMANUEL E. STREHLER AND DAVID A. ZACHARIAS
4. In situ hybridization
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 de-
tected 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).
5. Summary of the tissue and cell type expression
of the PMCA isoforms and their splice variants
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
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
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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).
Volume 81
TABLE
33
ISOFORMS OF THE PLASMA MEMBRANE CALCIUM PUMP
January 2001
2. Tissue distribution of PMCA1
Splice Variants at
Site C
Tissue
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹
⫹⫹
Site B
(⌬exon)
1a
1b
1c
1d
1e
⫹⫹
⫹⫹
⫹
⫹
⫹
⫺
⫺
⫺
⫺
⫺
⫹
⫹
⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹
⫹
⫹⫹
⫹⫹
⫹/⫺
⫺
⫹/⫺†
⫺
⫺
⫺
⫹/⫺
⫹/⫺
⫺
⫹
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹
ND
⫺
⫺
ND
⫺
⫺
⫺
⫺
⫺
ND
⫺
⫺
⫺
⫹/⫺†
⫺
⫺
⫺
⫺
⫺
⫹ (by Northern)
⫺
⫺ (by Western)
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹
⫺
⫺
⫺
⫹/⫺†
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹/⫺†
⫺
⫺
⫺
⫺
⫺
ND
⫺
⫺
⫺
ND
ND
⫺
⫺
ND
⫹⫹
⫺
⫺
ND
⫹/⫺*
⫹/⫺*
⫹/⫺*
The relative abundance of the different splice variants is given as follows: ⫹⫹, abundant; ⫹, present; ⫹/⫺, rare; ⫺, absent; ND, not
determined.
⌬Exon refers to optional exclusion of a 108-nt exon; in the default splice pathway, this exon is included.
* Very low abundance,
detected only by sensitive PCR (85, 87).
† Only detected by PCR in a single study in hamster (139).
The data were collected from the
following references: human (17, 85– 87, 127, 151, 153), rat (87, 95, 132, 151), gerbil (35), hamster (139), rabbit (99), mouse (37), and pig (38).
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 isoformspecific 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.
Downloaded from http://physrev.physiology.org/ by 10.220.33.4 on July 4, 2017
Brain
Spinal cord
Skeletal muscle
Heart
Aorta
Lung
Liver
Spleen
Pancreas
Stomach
Smooth
muscle
Mucosa
Small intestine
Colon
Kidney
Adrenal gland
Thymus
Uterus
Testis
Mammary gland
Placenta
Erythrocytes
Human platelets
Site A
(1x)
34
EMANUEL E. STREHLER AND DAVID A. ZACHARIAS
TABLE
Volume 81
3. Tissue distribution of PMCA2
Splice Variants at
Site A
Site C
2w
2x
2y
2z
2a
2b
2c
Brain
Spinal cord
Skeletal muscle
Heart
Lung
Liver
Spleen
Pancreas
Stomach
Small intestine
Colon
Kidney
Adrenal gland
Thymus
Uterus
Testis
Mammary gland (lactating)
Placenta
Erythrocytes
Human platelets
⫹
ND
⫹/⫺
⫹
⫹/⫺
⫹/⫺
ND
⫹
ND
⫹/⫺
⫹
⫹/⫺
⫹/⫺
ND
⫹/⫺*
ND
⫺
⫹/⫺*
⫺
⫺
ND
⫹⫹
ND
⫹/⫺
⫹
⫺
⫺
ND
⫹⫹
⫹
⫹/⫺
⫹/⫺
⫹/⫺
⫹
⫹/⫺
⫹/⫺
⫹/⫺ (?)
⫺
⫺
⫺
⫹/⫺ (?)
⫺
ND
ND
ND
ND
⫹/⫺
⫺
⫹
ND
⫹/⫺
ND
⫺
ND
⫺
ND
⫹
⫹
⫹/⫺ (?)
⫺
⫹
ND
ND
⫺
ND
ND
⫺
ND
ND
⫺
ND
ND
⫹
⫹
⫹/⫺
⫹
⫺
⫺
⫺
⫺ (by PCR)
⫺ (by Western and PCR)
⫹/⫺
⫺ (by PCR)
⫺
⫺
⫺ (by PCR)
⫹
⫺
⫺
⫺ (by PCR)
⫺ (by Western)
⫺ (by Western)
⫹
⫹
⫹⫹
⫹/⫺ (?)
⫹
⫺
The relative abundance of the different splice variants is given as follows: ⫹⫹, abundant; ⫹, present; ⫹/⫺, rare; ⫺, absent; ND, not
determined.
* Detected only by sensitive PCR in a single study in the rat (2). (?), Found only in some cases and/or questionable due to lack of
sequence identification.
Data were collected from the following references: human (17, 85, 86, 127, 151, 153), rat (2, 70, 95, 132, 151), and gerbil
(35).
TABLE
4. Tissue distribution of PMCA3
Splice Variants at
Site A
Site C
Tissue
3x
3z
3a
3b
3c
3d
3e
3f
Brain
Spinal cord
Skeletal muscle*
Heart
Lung
Liver
Spleen
Pancreas
Stomach
Small intestine
Colon
Kidney
Adrenal gland
Thymus
Uterus
Testis
Placenta
Erythrocytes
Human platelets
⫹
ND
⫹
⫹/⫺
ND
⫺
⫹
⫹
⫹
⫹/⫺(?)
ND
⫹/⫺(?)
⫹/⫺(?)
ND
⫺
⫹
ND
⫺
⫹/⫺(?)
ND
⫹
ND
ND
⫺
⫺
⫺
ND
⫺
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
⫺
⫺
⫺
⫹/⫺
⫹⫹
⫺
⫺
⫺
⫹/⫺
ND
⫺
⫺
⫺
⫺
ND
ND
ND
ND
ND
ND
⫺
⫺
⫺
⫺
ND
ND
ND
ND
ND
⫹⫹
⫹⫹
⫹/⫺
⫺ (by PCR)
⫹/⫺
⫺ (by PCR)
⫺ (by PCR)
⫺ (by PCR)
⫹/⫺
⫹/⫺
⫹/⫺
⫹/⫺
⫺
⫺ (by PCR)
⫹/⫺
⫹
⫺ (by PCR)
⫺ (by Western)
⫺ (by Western)
⫺
⫹/⫺
⫺
⫹/⫺
⫺
⫺
ND
ND
⫺
⫺
The relative abundance of the different splice variants is given as follows: ⫹⫹, abundant; ⫹, present; ⫹/⫺, rare; ⫺, absent; ND, not
determined.
