Physiol Rev 89: 1341–1378, 2009;
doi:10.1152/physrev.00032.2008.
Calcium Pumps in Health and Disease
MARISA BRINI AND ERNESTO CARAFOLI
Departments of Biochemistry and Experimental Veterinary Sciences, University of Padova, and Venetian
Institute of Molecular Medicine, Padova, Italy
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Brini M, Carafoli E. Calcium Pumps in Health and Disease. Physiol Rev 89: 1341–1378, 2009;
doi:10.1152/physrev.00032.2008.—Ca2⫹-ATPases (pumps) are key actors in the regulation of Ca2⫹ in eukaryotic cells
and are thus essential to the correct functioning of the cell machinery. They have high affinity for Ca2⫹ and can
efficiently regulate it down to very low concentration levels. Two of the pumps have been known for decades (the
SERCA and PMCA pumps); one (the SPCA pump) has only become known recently. Each pump is the product of
a multigene family, the number of isoforms being further increased by alternative splicing of the primary transcripts.
The three pumps share the basic features of the catalytic mechanism but differ in a number of properties related to
tissue distribution, regulation, and role in the cellular homeostasis of Ca2⫹. The molecular understanding of the
function of the pumps has received great impetus from the solution of the three-dimensional structure of one of
them, the SERCA pump. These spectacular advances in the structure and molecular mechanism of the pumps have
been accompanied by the emergence and rapid expansion of the topic of pump malfunction, which has paralleled
the rapid expansion of knowledge in the topic of Ca2⫹-signaling dysfunction. Most of the pump defects described so
far are genetic: when they are very severe, they produce gross and global disturbances of Ca2⫹ homeostasis that are
incompatible with cell life. However, pump defects may also be of a type that produce subtler, often tissue-specific
disturbances that affect individual components of the Ca2⫹-controlling and/or processing machinery. They do not
bring cells to immediate death but seriously compromise their normal functioning.
I. INTRODUCTION TO Ca2ⴙ SIGNALING
In the course of evolution, a number of agents have
emerged as carriers of signals that are essential for the
correct functioning of cell life. Ca2⫹ is the most versatile:
other messengers are normally committed to the regulation of a single cell function, or at most a few of them.
Ca2⫹ instead regulates a very large list of essential functions, beginning with the origin of new cell life at fertilization and ending with its termination in the process of
programmed cell death. Between these two events, Ca2⫹
transmits signals to functions as fundamental as gene
transcription, muscle contraction (and motility in general), secretion (including that of neurotransmitters), and
generation of fuels in various metabolic pathways (39).
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The involvement of Ca2⫹ in so many fundamental cell
processes naturally demands its efficient and precise control. The task is performed by specialized proteins that
bind Ca2⫹ with great specificity and affinity in the intracellular ambient, in which in most cases free Ca2⫹ oscillates around 100 nM. These proteins belong to two broad
classes: those that are intrinsic to membranes and move
Ca2⫹ across them, thus acting solely as Ca2⫹ buffers, and
those that are not membrane-bound but, in addition to buffering Ca2⫹, also decode the message it carries for the benefit
of protein (enzyme) targets. These proteins are thus also
appropriately called Ca2⫹ sensors. The discovery of Ca2⫹
sensor proteins can be traced back to the pioneering work
by Ebashi et al. (79) on troponin, whose C-subunit is the
Ca2⫹ receptor in the muscle molecule. Troponin C is the
0031-9333/09 $18.00 Copyright © 2009 the American Physiological Society
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I. Introduction to Ca2⫹ Signaling
II. Some Special Properties of the Ca2⫹ Signal
A. Autoregulation
B. Ambivalence
III. Ca2⫹ Pumps: General Introductory Concepts
A. The SERCA pump
B. The SPCA pump
C. The PMCA pump
IV. Concluding Remarks
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metric topography and operation of the respiratory chain
components (157).
The generation, processing, and control of the Ca2⫹
signal now define a large area of cell biochemistry and
physiology. This review will not discuss it in detail, as its
emphasis will primarily be on Ca2⫹ pumps and the role of
their dysfunctions in the disturbance of the Ca2⫹ signal. In
addition to those quoted in the paragraphs above, a number of other reviews deal in appropriate detail with other
important aspects of the Ca2⫹ signaling field: for instance,
the matter of the Ca2⫹ microdomains in cells (251) and of
the generation of polarized Ca2⫹ signals (238). They could
be consulted to complement the information offered in
this succinct preamble (19, 39, 43). In the next section,
this contribution will only discuss in some detail the
aspects of the Ca2⫹ signaling area that are especially
relevant to its focus.
II. SOME SPECIAL PROPERTIES OF THE
Ca2ⴙ SIGNAL
A. Autoregulation
In addition to versatility, the Ca2⫹ signal has a number of other distinctive properties. Some have been comprehensively discussed elsewhere (40); thus it will only be
necessary to discuss here the two that are especially
relevant to the topic of this article. One is the autoregulatory nature of the signal, which occurs both at the
transcriptional and posttranscriptional levels: autoregulation
means that the expression and the activity of a number of
proteins that control Ca2⫹ in cells are regulated by the
Ca2⫹ signal itself. That Ca2⫹ regulates the transcription of
a number of genes has long been known (259). Both early
and late response genes may be controlled by Ca2⫹: a
significant portion of the information now available on
the topic has been generated by the work on CREB, a
transcription factor of the bZIP family that binds to the
cAMP responsive element (CRE) and to the Ca2⫹ responsive element (CARE) on DNA and becomes phosphorylated and activated by calmodulin kinases. The Ca2⫹dependent phosphatase calcineurin then regulates the
phosphorylation state of CREB in a complex process also
involving other protein phosphatases. Other aspects of
the regulation of genes by Ca2⫹ are of general interest, but
in the context of this review, it will only be necessary to
discuss briefly the regulation of genes involved in the
control of Ca2⫹. This autoregulation has been studied
most extensively in neurons. A series of studies on cultured cerebellar granular cells have shown that the longterm survival of the cells depends on a modest increase of
cytosolic Ca2⫹, which in the cultures is achieved by the
partial opening of L-type Ca2⫹ channels (42). Even if
modest, the increase nevertheless demands a dramatic
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progenitor of the family of proteins that have later been
defined as EF-hand proteins. The family has now grown to
contain over 600 members (208).
The fine details of the process by which Ca2⫹ sensors
decode the Ca2⫹ signal have so far only been clarified at
the atomic level for very few proteins, among which
calmodulin is probably the most important. Essentially on
the basis of the work on calmodulin, and on a few other
Ca2⫹ sensor proteins, it is generally assumed that the
essence of the signal-decoding process is the conformational change of the sensor protein upon binding Ca2⫹ and
upon contacting the target enzymes.
The processing of the Ca2⫹ message by specialized
proteins indicates that, as a rule, Ca2⫹ does not transduce
signals per se: the intermediation of a sensor protein that
binds it and then contacts the targets of the message
appears essential. Even if this is indeed so in most cases,
a minority of proteins, enzymes or otherwise, may bind
Ca2⫹ to sites in their own structure and decode its signal
directly, without the intermediation of external sensor
proteins: as an important example, one could quote protein kinase C (216), which is thus for all purposes a bona
fide Ca2⫹ sensor enzyme itself. In still other cases, Ca2⫹regulated enzymes contain not only the calmodulin binding domains that accept the Ca2⫹ message after it has
been processed by calmodulin, but also a separate calmodulin-like subunit. This is, for instance, the case of calcineurin (6), an enzyme which is thus under dual Ca2⫹
regulation. An even more complex case is that of calpain,
which contains a covalently bound calmodulin-like structure that binds Ca2⫹ within the catalytic subunit, but also
a separate calmodulin-like, smaller subunit (20).
The decoding of the Ca2⫹ message demands that its
spatiotemporal distribution in the environment of the sensor proteins be very carefully modulated. This is made
possible by the operation of membrane-intrinsic Ca2⫹protein transporters and channels that move Ca2⫹ back
and forth across membrane boundaries, satisfying the
demands of the decoding proteins and, in turn, of the
targets of the Ca2⫹-signaling function. These membrane
intrinsic proteins belong to several subclasses that vary in
transport mechanism, affinity for Ca2⫹, and total transport capacity. The ATPases are high-affinity, low-capacity
systems; the exchangers (mostly Na⫹/Ca2⫹ exchangers)
have opposite properties, whereas the channels are proteinaceous structures that let Ca2⫹ flow passively across
membranes when a number of gating mechanisms induce
their opening (43). Voltage-gated (47) and ligand-gated
(281) channels have been known and studied for a long
time. Trp channels (309) and store-operated channels
(234) have joined the field later, but recent important
advances have raised interest in them. Lastly, an electrophoretic uniporter mediates the charge-uncompensated
entry of Ca2⫹ into mitochondria in response to the negative-inside membrane potential generated by the asym-
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Autoregulation of the Ca2⫹ signal also occurs at the
posttranscriptional level, the best known case being perhaps that of the PMCAs. They are all activated by Ca2⫹calmodulin (141), which interacts with a specific domain
in their COOH-terminal cytosolic tail (see below). A calmodulin binding domain has also been identified in the
main cytosolic loop of the NCXs (183), but no convincing
evidence has so far been provided that the regulation of
the exchangers by Ca2⫹-calmodulin occurs in the ambient
of the cell. Naturally, the autoregulation of the Ca2⫹ signal
by the interaction of calmodulin with the PMCAs (and,
possibly, with the NCXs) is reversible and is expected to
occur in response to the varying physiological demands of
cells. However, irreversible autoregulation, e.g., inhibition
under pathological conditions of Ca2⫹ overload, could
also occur, creating a vicious loop that augments the Ca2⫹
overload itself. This is, for instance, the case for NCX3,
which is cleaved and inactivated by calpain, which becomes activated under conditions of neuronal excitotoxicity that increase the influx of Ca2⫹ (15, 38). Calpain,
however, also cleaves COOH-terminally, and activates,
the PMCAs. The process has been extensively studied in
vitro but could occur in conditions of spontaneous pathology as well (258).
B. Ambivalence
Having chosen Ca2⫹ as a determinant for function,
cells benefit greatly from its practically unlimited availability in the external spaces. However, the choice forces
them to live in a perpetual situation of controlled risk,
that exposes them to the danger that amounts of Ca2⫹
much in excess of those they could control will penetrate
from the external spaces and persist in the cytosol.
Increases of the basal free concentration of Ca2⫹ in
the cytosol at rest significantly exceeding the optimal
concentration of 100 –200 nM could only be tolerated for
a short time. Transient increases of Ca2⫹ above (even
much above) the resting 100 –200 nM level may be necessary in some physiological circumstances and in selected
cell microdomains, but in these cases, the delivery of the
Ca2⫹ message occurs in the form of rapid repetitive transients. The oscillations are a convenient device to satisfy
the demands of cell functions that may require a brief
increase of Ca2⫹ concentration much above the normal
level. But sustained increases to such levels would lead to
the permanent activation of detrimental enzymes like proteases, phospholipases, and nucleases, and would lead to
various degrees of cell discomfort, up to death in extreme
cases. The negative outcome of the protracted global
increase of Ca2⫹ much above the normal level illustrates
very clearly the ambivalent nature of the Ca2⫹ signal,
which had already been recognized in early Ca2⫹ research: a number of now classical contributions (see Ref.