* Data for rat skeletal muscle; in humans, PMCA3 is expressed only in fetal skeletal muscle and is almost undetectable in neonatal
and adult skeletal muscle (17, 153).
(?), Found only in some cases and/or questionable due to lack of sequence identification.
The data were
collected from the following references: human (17, 86, 127, 151, 153), rat (22, 29, 70, 95, 151), and gerbil (35).
Downloaded from http://physrev.physiology.org/ by 10.220.33.4 on July 4, 2017
Tissue
ISOFORMS OF THE PLASMA MEMBRANE CALCIUM PUMP
January 2001
TABLE
5. Tissue distribution of PMCA4
Splice Variants at
Site C
Site A
4x
4z
Brain
Spinal cord
Skeletal muscle
Heart
Lung
Liver*
Spleen
Pancreas
Stomach
Small intestine
Colon
Kidney
Adrenal gland
Thymus
Uterus
Testis
Mammary gland (lactating)
Placenta
Erythrocytes
Platelets
⫹⫹
ND
⫹⫹
⫹
⫹⫹
⫹⫹
⫹⫹
ND
⫹⫹
⫹⫹
⫹⫹
⫹⫹
ND
ND
⫹⫹
⫹
ND
ND
ND
ND
⫺
ND
⫺
⫹⫹
⫺
⫺
⫺
ND
⫺
⫺
⫺
⫺
ND
ND
⫺
⫹⫹
ND
ND
ND
ND
⫹/⫺†
⫹/⫺†
⫹/⫺†
4a
4b
⫹⫹
⫹⫹
⫹
⫹⫹
⫹/⫺
⫺
⫺
⫹/⫺
⫹⫹
⫹⫹
⫹⫹
⫺
⫺
⫺
⫹⫹
⫹⫹
⫺
⫺
⫺
⫺
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹
⫹
⫹
⫹⫹
⫹⫹
⫹⫹
⫹
⫹
⫹
⫹⫹
⫹⫹
⫹/⫺
The relative abundance of the different splice variants is given as
follows: ⫹⫹, abundant; ⫹, present; ⫹/⫺, rare; ⫺, absent; ND, not
determined.
* Data for human liver; in rat liver, PMCA4 is expressed
at very low levels (85, 96).
† Very low abundance, detected only by
sensitive PCR (85, 87).
Data were collected from the following references: human (17, 85– 87, 127, 151, 153), rat (87, 95, 96, 151), and gerbil
(35).
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-mybmediated 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
A. Experimental Challenges in Determining the
Function of Individual PMCA Isoforms
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 [Vmax, Km(Ca2⫹)]
and regulation by calmodulin, phosphorylation, and phospholipids. However, they provide only limited information
concerning the true physiological properties of the iso-
Downloaded from http://physrev.physiology.org/ by 10.220.33.4 on July 4, 2017
Tissue
Site B
(⌬exon)
35
2z
⫹* ⫺
ND ND
ND ND
ND ND
2x
ND ND ND
ND ND ND
ND ND ND
⫹
ND
ND
ND
2w
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫺
⫹
2b
2c
3x
3z
3a
3b
3c
3d
4x
4z
⫺
⫹
⫺
⫹/⫺ ⫺
⫹/⫺
⫹/⫺
⫺
⫺ ⫹ ⫺
⫺
⫹/⫺
⫺
ND ND
⫹/⫺
⫹/⫺? ⫹/⫺? ⫺ ND ND
⫹/⫺
⫹ ⫹/⫺? ND ND
⫹/⫺
⫹/⫺? ⫹/⫺? ⫺ ND ND
⫺
⫹
⫺
ND ND
⫺
⫺
⫺
⫺ ND ND
(by PCR)
⫺ (by PCR)
ND ND
(by PCR)
⫺ (by PCR)
ND ND
(by PCR)
⫹ (by PCR)
⫹ (by
ND
ND
ND
ND ND
ND
ND
ND ND ND ND
ND
ND
ND
ND ND
ND
ND
ND ND ND ND
⫹/⫺
⫹ ⫹/⫺?
⫺ (by PCR)
ND ND
(by PCR)
⫺ (by PCR)
ND ND
(by PCR)
⫺ (by PCR)
ND ND
(by PCR)
ND ND ⫹/⫺
⫹/⫺ ⫹/⫺ ⫺ ND ND
(by Western)
⫺ (by Western)
⫺ (by
(by Western)
⫺ (by Western)
ND ND
(by Western)
⫺ (by Western)
ND ND
(by PCR)
⫺ (by PCR)
ND ND
(by PCR)
⫺ (by PCR)
ND ND
(by PCR)
⫺ (by PCR)
⫺ (by
(by PCR)
⫺ (by PCR)
⫹ (by
(by Western)
⫺ (by Western)
ND ND
2a
PMCA Isoform/Splice Variant
4b
⫺
⫹
⫺
⫹
⫹/⫺
⫹
⫺
⫹
⫺
⫹
⫹/⫺
⫹
PCR)
ND
ND
ND
ND
⫹/⫺
⫹
⫺
⫹
⫺
⫹
⫹
⫹
Western)
⫺
⫹
⫺
⫹
⫺
⫹
⫺
⫹
PCR)
PCR)
⫺
⫹
4a
EMANUEL E. STREHLER AND DAVID A. ZACHARIAS
Downloaded from http://physrev.physiology.org/ by 10.220.33.4 on July 4, 2017
The relative abundance of the different splice variants is given as follows: ⫹, present; ⫹/⫺, rare; ⫺, absent; ND, not determined.
* Expression regulated in a calcium-dependent
manner (187).
Data were collected from the following references: human cell lines (127, 133, 151, 187), rat primary cells and cell lines (1, 61, 74, 136), mouse cell lines (37, 186), and
monkey COS-7 cells (58, 127).
ND
⫹
⫹
⫹
⫹
⫹
ND
ND
ND
ND
⫹
⫹
⫺ ⫹
⫹/⫺
⫹/⫺
⫺ ⫹
⫺
⫺
⫺ ⫹
⫹/⫺
⫺
⫺ ⫹
⫺
⫺
⫺ ⫹
⫺
⫺
⫺ ⫹
⫹
⫹/⫺
⫹ (by PCR)
⫺ ⫹
⫺
⫺
⫺ ⫹
⫹
⫺
⫺ ⫹
⫹
⫹/⫺
⫺ ⫹
⫺
⫺
⫺ ⫹
⫺
⫺
⫺ ⫹
⫺
⫺
⫺ ⫹ (by Western)
⫺ ⫹ (by Western)
⫺ ⫹ (by Western)
⫺ ⫹
⫺
⫺
⫺ ⫹
⫺
⫺
⫹ (by PCR)
⫹ (by PCR)
⫺ ⫹/⫺ (by Western)
1d
⫹
⫹
⫹
ND
⫹
⫹
1c
Human IMR32 neuroblasts
Rat PC12 neuroblasts
Rat NGF-treated PC12 cells
Rat cortical astroctes
Rat L6 myoblasts
Rat L6 myotubes
Mouse embryonic stem cells
Mouse BC3H1 myoblasts
Mouse BC3H1 myotubes
Rat adult cardiomyocytes
Rat aortic smooth muscle cells
Rat aortic endothelial cells
Rat mesangial cells
Monkey COS cells
Human HeLa cells
Human HEK 293 cells
Human WI38 fibroblasts
Rat fetal skin fibroblasts
Rat ROS17/2.8 osteosarcoma cells
Rat UMR-106 osteosarcoma cells
Human DAMI megakaryoblastoid
cells
1b
1a
1x
6. PMCA expression in various cell types
Cell Type
TABLE
36
Volume 81
January 2001
ISOFORMS OF THE PLASMA MEMBRANE CALCIUM PUMP
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 COOHterminal 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.
Downloaded from http://physrev.physiology.org/ by 10.220.33.4 on July 4, 2017
forms 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).
37
38
EMANUEL E. STREHLER AND DAVID A. ZACHARIAS
2. Differential interactions with regulatory proteins:
G protein subunits as potential candidates?
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.
Alternative splicing affecting a limited region within
an intracellular loop has also been shown to be responsible for the differential interaction of the ␣-subunit of brain
Ca2⫹ channels with the presynaptic proteins syntaxin and
SNAP-25 (134). Similar differential protein interactions of
the alternatively spliced region in the first cytosolic loop
of the PMCAs could allow differential regulation of the
FIG. 5. A potential G protein ␤␥ binding site
is present in the first intracellular loop of the
PMCA. The location of a possible G protein
interaction site (black oval) immediately NH2
terminal to the alternative splice site A is indicated in a model of the PMCA similar to that in
Fig. 1B. Alternative splice site A is shown for
PMCA2, i.e., containing 3 separate exons
(striped boxes; see Fig. 2). The position of alternative splice site C is also indicated. The domains are labeled as in Fig. 1. The putative G
protein binding sequences in all 4 human PMCA
isoforms are shown and have been aligned with
the G protein ␤␥ binding sequences identified in
a number of other G protein-regulated proteins
(33). Conserved acidic and basic residues are
indicated in bold type. The position of alternative splice site A is indicated by a vertical line in
the PMCA sequences. The amino acid number of
the last residue shown (AA#) is also indicated
for each protein. ␤ARK, ␤-adrenergic receptor
kinase; AC, adenylyl cyclase; GIRK, G proteingated inwardly rectifying potassium channel.
Downloaded from http://physrev.physiology.org/ by 10.220.33.4 on July 4, 2017
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 membranebound 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-
Volume 81
January 2001
ISOFORMS OF THE PLASMA MEMBRANE CALCIUM PUMP
C. Splicing at Site C: Multiple Effects on the
Complex and Modular COOH-Terminal
Regulatory Domain
1. Calmodulin regulation
A) SEQUENCE DIFFERENCES BETWEEN “A” AND “B” SPLICE VARIANTS IN THEIR CALMODULIN BINDING DOMAIN.
The functional
implications of alternative splicing at site C have been
characterized to a much greater degree than those at sites
A and B. This is due in large part to the early realization
that much of the regulation of the PMCAs is mediated via
their COOH-terminal cytosolic tail (reviewed in Refs. 24,
128, 129). This tail is unique to the PMCAs in that it is
much longer (⬃100 residues in the “a” splice forms to
over 150 residues in the “b” splice forms) than any of the
COOH-terminal extensions in the other P2-type ion
pumps. Significantly, the amino acid sequence around
splice site C had previously been shown to correspond to
the calmodulin binding domain (90, 169). The amino acid
sequence of each of the four isoforms is completely conserved immediately preceding the location of the alterna-
tive splice site (see Fig. 6). Following the conserved
IQTQ, the sequence varies depending on whether a large,
conserved alternatively spliced exon (154 –175 nt) is included (a splice variants) or excluded (b splice variants)
(Fig. 3). Inclusion of the large exon in all isoforms effectively alters the COOH-terminal half of the calmodulin
binding domain and changes the reading frame for the
entire remaining COOH-terminal tail. The frame shift also
introduces an earlier translation stop site than in the b
splice variant, resulting in the PMCA a variants being
smaller by ⬃5 kDa than the b splice variants. In isoforms
1 and 3, the 154-bp alternatively spliced exon contains
internal donor sites, adding to the degree of splice form
diversity (see Fig. 3). The second half of the calmodulin
binding domain is similarly changed in splice variants 1c,
1d, and 1e as in 1a, although variants 1c and 1d continue
further downstream with the same reading frame as in 1b
(162). PMCA1e is identical to 1a except for the last three
residues (Fig. 6). An analogous situation is present in
PMCA3 for the splice variants 3c, 3d, and 3e.
B) DIFFERENCES IN RESPONSIVENESS TO CALMODULIN AND RESULTING EFFECTS IN CALCIUM SENSITIVITY. In PMCA1, alternative
splicing at site C was found to confer pH sensitivity to the
calmodulin binding properties of the different splice variants (98). hPMCA1b (which contains the “canonical” calmodulin binding domain) bound Ca2⫹-calmodulin at pH
7.2 and pH 5.9 with equal affinity, whereas variants 1a, 1c,
and 1d that contain histidine-rich inserts bound Ca2⫹calmodulin with higher affinity at the lower pH. It has thus
been suggested that the activation of PMCA variants 1a,
1c, and 1d could be pH sensitive. At acidic pH, these
splice variants would bind calmodulin (and thus be activated) more readily than at neutral pH. It is of interest
that acidic conditions (pH below 6.5) can (temporarily)
exist in certain tissues such as skeletal muscle (147).
Perhaps it is no coincidence that this is also one of the
tissues with the highest abundance of PMCA splice forms
1c and 1d (see Tables 2 and 6).
The impact of alternative splicing on the calmodulin
binding and activation properties of the PMCA has been
studied in most detail on the human 4a and 4b variants.