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rearrangement of the pattern of expression of a number of
plasma membrane Ca2⫹ transporters and of intracellular
Ca2⫹ channels. Under the culturing conditions that prolong the survival of the granules, two of the four basic
isoforms of the plasma membrane Ca2⫹ pump (PMCA, see
below) become slowly upregulated, whereas another
PMCA isoform (PMCA4) disappears instead rapidly. The
expression of still another PMCA isoform (PMCA1) remains quantitatively constant, but an alternative splicing
switch occurs that privileges a COOH-terminally truncated, probably less active variant. The rearrangement of
the pattern of expression also affects the plasma membrane Na⫹/Ca2⫹ exchanger (NCX): one isoform (NCX3) is
slowly upregulated, while another (NCX2) disappears
very rapidly. The intracellular inositol 1,4,5-trisphosphate
(InsP3)-gated Ca2⫹ channel also becomes slowly upregulated. The autoregulatory character of this complex transcriptional rearrangement is further emphasized by the
finding that the downregulation of PMCA4 and NCX2, as
well as the upregulation of the InsP3 receptor, as was the
case for CREB, are mediated by calcineurin, the protein
phosphatase which is itself the target of dual regulation
by Ca2⫹ (see above). At a first glance, it may seem surprising that such a modest increase of the set-point of
cytosolic Ca2⫹ should demand a complete reprogramming of the expression of Ca2⫹ transporters. But the
finding underlines the importance of controlling Ca2⫹
with utmost precision: to promote the survival of the
granule cells and, possibly, to fulfill the spatial and temporal Ca2⫹ signaling requirements of mature, as opposed
to immature cells, Ca2⫹ should evidently increase, but
only to a new modestly elevated set point, and no more.
To reach this goal, for reasons which we still do not fully
understand, several membrane transporters, not just one,
must be reprogrammed. Ca2⫹ can also influence gene
transcription without the intermediation of protein kinases and phosphatases, i.e., by interacting directly with
transcription factors. A very interesting example of this
type of transcriptional regulation is that mediated by the
downstream regulatory element antagonistic modulator
(DREAM), a Ca2⫹-binding protein that acts as a gene
repressor (45). In the Ca2⫹-free form, it represses transcription by binding to specific DNA sites in the promoters of several genes. The binding of Ca2⫹ removes
DREAM from the sites, restoring transcription. From the
autoregulation angle, it is interesting that one of the genes
controlled by DREAM in this way is that for NCX3, a
plasma membrane Na/Ca exchanger isoform that is particularly important to Ca2⫹ homeostasis in neurons (103).
This is not the only example of DREAM-mediated autoregulation of the Ca2⫹ signal, as another Ca2⫹-related
gene controlled by DREAM is that for the L-type Ca2⫹
channel, which is the most important entry path for Ca2⫹
in heart and other excitable cells (71).
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This review discusses in detail the conditions that originate from dysfunctions of Ca2⫹ pumps.
III. Ca2ⴙ PUMPS: GENERAL INTRODUCTORY
CONCEPTS
Three Ca2⫹-ATPases (pumps) have been described in
the cells of higher animals. They are located in the membranes of endo(sarco)plasmic reticulum, including the
nuclear envelope, which is an extension of the latter (the
SERCA pump), in those of the Golgi network (the SPCA
pump), and in the plasma membrane (the PMCA pump).
However, the Golgi membranes also contain the SERCA
pump. Interestingly, the SPCA of the Golgi membranes
also transports Mn2⫹, which is a cofactor of a number of
enzymes in the lumen of the Golgi compartment. Plant
cells, and cells of lower eukaryotes, e.g., yeasts, contain
additional Ca2⫹ pumps. They have interesting properties,
but are not discussed in this review, as its focus is on the
disease processes linked to the dysfunction of mammalian pumps.
All three animal Ca2⫹ pumps belong to the family of
P-type ATPases, which is characterized by the temporary
conservation of ATP energy in the form of a phosphorylated enzyme intermediate (hence P-type) (235) formed
between the ␥-phosphate of hydrolyzed ATP and an invariant D-residue in a highly conserved sequence in P-type
ATPases: SDKTGT[L/I/V/M][T/I/S]. The P-type ATPases have
now grown in number to form a large superfamily that
contains at least eight subfamilies, and hundreds of members. A large number of sequences are now available: they
have a low (⬃15%) degree of general identity to each
other. However, eight conserved regions that define the
core of the superfamily have been identified (14). Subfamilies have been identified based essentially on substrate
specificity, the appearance of which in evolution has been
accompanied by abrupt changes in sequence. Five
branches have been identified in the phylogenetic tree:
the Ca2⫹-ATPases belong to subgroups IIA (the SERCA
and the SPCA ATPases) and IIB (the PMCA ATPase)
(Fig. 1). Naturally, the three Ca2⫹ pumps share the essential basic properties, including membrane topology and
reaction mechanism, and probably also the general features of the three-dimensional (3D) structure. This, however, is only a plausible prediction, as only one 3D structure, that of the SERCA pump, has so far been solved
(296, 297). The three pumps, however, also display significant sequence differences, particularly in areas not directly related to the molecular mechanism of catalysis,
but related to regulation, action of some inhibitors, and
role in cellular processes.
A simplified reaction scheme, the basic lines of which
are valid for all three Ca2⫹ pumps, is shown in Figure 2.
Originally, the scheme envisaged two conformational
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90 for a detailed review of the topic) had indeed shown
cell death to be associated with abnormal increases of cell
Ca2⫹. The pioneering work on heart must specifically be
mentioned in this context (92), but the observations were
soon extended to other cell types (54, 211, 229).
The increase of cell Ca2⫹ much above the normal
resting level does not inevitably seal the fate of cells if it
only lasts for a limited time: a safety device helps cells to
temporarily cope with such situations. This is the Ca2⫹
uptake system of mitochondria, which has low Ca2⫹ affinity, and only becomes activated when the cytosolic
Ca2⫹ in their surroundings approaches the micromolar
range. Under normal conditions in most cells, the role of
the mitochondrial uptake system is that of regulating
three Ca2⫹-sensitive dehydrogenases in the matrix that
are essential for the operation of the citric acid cycle, and
thus for the synthesis of ATP. However, mitochondria can
store much larger amounts of Ca2⫹ in the matrix without
dangerously increasing its osmotic concentration. This is
so because they can accumulate inorganic phosphate
alongside with Ca2⫹, precipitating in the matrix deposits
of hydroxyapatite, thus making room for the uptake of
more Ca2⫹. Interestingly, the hydroxyapatite deposits in
the matrix do not crystallize but remain amorphous, thus
facilitating their dissolution and the release of Ca2⫹ from
mitochondria once the emergency that had caused the
cytosolic Ca2⫹ overload has disappeared. However, the
mitochondrial safety device only has temporary efficacy,
as mitochondria use the same energy (the membrane
potential across the inner membrane) to take up Ca2⫹ or
to synthesize ATP. As long as they are forced to continuously accumulate Ca2⫹, they will not synthesize ATP,
eventually depriving the Ca2⫹ pumps of the energy necessary to remove Ca2⫹ from the cytosol, thus triggering a
vicious loop that will exacerbate the Ca2⫹ overload (38).
Ca2⫹ catastrophe is the extreme case of the protected global Ca2⫹ overload. Naturally, the concept only
applies to the unwanted, toxic death of cells. It does not
apply to the programmed cell death, which is sometimes a
necessary outcome, for instance, in organ remodeling
and cell pruning. Programmed cell death is thus but one
of the meaningful ways to translate the Ca2⫹ signal.
However, apart from the unwanted (toxic) cell death,
the ambivalence of the Ca2⫹ signal is also illustrated in
less dramatic ways by conditions in which the disturbance of Ca2⫹ regulation is not massive, but subtler, as
it only destabilizes individual participants in the Ca2⫹controlling operation. Such conditions, which are
mostly genetic, are still compatible with cell life but
generate diverse paradigms of cell discomfort. They
may be caused by defects of Ca2⫹ sensor proteins, of
Ca2⫹ channels, or of the various Ca2⫹ transporters. A
number of reviews (38, 203, 252) and a recent book (41)
offer a general coverage of this rapidly expanding area.
CALCIUM PUMP DYSFUNCTIONS
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states of the pumps: the E1 state, in which the enzyme has
high Ca2⫹ affinity and interacts with Ca2⫹ at one side of
the membrane, and the E2 state, in which the lower Ca2⫹
affinity leads to the release of the ion at the opposite side
(60). More recent structural work on the SERCA pump
has increased the complexity of the conformational transitions that occur during the catalytic cycle. Upon binding
of Ca2⫹, a series of structural changes occur that involve
both the protruding cytoplasmic sector and the transmembrane domains, permitting the phosphorylation of
the catalytic D-residue by the ␥-phosphate of ATP. The
dissociation of Ca2⫹ from the enzyme follows the transition of the high Ca2⫹ affinity E1⬃P(Ca2⫹) enzyme to
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the lower affinity E2-P enzyme, the hydrolysis of which
then regenerates the Ca2⫹-free E2 ATPase, completing
the catalytic cycle. The three pumps are all inhibited
by the general inhibitors of P-type ATPases La3⫹ and
orthovanadate [VO3(OH)]2⫺. However, in the case of
La3⫹, differences exist: La3⫹ accelerates the dissipation
of the aspartyl phosphate in the SERCA and SPCA
pumps, but increases instead its steady-state concentration, i.e., it stabilizes it, in the PMCA pump. The
difference is experimentally useful, as it permits to
identify the PMCA pump by using 32P-gels in mixed
preparations contaminated by even large amounts of
the other two pumps.
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2⫹
FIG. 1. Phylogenetic tree of Ca -transporting ATPases. Protein sequences of P-type ATPases of different species were aligned using ClustalW
software, and a phylogenetic tree was generated using TreeView. The three different branches represent the three main Ca2⫹-ATPase: SERCAs and
SPCAs (type IIA) and PMCAs (type IIB).
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The solution of the 3D structure of the SERCA pump
has confirmed the existence and molecular nature of the
two Ca2⫹ binding sites in the transmembrane helices
which had been predicted by mutagenesis work (56, 57).
The two sites are formed by residues (many of them
acidic) in transmembrane domains 4, 5, 6, and 8 (see
below). However, the existence of two Ca2⫹ binding sites
is peculiar of the SERCA pump: the PMCA pump lacks an
essential acidic residue in transmembrane domain 5, and
thus only has one Ca2⫹ binding site that appears to correspond to site 2 in the SERCA1 pump. This explains the
difference in Ca2⫹/ATP transport stoichiometry, which is
2.0 in the SERCA pump, but only 1.0 in the PMCA pump.
The SPCA pump also lacks the essential acidic residue in
transmembrane domain 5, and thus only has Ca2⫹ (and
Mn2⫹, see below) binding site 2, and 1:1 stoichiometry
between hydrolyzed ATP and transported Ca2⫹ (or Mn2⫹).
A. The SERCA Pump
1. General properties
The first ATP-driven Ca2⫹ transport system, which
was later identified as the SERCA pump, was discovered
in 1961–1962 in a skeletal muscle fraction known at the
time as the Marsh relaxing factor. The fraction, which was
later shown to correspond to the vesicles of sarcoplasmic
reticulum (SR), accumulated Ca2⫹ at the expenses of the
hydrolysis of ATP (78, 123). The process was suggested to
involve an ATP-driven pump that was purified some years
later as a protein of ⬃110 kDa (193). Important features of
the transport process, including its reversibility, were
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FIG. 2. A simplified reaction scheme of the transport cycle of
Ca2⫹-ATPases. The pumps exist in two main conformational states: E1
binds Ca2⫹ with high affinity at the cytosolic site, and E2 has a low
affinity for Ca2⫹ and thus releases it to the opposite site of the membrane. The cycle has a number of other states that have been omitted for
simplicity, but that are discussed in the text. ATP phosphorylates a
highly conserved aspartic acid residue, allowing the translocation
of Ca2⫹.
established by the early studies: SR vesicles that had
accumulated Ca2⫹ mediated the synthesis of ATP when
incubated with ADP in the absence of Ca2⫹ (123). Initially,
the transport process was studied on the SR of skeletal
and cardiac muscles, but the work was later extended to
the endoplasmic reticulum (ER) of all cells. SR, however,
remained a preferred object of study, thanks to the abundance of the transporting pump in its membrane (up to
90% of the total protein), which facilitated its isolation
and purification.
The pump was soon recognized as the main system
for the control of cytoplasmic Ca2⫹ in muscle cells, where
its ability to remove Ca2⫹ from the cytosol induced relaxation. Studies in the early period showed that the pump
transported two Ca2⫹ per ATP hydrolyzed and that it
countertransported H⫹ in exchange for Ca2⫹. However,
fewer than four H⫹ were released to the cytosol per two
Ca2⫹ pumped, showing that the transport reaction was
partly electrogenic. This electrogenic mechanism could
thus be responsible for the alkalization of the luminal side
of the ER/SR (328). It was soon discovered that, as all
P-type pumps (see above), the SERCA pump was inhibited by La3⫹ and orthovanadate (284). Specific inhibitors
were soon also discovered: thapsigargin (isolated from
the roots of Thapsia garganica) (290), cyclopiazonic acid
(produced by Aspergillus and Penicillum) (272), and 2,5di(t-butyl)hydroquinone (227). Curcumin [diferuloylmethane, or 1,7-bis(4-hydroxy-3-methoxyphenol)-1,6-hepatadiene3,5-dione], a compound derived from the spice turmeric
which is presently attracting considerable attention for its
antitumoral action, also inhibits the pump, probably by
stabilizing the interaction between the nucleotide-binding
and phosphorylation domains, thus precluding ATP binding (22). However, its specificity is lower than that of the
other inhibitors, as it may also interfere with the intracellular Ca2⫹ release channels (77).
Cyclopiazonic acid (CPA) and 2,5-di(t-butyl)hydroquinone (tBHQ) have lower affinity for the pump than
thapsigargin (Kd in the low micromolar to high nanomolar
range). Although less is known about their mechanisms of
action than about that of La3⫹ and orthovanadate, a number of indications suggest that they probably compete
with ATP. Interestingly, recent studies on the structure of
the SERCA-CPA complex (205) have shown that CPA
occupies the Ca2⫹ access channel in the pump molecule.
While the inhibition by CPA and tBHQ is reversible, and
disappears following their removal, that by thapsigargin
(TG), is irreversible. TG is the most popular inhibitor of
the SERCA pump, and its mechanism of action is better
characterized than that of the other inhibitors. It binds
stoichiometrically to F256 in the M3 helix (327), locking
the pump in an irreversible inactive state (256). Its affinity
for the SERCA pump is very high: Kd values in the subnanomolar range have been measured (257). Thapsigargin
reacts preferentially with the Ca2⫹-free pump, a property
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which has been advantageous when solubilizing the pump
protein for structural work, as it prevents its denaturation. It has permitted to obtain the 3D structure of the
pump structure in the Ca2⫹-free (E2) state (297) and has
helped to clarify the conformational changes occurring
during the reaction cycle.
The cloning of the SERCA pump cDNAs (26, 34, 192,
194) has permitted to reach a number of conclusions on
the molecular mechanism of the pump. It has also led to
the proposal that the enzyme is organized in the membrane with 10 transmembrane helical domains (M1-M10)
and has allowed the manipulation of the pump by sitedirected mutagenesis. A number of predictions on the
structure and mechanism of the pump made in the early
studies have then been validated by the solution of its 3D
structure at the atomic level in 2000 (296). The structure
has permitted to decipher the details of the reaction cycle
that culminates with the transit of Ca2⫹ across the pump
protein. Its general aspects are briefly discussed here as
they are most likely paradigmatic for the molecular operation of the two other Ca2⫹ pumps. The structure has
shown that the single polypeptide chain of the pump folds
in four major domains (Fig. 3): a transmembrane (M)
domain, indeed composed of the 10 predicted transmembrane helices (M1-M10), and three cytosolic domains.
Two of them, the actuator domain (A) and the phosphorylation (P) domain, are connected to the M domain. The
nucleotide-binding (N) domain, instead, is connected to
the P domain.
During Ca2⫹ binding and translocation, a number of
large conformational changes switch the “compact” structure of the cytosolic portion of the pump to a more “open”
structure (Fig. 3) (294). Before translocation, Ca2⫹ becomes bound to two sites, (site I and site II) in the M
domain. As had been predicted, the binding sites, which
are located side by side closer to the cytoplasmic surface
of the membrane, exist in high- and low-affinity states.
The first allows access of Ca2⫹ from the cytosolic side (E1
state), and the second, in which the sites face the luminal
side of the membrane (E2 state), favors the release of
Ca2⫹. The binding of Ca2⫹ to the two sites occurs sequentially. Site I is located between M8 and M5, and site II
between M6 and M4. In both sites, Ca2⫹ coordinates seven
oxygens of residues in M4, M5, M6, and M8: side chain
oxygens of N768, E771 (M5), T799, D800 (M6), and E908
(M8) together with two water molecules form site I. Site
II is located slightly closer to the cytoplasmic surface
nearly on the M4 helix, which provides the two side-chain
oxygens of E309 and three carbonyl oxygens to the coordination of Ca2⫹. N796 and D800 (which contributes to
the formation of both sites) in M6 complete site II by
donating one side chain oxygen each. The molecular path
for Ca2⫹ across the pump protein has been mapped and is
shown in the cartoon of Figure 4: the first Ca2⫹ would
enter the binding cavity through gating residue Glu 309,
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FIG. 3. Three-dimensional structure of the SERCA pump showing
the open and closed conformation in the E1 2Ca2⫹ and E2 (thapsigargin)
states. The NH2 terminus is shown in blue and the COOH terminus in
red. The purple spheres (circled and numbered as I and II) represent
bound Ca2⫹. The three cytoplasmatic domains (A, N, and P), the ␣-helices in the A domain (A1-3), and those in the transmembrane domain
(M1-M10) are indicated. M1⬘ is an amphipathic part of the M1 helix. M2⬘
is the top part of M2 helix. Docked ATP and thapsigargin (TG), as
several key residues (some of them are mentioned in the text), are
shown. For details, see the text and Ref. 294. [Modified from Toyoshima
et al. (294).]
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MARISA BRINI AND ERNESTO CARAFOLI
positioning D800 to induce the rotation of M6 and, possibly, the straightening of the M5 helix. This would rearrange coordinating oxygens of site II to form a higher
affinity site (E23 E13 E1䡠2Ca2⫹ transition). At this point,
the side chain of E309 would cap site II Ca2⫹, mechanically transmitting the binding signal to the phosphorylation site ⬃50 Å away. The straightening of the M5 helix
would loosen up the compact configuration of the three
cytoplasmic domains, which prevents the delivery of the
ATP bound to the N domain to the catalytic D in the P
domain. The binding of Ca2⫹ also increases the tilting of
the N domain, separating the P and A domains, permitting
the delivery of ATP to the D315 phosphorylation site
Physiol Rev • VOL
(E1䡠2Ca2⫹3 E1P transition). The binding of ATP crosslinks the P and N domains, bending the former to make
contact with the A domain. The strain rotates the A domain, pulling up and bending the M1 helix, that closes the
cytoplasmic E309 gate occluding the two bound Ca2⫹.
The transfer of the ␥-phosphate of bound ATP to the
catalytic aspartate then dissociates bound ADP, opening
the N-P domain interface. The rotation of the A domain
rearranges helices M1-M6, opening the luminal gate for
the release of the occluded Ca2⫹ (E1P3 E2P transition).
H⫹ and water molecules stabilize the liberated Ca2⫹ binding sites, and at this point, a second rotation of the A
domain electrostatically locks it to the P domain, closing
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FIG. 4. A cartoon illustrating the conformational changes of the main domains of the SERCA pump during the reaction cycle based on the
three-dimensional structures by Toyoshima et al. (294). Full details of the transitions shown are given in the text. [Modified from Toyoshima et al.
(294).]
CALCIUM PUMP DYSFUNCTIONS
again the luminal gate (E2P3 E2䡠Pi transition). The A
domain contains the highly conserved TGES sequence:
the rotation of the A domain retracts the side chain of
E183 from the phosphorylation site, inducing a conformational change in the TGES loop that permits the entrance
of a water molecule in the phosphorylation site to hydrolyze the aspartylphosphate. The release of the phosphate
(and of Mg2⫹) then relaxes the P domain completing the
closing of the luminal gate.
2. Isoforms of the SERCA pump
FIG. 5. Generation of multiple SERCA isoforms by alternative splicing of the human ATP2A1-3 genes. Exons are represented by colored
boxes, introns by the black line. Thin box represents untranslated
pseudoexon. 5⬘D1, 5⬘D2, and 5⬘D3 indicate optional splice donor sites
for SERCA2a, SERCA2b, and SERCA2c transcripts, respectively. Sa-f
indicate the position of the different stop codons for the corresponding
protein isoforms. The size of the protein products is indicated on the left.
The boxes are not in scale. Details of the different splicings are described in the text.
Physiol Rev • VOL
The SERCA2a variant is selectively expressed in heart and
slow-twitch skeletal muscles, whereas the SERCA2b variant is expressed nearly ubiquitously and is thus considered the housekeeping isoform. SERCA3 is instead expressed in a limited number of nonmuscle cells. SERCA1
was the first pump to be studied, owing to the relative
ease with which it could be purified, and it was the first
member of the family to be cloned. Since it has been used
for the 3D work mentioned above, it is the best understood SERCA pump isoform. The SERCA1 gene contains
23 exons, and the two isoforms that have been described
result from the alternative splicing of exon 22 (42 bp), that
is removed in SERCA1b. In neonatal SERCA1b, a highly
charged sequence of eight amino acids replaces the E994
residue that is found in the adult form: the functional
meaning of this difference is still unknown. Two other
human transcripts have been recently described that originate from a splicing operation in the 5⬘ region. They
generate SERCA1T and SERCA1T⫹4, which are two truncated 46-kDa versions of the pump, understandably unable to transport Ca2⫹ (48). As mentioned, SERCA2 is
distributed widely, and this is probably why it has a larger
number of modulator factors. It is the oldest SERCA
pump, and its two isoforms are the product of four different mRNAs. One of them is translated into the
SERCA2a protein, while the other three, which only differ
in the 3⬘-untranslated region, encode SERCA2b. The human SERCA2 gene contains 22 exons. In SERCA2a, exon
21 is spliced out and the first part of exon 20 is joined to
exon 22. In SERCA2b, exon 20 is extended and contains
an alternative stop codon and three different noncoding
regions. Recently, the mRNA of a new SERCA2 isoform
(2c) has been detected in human monocytes and fetal
hearts (100). It results from the inclusion of a short coding
sequence (a new exon 21 or exon SERCA2c) containing
an in-frame stop codon. The SERCA2c protein could possibly have functional importance in (fetal) heart as its
expression predominates in the left ventricle (59); however, the variant has also been detected in skeletal muscles. The sequence of the three SERCA2 isoforms is identical for the first 993 amino acids. Then, four amino acids
in the COOH-terminal region of SERCA 2a are replaced
by 49 amino acids in the 2b variant and by 6 in the 2c protein. Interestingly, the extended COOH terminus of the
SERCA2b pump contains a highly hydrophobic segment
that has been proposed to form an additional (11th) transmembrane domain (16). The divergence in the COOHterminal portion of the three SERCA2 variants is responsible for functional differences (312). The expression of
SERCA2a and SERCA2b in model cells has shown that the
latter had higher affinity for Ca2⫹ but lower catalytic
turnover rate (68, 190, 310, 311). Both isoforms have the
same sensitivity towards phospholamban (191) (see below). SERCA2c has even lower Ca2⫹ affinity than the 2a
variant, possibly because it operates in an ambient of
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In mammals, three genes (human nomenclature
ATP2A1-3) encode three main SERCA proteins. Each of
the three transcripts undergoes tissue-dependent alternative splicing, increasing the number of pump variants (Fig. 5
offers a comprehensive view of all splicing processes).