The exon insertion responsible for generating the a variant results in a pump with a reduced affinity for calmodulin [measured as mean affinity constant (K1/2) for halfmaximal stimulation of the pump] and, as a result, an
apparent reduced affinity for Ca2⫹ (52, 142). At 1.5 ␮M
calmodulin, the K1/2 for Ca2⫹ was determined to be 0.25
␮M for hPMCA4b versus 0.54 ␮M for hPMCA4a (52).
These values are relevant because they are well within the
physiological range of free Ca2⫹ observed in living cells.
The difference in calmodulin affinity between the
PMCA4a and -4b splice variants is explained by sequence
and structural differences in the COOH-terminal portion
of their calmodulin binding domains. Using different
COOH-terminal truncation mutants of PMCA4a and
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splice variants (e.g., by G proteins as noted above) and/or
their differential recruitment to specific membrane domains. Differential regulation by phospholipids, heterotrimeric G proteins, and additional protein interactions need
not be mutually exclusive. In an interesting recent report,
Huang et al. (88) showed that inward rectifier K⫹ channels were activated by phosphatidylinositol 4,5-bisphosphate (PIP2) (as are the PMCAs) and that this lipid interaction was modified (stabilized) by G protein ␤␥. In the
absence of a sufficiently high concentration of PIP2, the
channel could not be stimulated by the G protein. Thus
there appeared to be synergism between the G protein
␤␥-subunits and PIP2 in effector activation. Conversely,
inhibition of an effector via G protein regulation of the
local phospholipid concentration could also be envisaged.
In the case of the PMCA, agonist-stimulated, G proteindependent activation of phospholipase C may decrease
the basal Ca2⫹ pump activity due to PIP2 hydrolysis (119).
Effective control of local PMCA activity could be exerted
if the phospholipase were held in close proximity of the
PMCAs. Scaffolding protein(s), perhaps the G protein
subunits themselves, may recruit the phospholipase to the
PMCA to ensure local control of signal transmission to the
pump. Given the mounting evidence that short variable
sequences generated by alternative splicing serve to provide alternative protein interaction interfaces within otherwise well-conserved protein isoforms, we predict that
splicing at site A in the PMCAs may fulfill such a function.
A systematic search for differential interacting partners,
e.g., by using the yeast two-hybrid interaction trap, may
well lead to exciting new insights into the functional role
of this variable region in the PMCAs.
39
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EMANUEL E. STREHLER AND DAVID A. ZACHARIAS
Volume 81
PMCA4b, Verma et al. (167) demonstrated that the calmodulin binding domain of PMCA4b is entirely contained
within a 28-residue sequence consisting of 18 residues
upstream and 10 residues downstream of the splice site.
In contrast, in PMCA4a, the calmodulin binding domain is
bipartite, spanning ⬃49 residues, including 18 residues
upstream of the splice site but extending at least 31
residues downstream of it (168).
As mentioned in section II and shown in Figure 2B,
the calmodulin binding sequence of the PMCA acts as an
autoinhibitory domain that keeps the pump in a relatively
inactive state in the absence of Ca2⫹ and calmodulin (25,
128). The detailed studies on PMCA4a and -4b have shown
that the calmodulin binding domain in PMCA4b does not
correspond to the entire autoinhibitory domain and that
additional, downstream sequences are necessary to confer full autoinhibition on the pump. In contrast, in
PMCA4a, the extended calmodulin binding domain of ⬃49
residues also corresponds to the entire autoinhibitory
domain (167, 168). Lower Ca2⫹-calmodulin affinity of the
a compared with the b splice form also characterizes
PMCA2 (47) and is probably a general finding for all a
versus b comparisons (49). Another finding of general
relevance in a comparison of the a and the b splice forms
is that the a forms show a significantly elevated basal
pumping activity (47, 142, 168). Because the a forms are
more restricted in their expression pattern than the b
forms and appear to be prevalent in excitable cells where
elevated free Ca2⫹ levels and frequent Ca2⫹ fluctuations
may prevail, they may be adapted to Ca2⫹ handling under
these specialized conditions. A higher basal activity and
hence a lesser dependence on calmodulin may enable
these splice forms to be active at moderately elevated
Ca2⫹ even when calmodulin is limiting.
C) CALMODULIN ACTIVATION OF PMCA4 IS SLOW AND IS INFLUENCED BY ALTERNATIVE SPLICING. Very recently, an improved in
vitro reconstitution assay was developed for measuring
PMCA activity in a continuous manner, allowing for the
first time the precise determination of calmodulin association and dissociation rates (30). Surprisingly, this study
revealed that activation of PMCA4b by calmodulin appears to be very slow with a half time (t1/2) of almost 1
min at a calmodulin concentration of 235 nM and at 0.5
␮M Ca2⫹. In contrast, activation of PMCA4a was significantly faster, with a t1/2 of ⬃12 s. The t1/2 for the rate of
inactivation upon removal of calmodulin was ⬍1 min for
PMCA4a but extremely long (over 20 min!) for PMCA4b,
consistent with the much higher Ca2⫹-calmodulin affinity
of isoform 4b than of 4a (30). Recent structural studies
using NMR and fluorescence spectroscopy have shown
that the COOH-terminal half of calmodulin binds with
much higher affinity to calmodulin binding peptides of the
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FIG. 6. COOH-terminal sequence comparison of human PMCA isoforms. Top: alignment of the sequences of the 4
human PMCA isoforms, starting after the last (10th) putative membrane-spanning domain and ending at splice site C. The
exon-intron boundary is indicated by a slash, and the last residue (a Gln) before the splice junction is numbered in
parentheses for each of the PMCAs. Residues conserved in all PMCAs are marked with a caret (∧). Middle: alignment of
the COOH-terminal sequences of the major alternative splice variants containing exon insertions at site C. Exon-intron
boundaries are indicated by a slash; the COOH-terminal residue (marked by an asterisk) is numbered for each splice
variant. #, Note that the last few amino acids of PMCA2a and 2c have been switched with respect to the previous
nomenclature; 2a now ends with the sequence . . .HPRREGVP*, which is closely related to the sequences of 1a, 3a, and
4a and is consistent with the general splicing pattern leading to the “a” splice forms (see Fig. 3). Residues conserved in
all a splice forms are indicated with a caret (∧). Bottom: alignment of the COOH-terminal sequences of the “b” splice
variants following splice site C. The exon-intron boundary is indicated by a slash, and the COOH-terminal residue is
numbered for each splice variant. Conserved amino acids in all 4 b splice variants are marked by a caret (∧).
January 2001
ISOFORMS OF THE PLASMA MEMBRANE CALCIUM PUMP
(129) that the calmodulin binding domain of the PMCA
bears some resemblance to the IQ motif originally identified as a light chain/calmodulin binding sequence in
unconventional myosins (34). The IQ motif takes its name
from its core consensus sequence IQxxxRGxxxR, although a more extended consensus is represented by the
sequence IQxxxRGxxxRRxxL, where x stands for any
amino acid (see Ref. 135 for a recent review of calmodulin
binding sequence motifs). The IQ-like sequence in the
PMCAs begins a few residues upstream of splice site C,
and the change in sequence affected by the splice leads to
a “weakening” in the IQ consensus in the a compared with
the b variants. Significantly, the nonconservative change
at the second position after the splice from a basic residue
(R, K) in the b variants to an acidic residue (D, E) in the
a variants replaces an important conserved residue in the
IQ consensus sequence (see Fig. 7). If this sequence is
indeed involved as an IQ motif in calmodulin binding, the
noted sequence difference may help explain the observed
difference in affinity between the a and the b variants of
the PMCA. Moreover, if the presence of an arginine residue at the second and eighth position after the splice is
crucial for high-affinity calmodulin binding, the lower
calmodulin affinity of PMCA4b compared with PMCA2b
becomes plausible. As shown in Figure 7, these two positions are occupied by a Lys and a His residue in
PMCA4b.
F) PMCA3F: A CONSTITUTIVELY ACTIVE, VIRTUALLY CALMODULININDEPENDENT PUMP. The splicing that occurs at site C of
PMCA3 is complex (see Fig. 3). Again, the theme of
exchange of a portion of the calmodulin regulatory domain by inclusion of a large exon (154 bp as in PMCA1)
remains consistent. Presumably, the overall calmodulin
regulatory properties of 3a and 3b differ similarly as those
in other a versus b splice pairs, although the lessons
learned with the PMCA2 suggest caution in further speculation until experimental evidence has been obtained. A
unique situation is presented by PMCA3f, where the insertion of a 68-nt exon before (or instead of) the 154-nt
exon at site C creates a pump with yet a different COOH
terminus. Not only is the second half of the calmodulin
binding domain replaced by a sequence completely different from that in the a and the b splice variants, but in
addition, the pump is truncated just 15 residues downstream of the splice (see Fig. 6). PMCA3f is thus by far the
smallest PMCA isoform, and it lacks the entire downstream regulatory region. A peptide corresponding to the
28-residue calmodulin binding sequence of rat PMCA3f
displayed an over 50-fold reduced affinity for calmodulin
(Kd of 15 nM) compared with a corresponding peptide of
PMCA2b (Kd of 0.2 nM) (60). A chimera of hPMCA4
carrying the COOH-terminal tail sequence of rPMCA3f
showed full activity in the absence of calmodulin, although it was still able to bind calmodulin. Similarly, the
full-length rPMCA3f bound to, but was only minimally
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PMCA than the NH2-terminal half and that a stable calmodulin-PMCA peptide complex may be formed that utilizes the COOH-terminal half of calmodulin alone (46,
163). It is conceivable, therefore, that once formed, such
a strong interaction between the PMCA and the COOHterminal portion of calmodulin may be difficult to disrupt,
leading to the observed slow off rates. The determination
of on rates and off rates for calmodulin activation allowed
calculations of the true calmodulin affinity of the PMCA4
splice variants, yielding dissociation constant (Kd) values
of 53 and 7.6 nM for PMCA4a and -4b, respectively (30).
Compared with several other calmodulin-regulated enzymes, both PMCA4a and -4b show a much slower on rate
(by 1–3 orders of magnitude), indicating a significant lag
time for activation of the pump by calmodulin. Considering the high affinity of PMCA4 for calmodulin, a possible
interpretation of these findings is that the calmodulin
binding domain of the (inactive) pump is “occluded,”
thereby decreasing its accessibility to calmodulin (30).
Conversely, once bound to calmodulin, the pump (particularly PMCA4b) may remain active for prolonged periods
of time. However, as pointed out by Caride et al. (30),
caution should be used in extrapolating from the in vitro
data to the physiological situation in a living cell (30). Of
note, the a splice form of PMCA4 is mainly found in cells
with fast response times to Ca2⫹ transients, such as in
muscle and nerve (see Tables 5 and 6).
D) PMCA2A AND -2B ARE EXTREMELY SENSITIVE TO CALMODULIN
AND SHOW ELEVATED BASAL ACTIVITY. A surprising finding was
the discovery of a much (5- to 10-fold) higher calmodulin
affinity in PMCA2b than in PMCA4b (47, 80). The sequence of the first 18 residues of the calmodulin binding
domain is identical in all PMCA isoforms (see Fig. 6), and
only 2 conservative differences (Arg for Lys and Arg for
His) are found in the 10 following residues in PMCA2b
versus PMCA4b. The immediate conclusion is that sequences outside of the 28-residue calmodulin binding domain must be responsible for this large effect on calmodulin affinity. This is supported by results showing that the
calmodulin affinity of PMCA2a, while smaller than that of
PMCA2b, is still higher than that of either PMCA4b or
PMCA4a (47). Thus PMCA2 (splice variants a and b)
remains activated at low Ca2⫹-calmodulin levels (in the
nanomolar range) and will be able to efficiently extrude
Ca2⫹ even when free Ca2⫹ drops to ⬍100 nM (47, 129).
This has led to the speculation that PMCA2 is constitutively active because the concentration of cytosolic calmodulin in the brain (where PMCA2 is most abundant)
may be as high as 50 ␮M (23). This is supported by the
finding that the basal activity of both PMCA2a and -2b in
the complete absence of calmodulin is remarkably high,
reaching Vmax values of 47 and 72%, respectively, of the
maximal Ca2⫹-calmodulin-stimulated activity (47).
E) SIMILARITY OF THE CALMODULIN BINDING SEQUENCE OF
PMCAS TO THE IQ DOMAIN. It has recently been pointed out
41
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EMANUEL E. STREHLER AND DAVID A. ZACHARIAS
Volume 81
FIG. 7. Alignment of the IQ domain consensus sequence with the IQ-like sequence at splice site C of the
PMCAs. The core of the IQ consensus sequence is shown
on top and has been aligned with the sequence of the PMCA
splice variants starting at their common IQTQ sequence
just upstream of the splice and extending 11 residues beyond the splice junction (indicated by a slash). Residues
matching the IQ consensus sequence are printed in bold
(perfect matches) or underlined (conserved substitutions).