The changes in the expression pattern of the variants
during development and tissue differentiation indicate
that each isoform is adapted to specific functions.
SERCA1a and SERCA1b variants are expressed in adult
and neonatal fast-twitch skeletal muscles, respectively.
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MARISA BRINI AND ERNESTO CARAFOLI
Physiol Rev • VOL
hydrophobic helical COOH-terminal portion intrinsic to
the SR membrane and a hydrophilic NH2-terminal portion
that protrudes into the cytosol (169): in its unphosphorylated form, it binds to the pump and inhibits it by reducing
its apparent Ca2⫹ affinity. The binding occurs to three
sites. One is the sequence K397DDKPV in the cytosolic N
domain of the pump, which interacts with the hydrophilic
portion of PLB (292). The sequence is conserved in the
SERCA1 and -2 proteins but not in SERCA3. Another site
of the pump is the L67 loop (9), which interacts with the
cytosolic portion of PLB and the M6 helix which interacts
with its transmembrane domain (10). A third site of interaction has been identified close to the M4 helix by crosslinking experiments between a PLB in which N30 had
been mutated to C, and C318 in the SERCA pump (144).
Phosphorylation of the hydrophilic portion of PLB by
protein kinase A (PKA) (S16) (and/or calmodulin-dependent kinase II, CaMKII on T17) is presumed to detach PLB
from the pump, increasing its affinity for cytosolic Ca2⫹
and reactivating Ca2⫹ uptake. However, it has also been
proposed that phosphorylated PLB would still remain
associated with the pump (209), possibly through the
interaction of its NH2-terminal domain with the site in the
N domain. The physiological importance of PLB regulation in muscle contractility is now well established and
has been repeatedly discussed in reviews (195). The generation of mice models with altered PLB expression levels
has indicated that the downregulation of PLB increases
the affinity of SERCA2a for Ca2⫹ and heart contractile
parameters, whereas its cardiac overexpression is associated with a significant impairment of heart function (146,
189) (see below).
Another small transmembrane protein, sarcolipin
(SLN), has recently received attention as a PLB homolog
that would regulate SERCA1 pump activity in skeletal
muscles (221). Recent observations have demonstrated
that SLN is coexpressed with SERCA2a and inhibits it.
The coexpression of SLN and PLB in model cells leads to
pump superinhibition. At variance with PLB, the physiological significance of the regulation of the SERCA pump
by SLN is still obscure.
The SERCA2 pump also plays a critical role in maintaining the Ca2⫹ concentrations in ER/SR. Changes in its
activity may thus affect a number of ER/SR functions,
including protein synthesis and maturation. In turn, a
number of Ca2⫹-binding luminal proteins are believed to
control the activity of the SERCA2 pump. Studies on
Xenopus laevis oocytes have demonstrated that the ER
chaperons calreticulin and calnexin regulate the activity
of the SERCA2 pump from the lumen, shaping the InsP3induced Ca2⫹ oscillations (36, 143). Calreticulin has been
suggested to functionally interact with SERCA2b but not
with SERCA2a (143). Unlike SERCA2a, SERCA2b contains an N-glycosylation site in its luminal COOH-terminal
portion. It was thus proposed that the functional differ-
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particularly high [Ca2⫹] concentration (59). The physiological meaning of the SERCA2a/2b diversity was recently
investigated (310) in transgenic mice in which the 2a
isoform was replaced by the 2b. The phenotype was characterized by cardiac malformations and by a 40% increase
in embryonic and neonatal mortality. The adult mice developed compensatory left ventricular hypertrophy with
impaired cardiac contraction and relaxation. Apparently,
then, the SERCA2a isoform is critical for the correct
development and function of the heart.
The SERCA3 pump is less well understood, and the
issue of its alternative splicing isoforms is rather complex, as documented by Figure 5. The numeration of the
exons in SERCA3 gene is also confusing, since, as in
SERCA2 gene, exons 8 and 9 are fused together, thus
generating one “double” exon. In humans, six possible
SERCA3 variants (a-f) have been described (23, 322). The
SERCA3 gene is organized in 22 exons, and the alternative
splicing of exons 20 and 21 generates the 6 variants,
which differ in the COOH-terminal portion. All transcripts
retain exon 22, but the a variant excludes exon 20 and 21,
the b-c variants retain a portion of or all exon 20 and
exclude exon 21, variants d-e retain a portion of or all
exon 20 and 21, and, finally, the f variant excludes exon
20. So far, only the a-c isoforms have been documented as
proteins.
All documented variants (a-c) have markedly lower
apparent affinity for Ca2⫹ than the other basic isoforms,
10-fold higher apparent affinity for vanadate, and do not
react with phospholamban (see below). The SERCA3
pump (see below) seems to be specialized for the control
of relaxation of vascular and tracheal smooth muscle. The
lower Ca2⫹ affinity suggests that the SERCA3 pump
would only become active when cytosolic Ca2⫹ reaches
abnormally high levels, e.g., after massive cell stimulation
with Ca2⫹-linked agonists. Interestingly, the SERCA3
pump is almost always coexpressed with the housekeeping SERCA2b, and no obviously pathological phenotype
follows the ablation of its gene (188).
If the structure-function relationships are best understood in the SERCA1 isoform, the regulation aspects are
instead best understood in the SERCA2 isoform. Therefore, in discussing modulatory protein partners for
SERCA pumps, most of the information necessarily concerns SERCA2 interactors. The best known interaction
with a regulatory protein is that with phospholamban
(PLB), which was described in the heart nearly 40 years
ago (13, 149, 156). The involvement of PLB in the regulation of the pump, and its site of interaction with it, which
was initially characterized by cross-linking experiments
(137), were later explored by site-directed mutagenesis
(293) and further defined through molecular modeling
approaches (135, 169, 233, 295). As mentioned, PLB interacts with SERCA1a, SERCA2a, and SERCA2b but not with
SERCA3. The tertiary structure of PLB shows that it has a
CALCIUM PUMP DYSFUNCTIONS
Physiol Rev • VOL
generation of the amyloid ␤ fragment. The finding that the
modulation of SERCA pump activity would alter amyloid
␤ production suggests a possible role of the SERCA pump
in the pathogenesis of Alzheimer’s disease.
3. Posttranslational modifications of the SERCA2
pump
Posttranslational modifications such as glycosylation, oxidation, and phosphorylation could also control
SERCA2’s pumping activity. The possible role of glycosylation in mediating the interaction of the SERCA pump
with calreticulin and calnexin, as well as oxidation effects, have been mentioned above. Both may be linked to
nitric oxide (NO), which can activate the pump in a
cGMP-independent manner, and thus potentiate the relaxation of vascular smooth muscle, and of cardiac and
skeletal muscles. The effect depends on the modification
of reactive thiol groups on the SERCA protein and appear
to require the presence of reactive oxygen species (ROS).
Accordingly, it is thus attenuated by the overexpression
of superoxide dismutase (SOD1). Indeed, the SERCA2
pump is remarkably sensitive to oxidative damage, and
even its expression is downregulated by the oxidative
stress (158).
A direct, CaMKII-dependent phosphorylation effect
has been described on the Vmax of the cardiac SERCA2
pump, but not on that of SERCA1, which lacks the CaMKII
consensus site RXXS/T (124). The kinase, however, only
phosphorylated a minor fraction of the pump molecules
(⬍20%), casting doubts on the physiological relevance of
the finding (124, 324). The doubts were strengthened by
other studies in which pump phosphorylation was not
detected or was not accompanied by stimulation (222).
4. The SERCA pumps in the disease process
Two human genetic diseases associated with mutations in the SERCA pump genes have been described so
far, but gene inactivation studies in mice have revealed
additional pathological phenotypes distinct from those of
the human diseases. The two human diseases (Brody’s
and Darier’s disease) will be described first. Other dysfunctions of the pump related to defects in its activity or
expression level, i.e., heart failure and cancer development, will then be discussed.
Brody’s disease is a rare recessive muscular condition characterized by impaired relaxation, painless cramps,
and stiffness following exercise (29). The clinical diagnosis is difficult, since the symptoms are rather heterogeneous and nonspecific. The activity of the SERCA1 pump
has been reported to be reduced in biopsies of the quadriceps muscle and in cultured muscle cells. In some cases,
decreased SERCA1 expression in fast-twitch muscles has
also been detected (18, 148). The disturbance of the
SERCA1 pump seems to be specific, since the fiber-type
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ences of the two isoforms could be related to the interaction of the lectin domain of calreticulin or of calnexin
with the glycosylated residue in SERCA2b. The matter is
interesting but controversial, as glycosylation of residue
N1036 has not been unambiguously demonstrated, whereas
glycosylation-independent protein-protein interaction of
SERCA2b may occur. In addition, the treatment with endoglycosidase H has failed to alter the electrophoretic
mobility of the SERCA2b protein, suggesting that it was
probably not glycosylated (253).
The ER oxidoreductase Erp57, which participates in
the quality control and the folding of newly synthesized
proteins, also interacts with the intraluminal loop 4 of
SERCA2b in a Ca2⫹-dependent manner. The interaction
probably occurs through calreticulin and results in the
inhibition of the pump, possibly through the modulation
of the overall redox state of ER, since Erp57 promotes
disulfide bond formation (182). Depletion of ER Ca2⫹
would displace Erp57 from SERCA2b, favoring disulfide
bond reduction and stimulation of ATPase activity.
SERCA2b might thus function as an ER redox sensor.
Other interactions of the SERCA pumps have also been
described. That with the luminal Ca2⫹ binding protein
sarcalumenin (SAR) in skeletal and cardiac muscle
(SERCA1a) (73) has no clear physiological role, and that
with the antiapoptotic protein Bcl-2 (SERCA1 and SERCA2)
(74, 166) has been proposed to reduce ER Ca2⫹ content
through the inhibition of pump activity (74). Other mechanisms
could also link the antiapoptotic function to the ER Ca2⫹ content, e.g., the formation of Ca2⫹ leak channels by Bcl-2, or the
role of Bcl-2 in the expression of SERCA pumps. The entire
matter is still controversial (166, 303), but the possibility of a
role of the SERCA pump in the regulation of the apoptotic
pathways cannot be discounted.
The insulin receptor substrate (IRS) proteins 1 and 2
also interact with the SERCA pump in a process that is
stimulated by insulin. The interaction occurs at a COOHterminal site of SERCA1a and -2a (1), which is not conserved in the SERCA3 pump. Nevertheless, isoform 3b has
also been proposed to interact with IRS proteins (24),
suggesting a general link between the tyrosine phosphorylation cascade and the effects of insulin on the homeostasis of Ca2⫹. IRS1 overexpression has been claimed
to inhibit the expression of SERCA2 (165) and -3 (165,
325) pumps. Lastly, the EF hand protein S100A1, which is
preferentially expressed in the myocardial tissue, coimmunoprecipitates with SERCA2 in a Ca2⫹-dependent
manner (155). The interaction with S-100 has been
claimed to enhance pump activity in vitro and in vivo
(207). Interestingly, it has recently been proposed that the
activity of the SERCA pump is physiologically regulated
by the interaction with presenilin, the membrane intrinsic
protein that localizes predominantly to the ER membrane
(109). Presenilin is the catalytic portion of the mutienzymatic complex of ␥-secretase, which is responsible for the
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MARISA BRINI AND ERNESTO CARAFOLI
tion in the SERCA1 gene has recently been found in
Belgian Blue cattle affected by congenital muscular dystonia 1 (51). Affected calves show impaired swallowing
fatigue upon muscular exercise or stimulation, inability to
flex limbs, injurious falling, and die after a few weeks
because of respiratory problems. The mutation affects
exon 14 of the gene and leads to the replacement of a very
conserved R (R559) with a C in domain N. No functional
analysis of the SERCA1 activity has so far been performed
on the muscles of affected Belgian Blue cattle, but the
role of the conserved R has been analyzed in rabbit muscles (131). R560 (corresponding to bovine 559 in rabbit
muscle) could interact with the ␤-phosphate moiety of
ATP (131) as it lies close to the nucleotide binding pocket
of the N domain. The positive charge of the R could thus
contribute to the stabilization of the substrate. Replacement of R560 with an A (131) produced a strong inhibition
of the ATPase activity and of the phosphoenzyme formation from ATP.