Note the better match of the “b” splice forms to the IQ
consensus sequence compared with the “a” (and 3f) splice
variants. x, Any amino acid.
2. Regulation by phosphorylation
A) OVERVIEW AND DATA ON PHOSPHORYLATION BY PROTEIN
KINASE A.
Phosphorylation of the PMCAs by a variety of
serine/threonine kinases, most notably by protein kinase
A (PKA) and protein kinase C (PKC), has been demonstrated for several isoforms and splice variants (reviewed
in Refs. 119, 129, 131). In addition, tyrosine phosphorylation has recently been shown to occur on the PMCA in
human platelets and to be responsible for downregulation
of PMCA activity (36). In all cases, phosphorylation is
thought to influence the regulation of the pumps either
directly (e.g., by deinhibiting the PMCA) or indirectly
(e.g., via interference with calmodulin regulation). Because the most striking differences among isoforms and
splice variants reside in their COOH-terminal tails, the tail
sequences are likely the relevant targets of differential
phosphorylation. Indeed, the entire COOH-terminal sequence of the PMCAs following splice site C is very rich in
Ser and Thr residues (see Fig. 6 and Ref. 129), but the
distribution and absolute position of many of these residues vary greatly between isoforms and splice variants.
PKA, for example, has been shown to phosphorylate Ser1178 in hPMCA1b within the sequence context KRNSS
(91). A comparable (but not identical) substrate sequence
(KQNSS) is found in only one other PMCA isoform
(PMCA2b), suggesting that PKA phosphorylation may be
specific for, and confined to, a certain subset of the PMCAs. Stimulation of PKA in heart sarcolemma has been
shown to activate the PMCA (32, 41, 123), and PKAmediated phosphorylation of Ser-1178 of PMCA1b in vitro
leads to an increase in the pump’s Ca2⫹ affinity and Vmax
(91). Because only some of the PMCA isoforms/splice
variants appear to be targets of PKA phosphorylation,
great variability exists in the potential for regulation of
Ca2⫹ extrusion by cAMP-dependent protein kinase, depending on the pattern of PMCA isoform expression in the
tissue/cell type of interest. It should also be noted that
induction of PKA activity need not lead to obligatory
activation of the PMCA. Most tissues express PMCA1b,
yet PKA stimulation does not activate pump-mediated
Ca2⫹ extrusion in all of them. Additional regulatory mechanisms likely exist that determine whether the PMCA will
be phosphorylated by PKA; these may include the regulated targeting of the kinase to the pump via anchoring
proteins (see Ref. 39 for a review).
B) PKC. Regulation of the PMCA by PKC has been
reported in a variety of cell types (see Refs. 119, 129 for
recent reviews). In general, stimulation of PKC leads to
increased Ca2⫹ extrusion by the PMCA. As in the case of
PKA, residues in the COOH-terminal tail region of the
PMCAs have been shown to be targets of direct phosphorylation by PKC (51, 174). Although an early in vitro study
of the purified erythrocyte PMCA identified the conserved
threonine within the calmodulin binding domain (just
before the splice site) as a major phosphorylated residue
(174), later studies showed that residues in the downstream region are more readily phosphorylated by PKC
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stimulated by, calmodulin (60). In the context of the
resemblance of the PMCA calmodulin binding sequence
to the IQ motif, the PMCA3f sequence would be expected
to be a much poorer calmodulin binding domain than the
b splice sequences (see Fig. 7). Because the expression of
rPMCA3f is essentially confined to skeletal muscle (22, 60,
95), it is tempting to speculate that its calmodulin-independent “constitutive” activity may be a physiological
adaptation to the unique Ca2⫹ fluxes occurring in skeletal
muscle.
January 2001
ISOFORMS OF THE PLASMA MEMBRANE CALCIUM PUMP
phosphorylation by PKC did not affect calmodulin binding or Ca2⫹-calmodulin activation of PMCA4a (170).
In conclusion, the picture that emerges from these
studies indicates that the different isoforms and splice
variants are very differently regulated by PKC. PMCA2a,
-3a, -4a, and -4b are readily phosphorylated by PKC,
whereas PMCA2b and -3b are poor substrates for this
kinase. The basal activity of the a splice forms is not
affected by PKC phosphorylation, whereas PMCA4b gets
partially activated through the release of downstream
inhibition. The Ca2⫹-calmodulin affinity and activating
properties are not influenced by PKC in PMCA4a and -4b,
whereas they are severely compromised in PMCA2a and
-3a. Although data on the in vivo effects of PKC stimulation on individual PMCA isoforms and splice variants are
still lacking (due mainly to the complex pattern of expression of multiple isoforms in most cells and tissues), the
above studies argue convincingly for the differential regulation of these pumps by PKC.
3. Differential targeting and assembly into
multiprotein signaling complexes
A) THE PMCA B SPLICE VARIANTS BIND TO PDZ DOMAIN PROTEINS.
Recent information on a new role for alternative splicing
at site C has come from the realization that the last four
COOH-terminal residues of the b splice forms conform to
the minimal consensus sequence for binding to the PDZ
protein interaction domain (Fig. 8). With the use of yeast
two-hybrid interaction assays, biochemical analyses, and
coimmunoprecipitation experiments, hPMCA4b was
shown to interact via its COOH-terminal sequence with
the PDZ1⫹2 domains of several members of the PSD-95
family of membrane-associated guanylate kinases
(MAGUKs) (100). The PMCA4b COOH-terminal sequence
shows a perfect match to the E(T/S)XV* sequence that
had previously been found to be critical for the interaction of the Kv1.4 potassium channel and the NR2 subunit
of the N-methyl-D-aspartate receptor with the PDZ domains of MAGUKs (42, 101, 102, 146). In contrast, the
PMCA4a splice variant, which contains a different COOH
terminus (Fig. 6), is unable to interact with the PDZ
domain (100). The COOH-terminal sequence of the other
PMCA b splice variants (ETSL*) also fits the broad consensus sequence for class I PDZ ligands (146 and see Fig.
8). Accordingly, a recent yeast two-hybrid screen using
the COOH-terminal tail of PMCA2b as bait identified a
number of PDZ domain containing proteins as potential
binding partners of this pump (161). Among these are the
known MAGUK SAP97/hDlg (114, 121), ␤2-syntrophin
(68), and a protein called NHERF for Na⫹/H⫹ exchanger
regulatory factor (176). Subsequent studies in our laboratory have shown that both PMCA2b and -4b interact with
high affinity with several members of the PSD-95 family of
MAGUKs (such as SAP90/PSD95, SAP97/hDlg, and
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(51). Indeed, differential effects of PKC on PMCA activity
could not be explained easily if the conserved threonine
residue were the major site of phosphorylation. Using the
COS cell expression system and different COOH-terminal
truncation mutants of PMCA4b, Enyedi et al. (51) found
that phosphorylation by PKC affected a region of ⬃20
residues located downstream of the calmodulin binding
domain. This region overlapped with the downstream
portion of the autoinhibitory domain of the pump. Phosphorylation by PKC partially stimulated the PMCA by
releasing the inhibition conferred by that region; however, full activation of the pump was only achieved when
the inhibitory effect of the calmodulin binding domain
was also relieved. Thus, in PMCA4b studied in a native
membrane environment, PKC phosphorylation does not
interfere with Ca2⫹-calmodulin binding, and its positive
effect on pump activity appears to be confined to releasing a part of the autoinhibition. These findings are in
agreement with in vivo data demonstrating that PKC stimulation only partially activates the pump and that the
effects of PKC and Ca2⫹-calmodulin are additive (145). In
contrast, phosphorylation of the conserved Thr residue in
the calmodulin binding domain interferes with calmodulin binding and is expected to substantially activate the
pump in a calmodulin-independent way (83).
In accordance with the considerable COOH-terminal
sequence variation among isoforms and splice variants,
recent studies have shown that PKC affects the different
PMCAs in very different ways. PKC was found to readily
phosphorylate PMCA2a and -3a in microsomal membranes, whereas PMCA2b and -3b were very poor substrates for the kinase (48). Contrary to the results with
PMCA4b, PKC-mediated phosphorylation of PMCA2a and
-3a did not change their basal activity; rather, it inhibited
calmodulin binding and thus prevented Ca2⫹-calmodulin
stimulation of pump activity. On the other hand, prior
incubation with Ca2⫹-calmodulin inhibited subsequent
phosphorylation by PKC. Taken together, these data show
that PMCA2a and -3a are phosphorylated by PKC in a
downstream region that overlaps with the extended calmodulin binding domain in these variants. The finding by
Enyedi et al. (48) that PMCA2b and -3b were not significantly phosphorylated by PKC provides further evidence
that the conserved Thr residue present in all PMCA isoforms is not a likely site for PKC phosphorylation in vivo.
Whereas the precise residues phosphorylated by PKC in
PMCA2a, -3a, and -4b are not yet known, a recent study
identified Ser-1115 in the downstream part of the bipartite
calmodulin binding domain of PMCA4a as the likely site
of PKC phosphorylation (170). As was the case for
PMCA2a and -3a, PKC phosphorylation of PMCA4a did
not activate the pump’s basal activity, and prior incubation with Ca2⫹-calmodulin inhibited phosphorylation. In
contrast to the results with PMCA2a and -3a, however,
43
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EMANUEL E. STREHLER AND DAVID A. ZACHARIAS
Volume 81
SPECIFIC MEMBRANE DOMAINS MAY BE MEDIATED BY PDZ PROTEIN
INTERACTIONS.
FIG. 8. The COOH-terminal sequences of the PMCA “a” and “b”
splice variants feature consensus motifs suggesting their involvement in
protein-protein interactions. Top: alignment of the COOH-terminal sequences of the known b splice variants from different mammalian
species. For each PMCA, the human sequence is shown in the first line,
and residues identical in the other species are indicated by a dash. The
COOH-terminal residues are marked by asterisks. The derived PMCA “b
splice” consensus sequence is also shown and compared with the consensus sequence of ligands for the class I PDZ domain. Lowercase letters
in the PMCA consensus sequence indicate residues less frequently found
at a given position. ␾ stands for a hydrophobic amino acid, and X stands
for any residue. Bottom: alignment of the COOH-terminal sequences of
the known a splice variants, and comparison of the PMCA “a splice”
consensus sequence to the COOH-terminal sequence of an alternatively
spliced isoform of G␣i-2, which localizes to the Golgi apparatus (120). All
symbols are as for the alignment on the top.
All the PDZ proteins mentioned above as
potential binding partners of the PMCA b splice variants
share the property of linking membrane channels, receptors, and transporters to the underlying membrane cytoskeleton. MAGUKs are thought to be involved in clustering and anchoring channels and receptors at sites of
membrane specialization such as the postsynaptic density
(see Refs. 57, 103, 130, 137, 143 for reviews). In addition,
because most MAGUKs contain multiple PDZ domains as
well as other protein interaction modules (e.g., SH3 and
guanylate kinase-like domains), they serve as scaffolds
for the assembly of multiprotein complexes linking membrane proteins to diverse signaling pathways (122, 130,
165). This may include the recruitment of specific kinases
(PKA, PKC) to a particular target protein such as a Ca2⫹
channel, N-methyl-D-aspartate receptor, or PMCA. Specific PMCA-PDZ protein interactions may be responsible
for the known concentration of the pump at subcellular
sites such as in caveolae (63), basolateral membranes of
Ca2⫹-transporting epithelia (12, 13), synaptic spines of
cerebellar Purkinje cells (82, 152), or the stereociliary tips
of auditory hair cells (6, 182). In addition, clustering of
PMCAs via PDZ domain containing scaffolding proteins
could lead to high local concentrations of the pump and
thus allow the formation of pump dimers. As mentioned
in section II, dimerization (oligomerization) of the PMCA
via its calmodulin binding COOH-terminal region has previously been shown to be a mechanism for calmodulinindependent pump activation (104, see Ref. 128 for review).
C) DIFFERENTIAL TARGETING OF COOH-TERMINAL SPLICE FORMS
OF THE PMCA VIA DIFFERENTIAL PROTEIN-PROTEIN INTERACTIONS?
These recent findings indicate a new role for alternative
splicing at site C of the PMCA: differential localization of
pump variants to sites of membrane specialization where
they may participate in local Ca2⫹ signaling as part of a
multifunctional protein complex. The interaction with
PDZ domains is apparently limited to the b splice forms;
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PSD93/Chapsyn-110), whereas the interaction with syntrophin is much weaker (S. J. DeMarco and E. E. Strehler,
unpublished data). Although all b splice variants of the
PMCA are predicted to be able to interact with class I PDZ
domains, there are clearly variant-specific differences in
the preferred PDZ domain proteins. For example, the
interaction of hPMCA2b with NHERF appears to be much
more robust than that of hPMCA4b, suggesting that residues upstream of the COOH-terminal consensus PDZ
binding sequence are involved in conferring selectivity to
the interaction. Thus, although there will almost certainly
be some promiscuity among the PMCA b splice variants in
their PDZ protein partners, a search for specific partners
may yield interesting candidates with the requisite binding selectivity.