Darier’s disease (or Darier-White’s disease) is a rare
autosomal dominant skin disorder characterized by loss
of adhesion between epidermal cells and abnormal keratinization (33). The disease is characterized by keratotic
papules in seborrhoic areas of the skin and in the skin
flexures which can then form confluent plaques. The first
lesions often appear at puberty, have a chronic relapsing
course, and are exacerbated by sunlight or ultraviolet
irradiation, heat, sweating, friction, and infections. The
histology of the condition shows loss of cell-to-cell adhesion in the suprabasal layer (suprabasal acantholysis) and
abnormal keratinization (dyskeratosis) (Fig. 6). Apoptotic
keratinocytes are thought to become eosinophylic dyskeratotic cells (grains) (101, 108, 161, 236, 319).
More than 130 mutations in the SERCA2 gene have
now been reported in human patients. The mutations are
distributed across the entire pump molecule, with no
evidence of clustering or of mutation hot spots. Interestingly, while the majority of Darier’s disease patients harbor mutations in a region which is common to 2a and 2b
FIG. 6. A: histology of Darier’s disease skin. The
suprabasal cleft of the epidermis contains acantholitic
cells (indicated by arrowheads), associated with hyperkeratosis (indicated by white arrows) and rounded
dyskeratotic cells (indicated by yellow arrows).
B: histology of normal skin is shown for comparison.
The four layers or strata of epidermis are indicated:
the stratum germinativum (SG), the stratum spinosum
(SS), the stratum granulosum (SGR), and the stratum
corneum (SC). The stratum lucidum in not shown.
(Modified from DermAtlas, copyright Drs. Bernard A.
Cohen and Christoph U. Lehmann, Johns Hopkins
University, 2000 –2008.)
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distribution and the total protein content of the muscle
are modified with respect to control muscles. Three mutations in the SERCA1 gene had been originally identified
in two affected families (223). One occurred at the splice
donor site of intron 3, and the other two created premature stop codons, truncating the pump and thus deleting
essential domains. Three other mutations leading to premature stop codons and an inactivating missense mutation were also later identified in a total of six families
(220, 224). The patients could still relax fast-twitch muscles, although at a significantly reduced rate. This suggests that some compensation by ectopic expression of
SERCA2 or SERCA3, and/or by Ca2⫹ removal from the
cytosol by the PMCA pump and the Na⫹/Ca2⫹ exchanger
could limit the defect in the Ca2⫹ clearing activity of the
SR. The recovery phase of resting cytosolic [Ca2⫹] after
acetylcholine stimulation in cultured muscle cells was
slower than in control cells.
Interestingly, a missense mutation (in exon 6 of the
gene) leading to the R164H replacement has been very
recently identified in Chianina cattle suffering from congenital pseudomyotonia, a muscle function disorder very
similar to that of Brody’s disease (75). The disorder is
characterized by muscle contractures that occur when
the animals are forced to move fast, or are exposed to
stresses that induce escape behavior. The content of the
SERCA1 protein in affected muscles was found to be
sharply reduced (up to 90%), whereas that of its mRNA
was not; thus a defect in transcription was apparently not
involved (255). R164 is located in the A domain of the
pump, where its side chain could neutralize the negative
charge of neighboring E2 during the large movement of
the A domain in the E1-E2 transition. The replacement of
the R side chain with the imidazole side chain of H would
destabilize the E2 state, possibly enhancing the susceptibility of the protein to degradation (G. Zanotti, personal
communication) (172). It is of interest that an R164S
mutation has also been found in a Darier’s disease Hungarian patient (see below) (243). Another missense muta-
CALCIUM PUMP DYSFUNCTIONS
Physiol Rev • VOL
below for details). The Ca2⫹ gradient and the role the
SERCA pump defect could have in its possible disruption
in Darier’s disease have not been studied as yet.
The reason for the restriction of the clinical symptoms of Darier’s disease to the epidermis and to certain
areas of the skin is not clear: it is probably related to the
fact that the SERCA3 pump, which is coexpressed with
SERCA2b in a large variety of cells, is not expressed in the
epidermis and may thus fail to provide compensation in
cutaneous tissues. Darier’s disease shares features with
Hailey-Hailey’s disease, which is also characterized by a
loss of adhesion between keratinocytes and by abnormal
keratinization of the epidermis (see below). Thus it appears that the SERCA and SPCA pumps cooperate in
maintaining Ca2⫹ homeostasis in the keratinocytes and
that the imbalance of their activity compromises Ca2⫹
homeostasis and, in the absence of compensatory factors,
leads to clinical phenotypes.
5. Other diseases related to alterations of the SERCA
pumps
Other disease phenotypes linked to alterations in the
expression of the SERCA pump have also been described.
The SERCA pump is functionally decreased in nearly all
models of heart failure (see Ref. 163 for review), and
decreased SR Ca2⫹ transport and SR Ca2⫹ content have
been detected in animal heart failure models. Although
heterogeneity in the expression level of the SERCA pump
has been observed in failing hearts, it is generally accepted that a reduction of the SERCA2a protein levels
(and/or activity) plays a role in the development of the
failure condition. The level of the SERCA2a regulator PLB
is not altered in heart failure, but data have been reported
suggesting that its phosphorylation state may be reduced
(132, 249, 270). This would explain the lower activity of
the SERCA pump. Surprisingly, only one report has described naturally occurring mutations in the human
SERCA2 gene that may predispose to heart failure (262).
However, the mutations, which occur in the exons corresponding to the SERCA2-PLB interaction domains, did
not result in sequence alterations in the expressed protein, suggesting that the SERCA2 gene is tightly regulated
to maintain low genetic variation. In contrast, three mutations in the coding region of the PLB gene have been
described which appear to be inherited in a familial manner. The R9C mutation was associated with the inheritance of dilated cardiomyopathy (263), resulted in the
decrease of phosphorylation of PLB, and induced dilated
cardiomyopathy and early death when introduced in
transgenic mice. A second mutation (L39stop), detected
in two large families (94), was also associated with dilated
cardiomyopathy. Several lines of evidence have indicated
that the L39stop mutation had no effect on SERCA2a
activity, nor on the mechanics and Ca2⫹ cycling of the
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isoforms, three families with classical Darier’s disease
were heterozygous for a mutation located in a region of
exon 20 which is specific for SERCA2b (67, 136). These
mutations led to premature termination codons and inactivated the pump, indicating that the suppression of
SERCA2b activity was sufficient to determine the pathological phenotype, i.e., the expression of SERCA2a could
not compensate for it. The overexpression of recombinant mutated pumps in model cells has revealed that in
the large majority of cases the mutations indeed impaired
their ability to transport Ca2⫹. However, reduced pump
expression, probably due to protein instability, has also
been observed in some cases.
Symptoms like behavior and learning difficulties,
which have been observed in some patients with Darier’s
disease, are probably secondary. Surprisingly, instead, no
reports have described heart defects, in spite of the finding that the large majority of patients carry mutations in
the region of SERCA2 gene which is common to both
SERCA2a and -2b isoforms.
Keratinocytes from Darier disease patients have
lower ER Ca2⫹ content but show a normal Ca2⫹ response
to raised extracellular Ca2⫹ concentration, a condition
that determines their differentiation. This has been attributed to the compensatory overexpression of the SPCA
pump (93). The resting cytosolic Ca2⫹ concentrations
varied in different patients (93), but in one report, cytosolic Ca2⫹ in cultured keratinocytes was instead found to
be increased (179). These keratinocytes were practically
incapable of lowering cytosolic Ca2⫹ after treatment with
thapsigargin and responded to the stimulation of purinergic receptors with a reduced elevation of cytosolic Ca2⫹
(179).
The depleted ER Ca2⫹ store in Darier’s keratinocytes
could alter the posttranslational processing of proteins in
the secretory pathway and limit their sorting to the
plasma membrane, thus contributing to the triggering of
the ER stress response. This would be in keeping with the
exacerbation of lesions induced by stressing factors. The
sorting of the tight junction protein ZO-1 and of the desmosomal protein desmoplakin were indeed strongly inhibited by thapsigargin in a MDCK cell model for intracellular junction assembly (282). A study on Darier’s keratinocytes has confirmed the inhibition of the trafficking
of desmoplakin to the plasma membrane. Interestingly,
the defect was found to be specific for desmoplakin, as
two other desmosomal proteins, desmoglein and desmocallin, were instead efficiently sorted to the plasma membrane (66).
An important factor to be considered in the molecular pathology of Darier’s disease would be the epidermal
Ca2⫹ gradient, which could become disrupted as in
Hailey-Hailey’s disease, a similar acantholytic skin disorder linked to inactivating mutations in the gene of SPCA
pump and/or to the reduced level of its expression (see
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Physiol Rev • VOL
mia cell types during differentiation. The SERCA3 pump
was significantly reduced or lost in colon carcinomas
(99). Treating colon and gastric cancer cells with differentiation-inducing agents markedly stimulated the expression of SERCA3 (not of SERCA2) (99), and a similar
overexpression of all three SERCA2 splice isoforms was
detected during the spontaneous differentiation of Caco-2
cells. Treatment of KATO-III gastric cancer cells with
differentiating agents concomitantly restored the increased cytosolic Ca2⫹ to normal (99). A similar study on
colonic epithelium has shown that the level of SERCA3
pump expression correlated with the degree of malignancy, being high in normal cells, moderately decreased
in adenomas, and barely detectable, or absent, in poorly
differentiated tumors (30). Similar observations, but on
SERCA2, have been made in prostate cancer cells. The
transformation of the human prostate cancer LNCaP cell
line to androgen-independent phenotypes, which are characterized by increased malignancy and apoptotic resistance,
resulted in the decreased expression of SERCA2b (305).
In an interesting approach, SERCA pump inhibition
by TG has been proposed as a potential targeted therapy
for prostate cancer. As is well known, TG (or similar
agents) induces the activation of apoptotic pathways
within the ER and the mitochondria. Obviously, as
SERCA is an ubiquitous protein, TG would be toxic and
thus impossible to use in therapy. The toxicity problem
has been eliminated in the approach by coupling TG to a
carrier peptide, producing an inactive prodrug that would
only become activated in the environment of prostate
malignant cells. This is due to the fact that prostate cancer cells produce a specific antigen (PSA) that is a serinethreonine protease. In the prodrug, the carrier peptide
that shielded the toxicity of TG was the peptide substrate
of PSA, which would only be cleaved off efficiently from
the inactive complex in the environment of the prostate
cancer cells, where the PSA protease is abundant, liberating TG: prodrugs of this type, which are currently under
preclinical evaluation, have yielded promising initial results (64). Curcumin has also been proposed as an anticancer drug: thanks to its low toxicity it is now in phase
II of clinical trials for advanced treatment of pancreatic
cancer (2, 65).
Interesting findings have demonstrated that the human hepatitis B virus (HBV) DNA integrates into the gene
of SERCA1 pump in a liver tumor causing the expression
of mutated proteins, which resulted in decreased ER Ca2⫹
content and in the increase in apoptosis (49). Viral integration was shown to cis-activate SERCA2 chimeric transcripts with the splicing of exon 4 and/or exon 11. The
splicing of exon 11 created a frame shift and generated a
premature stop codon in exon 12. The encoded protein
lacked the main portion of cytosolic domains N and P and
transmembrane segments M5 to M10. Predictably, it was
unable to pump Ca2⫹. Splice variants similar to the mu-
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myocytes; however, the mutant PLB was not detected in
human homozygous ventricles. A third human mutation
(deletion of R14) has also been recently associated with
dilated cardiomyopathy (quoted in Ref. 163). As discussed
in Kranias and Bers (163), the human PLB mutations
resulted in dilated cardiomyopathy and eventual heart
failure from the inhibition of the basal or the ␤-adrenergic
stimulated SERCA2a activity in the R9C and R14del mutations. In the L39stop mutation, which did not alter the
activity of the SERCA pump but caused the absence of
PLB, heart failure would be caused by the fact that in
humans hearts the regulation of SERCA pumping by PLB
is essential for normal function, due to the large cardiac
reserve required for the flight-or-fight situations that
would allow two- to threefold increases in heart rate.
A relationship between cancer and SERCA pump
dysfunction has been repeatedly documented: it is acquiring special interest, as accumulating evidence now indicates that the altered cellular homeostasis of Ca2⫹ may be
involved in the abnormal cell proliferation that is a hallmark of the malignant transformation. The matter has
aspects that are seemingly paradoxical: on one hand, the
increase of cytosolic Ca2⫹, e.g., by hyperactivated plasma
membrane channels, promotes cell proliferation. On the
other hand, situations of Ca2⫹ overload trigger apoptotic
death pathways. Somehow, therefore, tumor cells regulate the cell signaling machinery to promote proliferation
while at the same time protecting themselves from apoptosis. A remodeling of Ca2⫹ homeostasis is thus increasingly considered important in the process of malignant
transformation (see Ref. 206 for a review), and it is thus
only to be expected that alterations of the expression
and/or function of Ca2⫹ regulators such as the Ca2⫹
pumps should have a role in the process of tumorigenesis.
Several reports have shown that ATP2A2 (SERCA2) haploinsufficient mice often developed cancer. Mutations in
the ATP2A2 gene could be responsible for human cancer
development. Thirteen alterations of the gene were found
in patients with colon or lung cancer. They were missense
mutations, intronic deletions or insertions, and singlenucleotide alterations (2 in the coding region, 3 in the
intronic region, and 3 in the promoter region). Loss or
reduced expression of SERCA2 was also detected in all
patients with alterations in the promoter region of the
gene, as well as in patients with combinations of gene
alterations (159). Similarly, eight different mutations have
been found in the ATP2A2 and ATP2A3 genes in 11
patients with head and neck squamous cell carcinoma
(160), suggesting that germline alterations of these genes
may predispose to cancer and that impaired SERCA genes
might be involved, directly or indirectly, as an early event
in carcinogenesis.
Additional evidence for the involvement of SERCA
pump defects in cancer comes from studies on the expression of pump isoform in some carcinoma and leuke-
CALCIUM PUMP DYSFUNCTIONS
6. Genetic manipulation of the SERCA pumps
Knockout mice have been developed for all three
SERCA pump isoforms. The study of their phenotypes has
revealed unexpected differences between human and
mice. Mice lacking the SERCA1 pump (231) were born
alive and appeared to be initially normal. However, at
variance with humans, in whom Brody’s disease patients,
which have no SERCA1 or markedly lower levels of it,
have no respiratory ailments, null mice exhibited instead
respiratory failure and died shortly after birth. They also
exhibited slow limb movements and evidence of cramping. The Ca2⫹ uptake activity in homogenates obtained
from the diaphragm or hindlimb muscles of these mice
was severely impaired, without compensation by SERCA2
and -3 isoforms. This confirmed the major role of the
SERCA1 pump in maintaining the SR Ca2⫹ stores needed
for muscle excitation-contraction coupling. The differences in phenotypes with respect to humans could be due
to the fact that ⬃40% of human diaphragm muscle fibers
Physiol Rev • VOL
are slow-twitch type I, whereas in mice these fibers account for ⬍10% of the total (231): the higher levels of
SERCA2 in human diaphragm could thus explain the absence of respiratory failure in Brody’s disease patients.
Consistent with the role of the SERCA2a pump in heart,
and of the SERCA2b pump as an ubiquitous pump, breeding of heterozygous null SERCA2 mutant mice yielded
only wild-type and heterozygous offspring, which developed and grew with no apparent pathological phenotypes
(5, 133, 142, 237, 268, 310).
Numerous studies have shown that the reduction in
SERCA2a activity or expression may contribute to the
development of cardiac diseases; thus cardiac physiology
studies were performed on heterozygous mutants, which
exhibited a 65% reduction of the SERCA2a protein levels.
Despite the development of compensatory mechanisms,
i.e., upregulation of the Na⫹/Ca2⫹ exchanger and increased phosphorylation of PLB, the mutants had deficits
in relaxation and contractility. Interestingly, the mean
arterial blood pressure and the left ventricular systolic
pressure were reduced under basal conditions and at
lower levels of ␤-adrenergic stimulation, but not under
conditions of maximal stimulation. Studies on isolated
cardiac myocytes from heterozygous mice (142) have
confirmed the deficit in contractility and have revealed
reduced Ca2⫹ transients induced by agonists. The reduction of SERCA pump levels reduced the size of the SR
Ca2⫹ store and the amount of Ca2⫹ released at the beginning of each contractile cycle. However, even if the impairment of cardiac performance was clear, no evidence
of heart failure or cardiac hypertrophy was observed (it
was also not observed in Darier’s disease patients, see
above) showing that the loss of one copy of the gene was
insufficient to trigger the heart defect (33, 288). In another
mouse model, the exon encoding the COOH terminus of
SERCA2a was eliminated (310) so that the mouse expressed only the SERCA2b variant. The level of expression of SERCA2b in the hearts was ⬃60% of that of
SERCA2a in wild-type hearts, and even if some embryonic
lethality was observed, most of the homozygous mutants
survived to adulthood and developed a compensatory
cardiac hypertrophy (310), which was accompanied by a
reduction in contractility and relaxation.
However, the most striking phenotype observed in
SERCA2 heterozygous mutant mice in which the activity
of the SERCA2 pump was reduced was a very high incidence of squamous cell tumors (187), which occurred at
53 to 81 wk of age. The tumors were restricted to regions
with keratinized, stratified epithelium and included carcinomas and papillomas of the oral mucosa, tongue, esophagus, forestomach, genitalia, and hairless areas of the
skin. Darier’s disease patients deficient in SERCA2 activity could be mentioned in this context: they do not exhibit
strong propensity to cancer, but an association with squamous cell carcinoma may nevertheless exist (275). Con-
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tated proteins were also expressed in normal liver, their
overexpression increasing Ca2⫹ leakage from the ER, and
inducing apoptosis (48). The findings reinforce the suggestion of a role of the SERCA pump in the control of cell
death.
The expression of the SERCA3 pump in pancreatic
␤-cells, and the association of sequence variants of the
pump with type II diabetes (306), as well as the finding
that SERCA pumping activity was impaired in diabetic
rats model (181) have suggested that the SERCA3 pump
could be involved in the Ca2⫹ deregulation linked to the
onset of diabetes and that it could contribute to the
genetic susceptibility to type II diabetes. However, a direct link to insulin secretion, which could be mediated by
modulating cytosolic Ca2⫹, has not yet been demonstrated.
A few words are in order on the recent finding that
the most important class of antimalarial drugs, the sesquiterpene lactone artemisinins, would target the Plasmodium falciparum SERCA pump ortholog PfATP6 (80).
The drug was found to inhibit the Plasmodium pump, but
not mammalian SERCA, with the same potency and stoichiometry of TG. The specificity of artemisinin for the
plasmodium enzyme was found to be due to the replacement of a glutamic acid in transmembrane domain 3 of the
mammalian pump with a leucine (L263E) (299). If L263 of
PfATP6 was replaced by an E, the pump became resistant
to artemisinin, but retained the sensitivity to TG. Considering the social importance of malaria, these studies, even
if still preliminary, deserve to be developed and expanded. The finding that artimisinin resistance can be
introduced in PfATP6 is a matter of concern, and it will
thus be important to identify the full repertoire of ATP6
residues that interact with artemisinins.
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B. The SPCA Pump
1. General properties
The Ca2⫹ pump of the secretory pathway (SPCA) is
the newest addition to the family of Ca2⫹-transporting
ATPases. The first member of the subfamily (Pmr1,
plasma membrane ATPase related pump) was identified
in yeast (254) and was located in the Golgi membranes
(4). The first mammalian member of the subfamily was
cloned from rat in 1992 (116). In humans the first SPCA
pump (SPCA1) was identified less than 10 years ago in
two studies that also described mutations leading to
Hailey-Hailey disease (130, 283). A second human SPCA
pump (SPCA2) was described a few years later (304, 323).
One distinctive property of the SPCA pumps soon became
evident: in addition to Ca2⫹, these pumps also efficiently
transport Mn2⫹ (302). This peculiarity of the SPCA pumps
is evidently related to the presence of a number of Mn2⫹requiring enzymes in the lumen of the Golgi compartment,
the most important being the glycosyltransferases. Among
them, galactosyl transferases have been most extensively
studied. N-acetyl-glucosamino transferases, mannosyl transPhysiol Rev • VOL
ferases, glucoronyl transferases, and fucosyl transferases
are also Golgi enzymes that have Mn2⫹ as a cofactor. The
transport of Mn2⫹ by the pump is also important for the
regulation of the Mn2⫹ concentration in the cytosol. Oxidative damage in yeast cells lacking SOD was suppressed
in cells lacking functional Pmr1: they accumulated Mn2⫹,
which replaces SOD as a scavenger of ROS in the cytoplasm (170). However, Mn2⫹ can also become toxic to
cells. Since the SPCA pump is the major route for removing Mn2⫹ from the cytoplasm, it has a major role in the
process of Mn2⫹ detoxification. Two isoforms of the
SPCA pump have been described: SPCA1 is expressed
ubiquitously and is thus considered a housekeeping enzyme. Its expression level varies with the cell type and is
especially high in human epidermal keratinocytes (130).
The expression of SPCA2, instead, is more restricted.
Transcript analysis has revealed high levels of expression
of the SPCA2 pump in the mucus-secreting goblet cells of
human colon (304), suggesting a role for this pump in the
secretion of mucus. Whereas tissue fractionation and immunofluorescence work has unambiguously located the
SPCA1 pump in the Golgi compartment of mammalian
cells (247, 301), the subcellular localization of SPCA2
pump is less certain: in some cell types it colocalizes with
Golgi markers (304); in others, e.g., hippocampal neurons,
it colocalizes instead with trans-Golgi markers (323). The
SPCA1 is not the only pump that transports Ca2⫹ into the
lumen of the Golgi compartment. This was first indicated
by the finding that the uptake of Ca2⫹ into Golgi vesicles
was partially inhibited by the SERCA pump inhibitor TG,
which has very poor affinity for the SPCA pumps (5 orders
of magnitude lower than for the SERCA pump). This has
shown that a SERCA pump also contributes to the uptake
of Ca2⫹ in the Golgi compartment (289). The contribution
of the two pumps varies with the cell type, the SPCA
pump taking primacy in human keratinocytes, where it
contributes ⬃70% of the total Ca2⫹ uptake into the Golgi
vesicles (35).