B) CLUSTERING/RETENTION OF PMCA B SPLICE VARIANTS IN
January 2001
ISOFORMS OF THE PLASMA MEMBRANE CALCIUM PUMP
IX. CONCLUSIONS AND
FUTURE PERSPECTIVES
Four nonallelic PMCA genes are expressed in rats
and humans and, presumably, in all other mammals.
When rat and human protein sequences are compared, the
corresponding isoforms are ⬃99% identical. A sequence
comparison between protein isoforms within a species
shows only 75– 85% identity and 85–90% similarity (157).
The existence of four PMCA isoforms performing essentially the same function (i.e., expulsion of Ca2⫹ from the
cell against a large concentration gradient) might suggest
that they are largely redundant. However, given the high
degree of corresponding isoform homology between species and the lower degree of similarity between isoforms
within a species, there appears to be selective pressure to
maintain all four isoforms in their present form. This
indicates that the differences between the isoforms are
significant and that all four are somehow necessary for
the success of the organism. Moreover, the amino acid
sequences of the four PMCAs include regions of variable
or lower homology. Regions that are conserved between
the isoforms almost certainly represent domains that are
essential to the basic structure as well as catalytic and
transport functions of the pumps. The regions of higher
diversity are likely to reflect isoform-specific regulatory
and functional specializations that allow each pump to
fulfill a unique role in the specific cell or tissue in which
it is expressed. This is particularly obvious for the differences resulting from alternative splicing where otherwise
identical PMCA variants are created that differ only in a
small, precisely defined region of their primary sequence.
The differences among splice variants include differential
protein-protein interactions with scaffolding and/or regulatory proteins (e.g., via differential binding to PDZ motifs), differential binding to and regulation by Ca2⫹-calmodulin, as well as differential regulation by kinases. In
recent years, several other examples have been reported
where alternative splicing profoundly alters a protein’s
spatial distribution and/or regulation by differential protein interaction: localization of NMDA receptor subunits
NR1A and NR1D to the postsynaptic density (45), targeting of multifunctional Ca2⫹/calmodulin kinase to the nucleus (148), interaction between neuroligin-I and ␤-neurexins (89), or the functional identity (P type vs. Q type) of
voltage-gated Ca2⫹ channels (14). Thus it has become
clear that inclusion/exclusion or exchange of a protein
module or domain as a result of alternative RNA splicing
can have profound functional consequences for the encoded protein variants, a situation reflected in an exemplary manner in the PMCA family.
It is still possible that the function and specific membrane localization of some of the PMCA isoforms and
splice variants overlap enough that the malfunction or
absence of one isoform may be compensated for, at least
in part, by another expressed in the same cell. One potentially effective way to address the issue of redundancy
is to create mice carrying a null mutation for one or more
of the PMCA isoforms or splice variants. This strategy has
already been successfully used for the recently reported
PMCA2 knockout mice (106). These mice survive into
adulthood but are deaf and severely impaired in maintaining their balance. Interestingly, mice of the spontaneous
“deafwaddler” and “wriggle mouse Sagami” mutant
strains carry specific mutations in the PMCA2 gene that
either abort the production of the protein or impair its
function due to nonconserved amino acid substitutions.
As their name suggests, these mice are deaf and display
abnormal movements (155, 164). These data thus strongly
argue for a specific role of PMCA2 in particular cells and
tissues and against the general redundancy of the PMCAs
(recently reviewed by Garcia and Strehler, Ref. 66). In the
near future, similar studies on the other PMCAs are expected to shed light on their respective roles in tissue and
cell function. However, the functional significance of a
given PMCA isoform or splice variant in a particular tissue
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the a forms end in a completely different COOH-terminal
sequence that does not fit any known PDZ binding consensus. We note, however, that there is significant similarity among the COOH-terminal sequences of the a forms
and that their last four residues conform to a consensus
motif ES(V/I)(P/S)* (Fig. 8). Of interest, the COOH-terminal sequence DSVP*, which closely matches this consensus motif, has previously been identified in an alternatively spliced G␣i-2 ␣-subunit of heterotrimeric G proteins
(120). In this G protein, alternative splicing analogous to
that at site C of the PMCAs results in ␣i-2 subunits with
completely different COOH-terminal tails. As a consequence, the splice forms show different subcellular localization. Specifically, the variant with the DSVP* COOH
terminus has been shown to be targeted to the Golgi
apparatus, where it may interact with compartment-specific receptors (120). Based on these data and on similar
studies demonstrating that alternative splicing frequently
affects protein interaction domains, we predict that the
COOH-terminal tails of the PMCA a splice forms also
interact with specific target proteins. As in the case of the
b splice forms, these interactions may allow the specific
targeting of the a variants to subcellular membrane compartments. It will be interesting to see if these include the
Golgi or some other vesicular compartment. Retention of
specific PMCA splice variants in nonplasma membrane
compartments via novel protein interactions may also
explain the observation of strong PMCA immunoreactivity in cholinergic synaptic vesicles (62). Of note, the a
splice forms of the PMCA are most abundant in excitable
cells and appear relatively late in neuronal development
(16).
45
46
EMANUEL E. STREHLER AND DAVID A. ZACHARIAS
We thank the members of the Strehler lab, both past and
present, for many fruitful discussions and experimental contributions. Special thanks go to Billie Jo Brown, Steven J. DeMarco, and Michael S. Rogers for their continuous experimental
and intellectual input. Thanks are also due to Dr. John T. Penniston and members of his group, especially Dr. Ágnes Enyedi,
Adelaida G. Filoteo, and Dr. Anil K. Verma, for stimulating
discussions and long-term collaborations.
This work was supported by National Institute of General
Medical Sciences Grant GM-58710 (to E. E. Strehler) and the
Mayo Foundation for Medical Education and Research.
Address for reprint requests and other correspondence:
E. E. Strehler, Dept. of Biochemistry and Molecular Biology,
Mayo Clinic/Foundation, 200 First Street SW, Rochester, MN
55905 (E-mail: [email protected]).
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