SPCA pumps are predicted to be organized in the
membrane with 10 transmembrane helices and to protrude into the cytosol with a large headpiece, which is
predicted to be divided in the three A, P, and N units
identified in the SERCA pump (Fig. 7). The sequence of
SPCA pumps shares the critical consensus sites, e.g., the
sequence (SDKTGTLT) surrounding the catalytic D residue with those of the SERCA pump (and of the PMCA
pump, see below). SPCAs are shorter than the SERCA
pumps (and, especially, than the PMCA pumps, see below): this is due to the shorter luminal loops that connect
some of the transmembrane domains, and to the absence
of a long cytoplasmic tail. The NH2-terminal domain of
human SPCA2 is 32 residues longer than that of human
SPCA1 (323): it may be involved in specific interactions
with partner proteins and in domain rearrangements that
could modulate the affinities for the transported ions, in
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sidering the wide tissue distribution of the SERCA2b
pump, it is surprising that no other clear pathological
phenotype has emerged in mice lacking one copy of the
gene. Evidently, an unusual compensation occurs, since
in several tissues of SERCA2 heterozygous mutant mice
the SERCA2 protein levels were only reduced by 20%.
This suggests mechanisms for upregulating SERCA2
pump expression in response to a deficit in its level, or in
its activity.
Null SERCA3 gene mutants exhibited no structural
malformations and showed no apparent disease phenotype (188), in agreement with the notion that the SERCA3
pump does not have a housekeeping function, as it is invariably coexpressed with the SERCA2b variant. SERCA3 pump
is widely expressed in many tissues (198). Considering
that the expression of the SERCA3 pump is prominent in
vascular endothelial cells (3, 322) and in pancreatic
␤-cells (8), smooth muscle relaxation and diabetes were
investigated with particular attention. Smooth muscle relaxation defects were observed in SERCA3 null mutant
mice in vitro; however, a corresponding in vivo phenotype
was not observed. As for pancreatic ␤-cells, several missense mutations in the human SERCA3 gene have been
associated with type II diabetes (306), and reduced Ca2⫹ATPase activity was found in a rat diabetes model (181).
Although alterations in Ca2⫹ signaling have been found in
null mutant ␤-cells, no defects in insulin secretion or
glucose levels in the blood were detected, suggesting that
the loss of SERCA3 activity did not cause diabetes, probably because of compensatory mechanisms that became
activated.
CALCIUM PUMP DYSFUNCTIONS
analogy with what proposed for the copper transporting
ATPase (134). SPCA2 also diverges substantially from
SPCA1 in the COOH-terminal portion. A putative retrieval
motif from the plasma membrane (L925L) (323) and a
COOH-terminal PDZ binding motif (P943EDV) have been
identified (125, 315), which could be involved in the intracellular trafficking and location of the SPCA2 pump
(110). A EF-hand Ca2⫹ binding motif has been identified
in the NH2-terminal domain of the yeast Golgi Pmr1. It
could be involved in the regulation of the transport function of the pump (317), much as the COOH-terminal Ca2⫹
binding sites that have been identified in the PMCA pump
would do (128) (see below). Sequence comparison with
the SERCA pump predicts that the SPCA pump only has
one Ca2⫹ transport site, corresponding to SERCA site II,
consistent with a probable 1:1 Ca2⫹/ATP transport stoichiometry. The site appears to be formed by oxygens of
residue E329 in transmembrane domain 4 and N796 and
D800 in transmembrane domain 6 (316). Work on the
Pmr1 yeast enzyme (196, 197) has shown that the Mn2⫹
selectivity of the pump is instead defined by residue Q783
in transmembrane domain 6, and by conformation-sensitive packing interactions between Q783 and V335 in transmembrane domain 4. However, G309 could also be involved in the conferral of Mn2⫹ binding selectivity at the
cytosolic side (86).
The SPCAs are sensitive to the general P-type pump
inhibitors La3⫹ and orthovanadate and only very poorly
sensitive to the specific SERCA pump TG. The other two
commonly used specific SERCA pump inhibitors (CPA
and tBHQ) are nearly ineffective (204). The very poor
sensitivity to thapsigargin is consistent with the absence
of the F residue equivalent to F256, which is essential for
Physiol Rev • VOL
the binding of TG in the SERCA pump (see above). No
specific SPCA inhibitors have so far been described, and
endogenous activators are also unknown.
The two SPCA pumps have higher affinity for Ca2⫹
than the two other Ca2⫹ pumps: the Kd (Ca2⫹) of SPCA1
is ⬃10 nM (69) and is ⬃2.5 times lower in SPCA2 (70): the
higher Ca2⫹ affinity of the SPCAs with respect to the two
other Ca2⫹ pumps has been related to a slower E1P(Ca2⫹)/E2P transition in the reaction cycle (69). The
extremely high Ca2⫹ affinity of the SPCA pumps, which
have a Kd for Ca2⫹ below its presumed concentration in
most cytosols at rest, ensures that the Golgi compartment
will be constantly refilled with Ca2⫹, which is evidently
needed inside the vesicles for the optimal activity of a
number of important enzymes, most notably the endoproteases that perform the proteolytic processing of prohormones (50, 219), even in the absence of agonist-induced
cytosolic Ca2⫹ transients. As in the ER Ca2⫹ store, a large
fraction of the Ca2⫹ in the Golgi lumen is sequestered by
binding proteins. Some of those typical of the ER lumen
have also been detected in the Golgi compartment (31,
314), but others appear to be restricted to it, e.g., nucleobindin (184) and Cab45 (261).
At variance with the SERCA and PMCA pumps (see
below) which function as partially electrogenic H⫹ exchangers (however, see below for very recent findings
on the PMCA pump), the SPCA pumps do not appear to
countertransport H⫹. Protons are needed for a number
of important functions in the lumen of the Golgi vesicles and must thus be kept inside. Both SPCAs transport Mn2⫹ with an affinity that is as high as that for
Ca2⫹. As mentioned, the transport of Mn2⫹ is demanded
by the needs of Mn2⫹ requiring N- and O-linked glycosylations of a number of proteins (150, 308) which
occur inside the Golgi compartment. It is also required
for optimal activity of a Golgi-located casein kinase
(171, 318).
Alternative splicing only occurs in the primary transcripts of SPCA1. The primary transcript of SPCA2, instead, is not known to undergo alternative splicing, i.e.,
only one SPCA2 isoform (946 amino acids) is known. In
the SPCA1 pump, alternative splicing at the 3⬘-end of the
primary mRNA results in the generation of four transcripts resulting from the coupling of exon 26 to exon 27,
which contains two internal splice donor sites, or to exon
28, one transcript containing exon 28 and the portion of
exon 27 up to one donor site, the other exon 28 and the
portion of exon 27 up to the second donor site. The
resulting four isoforms (SPCA1 a-d) have COOH-terminal
domains of different length and sequence (Fig. 8) (300).
Information on possible differential properties of SPCA1
isoforms is still very scarce, although some kinetic differences have been detected.
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FIG. 7. Deduced structure of the SPCA pump (in white) superimposed on that of the SERCA pump (in red). The image is a kind gift of
Dr. L. Raeymakers (Leuven, Belgium).
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MARISA BRINI AND ERNESTO CARAFOLI
2. The Hailey-Hailey’s disease
In 1939 two dermatologist brothers described a familial benign chronic pemphigus, a skin disorder that was
eventually named after them (117). The disorder affects
both sexes and is inherited in an autosomal dominant
way, with symptoms typically arising in the third or fourth
decade of life. The disease has similarities to Darier’s
disease. It also presents with blisters and itchy erosions
mainly located at sites of sweating and friction like the
groin and the axillar regions, with pain and unpleasant
smell of the skin following the development of macerations. The disease proceeds through remissions and exacerbations caused by a number of external factors like
friction, sweating, infections, and ultraviolet exposure. In
2000, inactivating mutations in the disease were discovered in one allele of the gene coding for SPCA1 (ATP2C1)
(130, 283). The disease is essentially benign, but squamous cell carcinomas may develop from the skin lesions.
This point is important, as the disruption of SPCA1’s gene
in mice instead causes a number of squamous cell carcinomas, but not the acantholytic skin disorder (225) it
FIG. 9. A: histology of Hailey-Hailey’s disease’s skin,
showing widespread loss of adhesion between keratinocytes (acantholysis, indicated by arrowheads) with abnormal keratinization (dyskeratosis, indicated by white
arrows) near the granular layer. B: histology of normal
skin is shown for comparison. (Modified from DermAtlas, copyright Drs. Bernard A. Cohen and Christoph U.
Lehmann, Johns Hopkins University, 2000 –2008.)
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FIG. 8. Generation of multiple SPCA1 isoforms by alternative splicing of the human ATP2C1 gene. Exons are represented by filled gray
boxes, introns by the black line. 5⬘D1 and 5⬘D2 indicate splice donor
sites in exon 27 for SPCA1b and SPCA1d, respectively. Sa-d indicate the
position of the different stop codons in the a-d variants. Details of the
different splicings are described in the text. The relative sizes of each
splice variant are on the left.
causes in humans. It has thus been proposed that a prosurvival response would predominate in the Golgi (but
also in the ER) of mice keratinocytes, leading to cancer,
whereas proapoptotic responses would predominate in
the Golgi and ER of keratinocytes in humans leading to
the acantholytic skin disorder (225). Interestingly, the
SPCA may be involved in breast cancer as well: SPCA2
may have a role in the normal functioning of the mammary gland, as a pronounced increase in the SPCA2
mRNA levels was detected in the gland during lactation,
in analogy to what was found for isoform 2 of the PMCA
pump (85) (see below). A similar increase has also been
found in breast cancer.
Histologically, Hailey-Hailey’s disease is characterized by the loss of adhesion between suprabasal keratinocytes (acantholysis) and by the abnormal keratinization
of the epidermis (dyskeratosis) (Fig. 9). Aggregates of
keratin filaments in the perinuclear region of keratinocytes are a distinctive feature (120, 122, 201). Following
the first description of the defect in the SPCA gene, ⬃100
additional mutations were identified, randomly scattered
in the gene (300). The mutations downregulate the expression of the SPCA1 pump in the keratinocytes (17,
240), a finding that has been confirmed by expressing
mutated versions of the SPCA1d pump in model cells (86,
87): about half of the mutations produced low levels of
SPCA expression (the amount of transcript, however, was
normal). In addition to the downregulation, a number of
mutants displayed instead deficient binding of Ca2⫹ and
of Mn2⫹ to the pump, or a dysfunction in the E1-P(Ca2⫹)
to E2-P conformational transition in the reaction cycle.
The molecular link between the impaired expression
and/or the functional defect of SPCA1 and the skin lesions
is still not understood. Since the SPCA pump also transports Mn2⫹, it would in principle be possible to relate the
disease to a defect in the transport of Mn2⫹. However, the
close similarity between Hailey-Hailey’s and Darier’s diseases convincingly indicates that the inability of the SPCA
pump to efficiently transport Ca2⫹ is the causative factor
CALCIUM PUMP DYSFUNCTIONS
Physiol Rev • VOL
remains to be understood, however, how the SPCA pump
defect would mechanistically exacerbate the cell adhesion problem. The defects in the posttranslational processing of proteins that will eventually contribute to the
formation of desmosomes which has been described in
Darier’s disease (see above) could possibly have a role in
the Hailey-Hailey’s disease. Other observations that could
be relevant to the problem deserve to be mentioned here
even if their involvement in the molecular etiology of the
disease is at the moment purely speculative: for instance,
the finding that the Ca2⫹-sensing receptor would be also
expressed in the Golgi network (298) and the discovery
that one of the Trp channels (TrpV6) is an important Ca2⫹
influx path in keratinocytes (175). As for the rise in extracellular Ca2⫹, which would be reflected in the increase
of cytosolic Ca2⫹, it could lead to the translocation to the
nucleus of transcription factors that would activate the
SPCA gene (151, 200). Others, however, have found no
changes, or even a decrease, in SPCA1 pump expression
when extracellular Ca2⫹ was increased (245, 326). Eventually, the SPCA defect must be causatively related to the
inability of keratinocytes to increase external Ca2⫹ in the
granular layer: this could be possibly linked to the special
functional properties of the SPCA pump, which are different from those of the SERCA and PMCA pumps, and
which are evidently uniquely qualified to maintain the
proper function of keratinocytes (including their ability to
build the epidermal Ca2⫹ gradient). It is interesting that
Darier’s disease, which causes an apparently very similar
keratinocytic defect, is instead related to mutations in the
SERCA pump. Long-term transcriptional effects are also
likely to be important, but the finding that the HaileyHailey rash that follows physical trauma develops very
rapidly is difficult to reconcile with transcriptional effects, and with other relatively slow processes.
C. The PMCA Pump
1. General properties
The PMCA pump was discovered in 1966 by Schatzmann (260) as an ATP-driven system that expelled Ca2⫹
from erythrocytes. Initially, most of the work concentrated on erythrocytes, but then spread gradually to other
cells, including those of excitable tissues, where a larger
Ca2⫹ extrusion system, the Na⫹/Ca2⫹ exchanger, predominates quantitatively. The pump was isolated and purified
in 1979 using a calmodulin column (214) that exploited
the finding (106, 140) that the pump was a target of
calmodulin regulation. It belongs to the PIIB subfamily of
P-type pumps. The pump operates with high Ca2⫹ affinity
and low transport capacity, with a 1:1 Ca2⫹/ATP stoichiometry. It operates as a Ca2⫹/H⫹ exchanger, but the
matter of Ca2⫹/H⫹ stoichiometry is still controversial.
Work on the purified pump reconstituted in liposomes
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in the disease (the SERCA2 pump has essentially no Mn2⫹
transport ability). The Ca2⫹ transport defect is expected
to decrease Ca2⫹ in the Golgi lumen of keratinocytes, as
the defect of the SERCA pump decreased it in the ER of
Darier keratinocytes, thus impairing the posttranslational
processing and targeting of proteins which are essential
for the biogenesis of the cell adhesion structures. A molecular pathogenesis of the skin defect similar to that of
Darier’s disease could be envisaged in the Hailey-Hailey’s
disease; however, the picture is likely to be more complex
and not limited to processes taking place in the Golgi
lumen: an increase of basal cytosolic Ca2⫹ has also been
reported in Hailey-Hailey keratinocytes (130), which
would be compatible with cytosolic Ca2⫹ signaling defects. Others (179), however, have not confirmed the cytosolic Ca2⫹ increase in cultured keratinocytes, but have
reported a decrease in the amount of Ca2⫹ released by TG
and a reduced response of cytosolic Ca2⫹ to the stimulation of purinergic receptors. An important factor to be
considered in the molecular pathogenesis of HaileyHailey’s disease (possibly, of Darier’s disease as well) is
the disruption of the Ca2⫹ gradient: in the layers of normal epidermis, Ca2⫹ is lowest in the basal layer and
highest in the granular layer (82, 199). It is generally
accepted that the gradient is due essentially to extracellular Ca2⫹, which is necessary for the assembly of desmosomes and adherent junctions in the suprabasal layers
of the epidermis. The concentration of extracellular Ca2⫹
in the basal layer where proliferation occurs is actually
lower than in most other extracellular fluids. Increases of
external Ca2⫹ in the latter layer above 0.1 mM activate the
Ca2⫹ sensing receptor (21) that is present in the plasma
membrane of keratinocytes, blocking their proliferation
and triggering instead their differentiation. The differentiated keratinocytes would then move upwards in the
epidermis, eventually ending up in the cornified layer. No
Ca2⫹ gradient has instead been found in the skin of
Hailey-Hailey’s disease patients, even in nonaffected areas (17, 130), i.e., the basal and suprabasal layers of the
epidermis contain the same low amount of Ca2⫹. According to an attractive proposal (153), the disruption of the
Ca2⫹ gradient would be crucial to the molecular etiology
of Hailey-Hailey’s disease: in normal skin, insults that
disrupt the skin permeability barrier upon wounding eliminate the Ca2⫹ gradient. The resulting increase of Ca2⫹ in
the extracellular space of the more profound layers of the
epidermis directly stabilizes the desmosomes. However, it
would also activate the plasma membrane Ca2⫹ sensing
receptor to trigger cell-to-cell adhesion and differentiation of the cells in the granular layer, repairing the wound
and reconstituting the Ca2⫹ gradient. In the Hailey-Hailey
skin, instead, noxious stimuli would somehow fail to increase extracellular Ca2⫹ in the suprabasal (granular)
layers of the epidermis, failing to stabilize desmosome
integrity and to activate the Ca2⫹-sensing receptor. It still
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MARISA BRINI AND ERNESTO CARAFOLI
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FIG. 10. Deduced structure of the PMCA pump (in blue) superimposed on that of the SERCA pump (in red). The long COOH-terminal tail
of PMCA pump (absent in the SERCA pump) is not included in the
superimposition. The image is a kind gift of Dr. S. Pantano (Montevideo,
Uruguay).
single PMCA site are part of transmembrane domains 4
and 6 (115). Interestingly, the insertion of the missing
acidic residue in transmembrane 5 of the PMCA pump by
a mutagenesis approach raised its Ca2⫹/ATP stoichiometry to 2, suggesting the formation of a second Ca2⫹ binding site which corresponds to site 1 of the SERCA pump
(115). The long COOH-terminal tail of the PMCA pump
contains most of the regulatory domains, the most prominent among them being the calmodulin binding domain,
which has the canonical amphipathic helix secondary
structure of calmodulin binding domains (138). Ca2⫹
binding motifs identified upstream and downstream of the
calmodulin binding domain (128) may have regulatory
function on the activity of the pump.
Under nonactivated conditions, the COOH-terminal
tail of the pump is proposed to fold over to interact with
two sites in the first and second cytosolic loops of the
enzyme, compromising the access to the active site (88,
89). Calmodulin then interacts with its binding domain,
removing it from its docking sites next to the active
center, freeing the pump from autoinhibition. At variance
with all other targets of calmodulin regulation, the PMCA
pump can be activated by the COOH-terminal half of
calmodulin isolated after trypsin cleavage (113). This is in
line with the 3D structure of the complex of calmodulin
with a synthetic peptide corresponding to the main portion of the PMCA calmodulin binding domain, which has
shown that the PMCA only interacts with the COOHterminal portion of calmodulin (83). One important distinctive property of the PMCA pump is the multiplicity of
activatory mechanisms. Next to calmodulin, the most in-
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had indicated electroneutrality (215), whereas other experiments had instead indicated a partially electrogenic
1:1 Ca2⫹:H⫹ transport process (119). Very recently, the
activity of the pump in native membranes has been found
to be insensitive to the variation of the membrane potential, indicating an electroneutral H⫹/Ca2⫹ reaction (291).
Under optimal conditions, the Kd of the pump for Ca2⫹
drops from the 10 –30 ␮M range in the pump at rest to the
0.2– 0.5 ␮M range routinely measured under optimally
activated conditions. Even under optimal conditions the
Ca2⫹ affinity of the PMCA pump is thus lower than that of
the SPCA pumps, but is high enough to enable it to
operate with reasonable efficiency even under the Ca2⫹
concentration conditions prevailing in most cytoplasms at
rest; accordingly, the PMCA pump is conventionally defined as the fine tuner of cytosolic Ca2⫹. As all P-type
pumps, the PMCA pump is inhibited by La3⫹ and vanadate. No really specific inhibitor comparable to TG exists,
even if carboxyeosin is frequently mentioned (98). More
recently, however, synthetic peptides of the caloxin family have been claimed to specifically inhibit the PMCA
pump (285). Interestingly, caloxin peptides, of which several have now been tested, bind to the extracellular domains of the pump (129) and could thus in principle be
used to inhibit the PMCA pump in intact cell systems.
They have also been claimed to have isoform-specific
inhibitory properties: one of them (caloxin 1C2) has
strong preference for PMCA isoform 4 (Ki ⫽ 2.35 ␮M,
compared with 10 times higher Ki values for PMCA1, -2,
and -3) (232).
The PMCA pump was cloned in 1988 (276, 313),
showing the same essential membrane organization and
topology properties of the SERCA pump, which were
already known at the time of the cloning. The 3D structure of the pump has not yet been solved, but molecular
modeling work based on the structure of the SERCA
pump predicts the same general features, with 10 transmembrane domains and the large cytosolic headpiece
divided into the three main A, N, and P domains (Fig. 10).
As expected, the catalytic phosphorylation site (SDKTGLT) is conserved as are other important consensus domains, but prominent sequence/domain differences with
respect to the other two Ca2⫹ pumps also exist, for instance, in the first cytosolic loop that contains an acidic
phospholipid binding domain (see below), and, especially
in the long cytosolic COOH-terminal tail, which is absent
in the SERCA and SPCA pumps. The comparison with the
sequence of the SERCA pump also reveals important
differences in the amino acids that contribute to the formation of the Ca2⫹ binding sites. As already mentioned,
one essential acidic residue (an E) that contributes coordinating oxygens to site 1 of the SERCA pump is missing
in transmembrane domain 5 of the PMCA pump, which
thus does not have site 1. As in the case for site 2 of the
SERCA pump, the residues that coordinate Ca2⫹ in the
1361
CALCIUM PUMP DYSFUNCTIONS
2.
and -4
TABLE
Ca2⫹-dependent ATPase activity of PMCA2
Ca2⫹-Dependent ATPase Activity, nmol ATP 䡠 mg
protein⫺1 䡠 min⫺1
PMCA2
PMCA4
⫺Calmodulin
⫹Calmodulin
4.21
1.45
5.87
6.15
From Hilfiker et al. (126) and Elwess et al. (84).
pump). The four gene products (PMCA pump isoforms
1– 4) differ in tissue distribution and calmodulin affinity
(Table 1). Pumps 1 and 4 are ubiquitous and have poor
calmodulin affinity, and pumps 2 and 3 have higher calmodulin sensitivity and their expression is restricted to
some tissues: PMCA2 is expressed prominently in the
nervous system and in the mammary gland and PMCA3 in
the nervous system (280). These are not the only distinguishing properties of the isoforms; for instance, Table 1
shows that PMCA1 has peculiar calpain sensitivity: additional differences have also been discovered in the interaction of the isoforms with protein partners (see below).
The isoform work has gradually made clear that the
PMCA2 pump has properties that single it out from the
other three basic isoforms. For instance, it has peculiarly
high resting activity so that calmodulin, which normally
stimulates the activity of the other three pumps four- to
sixfold, only increases its activity by 20 or 30% (Table 2)
(84, 126). This peculiarity of PMCA2, as well as others to
be discussed below, could explain its predominance in
cells that have special Ca2⫹ homeostasis demands.
Alternative splicing occurs in the transcripts of all
four pump isoforms. It occurs at two sites that do not
involve, as expected, the catalytic core of the ATPase. Site
A is located upstream of the phospholipid binding domain
in the first cytosolic loop of the pump, and site C inside
the calmodulin binding domain in the COOH-terminal tail
(Fig. 11). Two nomenclature systems have been proposed
to identify the splice variants (37, 152). In the most commonly used, site A inserts are designated with letters from
z to w according to the number of the exons inserted. The
splicing operation at this site shows once more the peculiarity and special complexity of PMCA2: three exons may
TABLE 1. Tissue distribution, calmodulin affinity, and calpain sensitivity of the four basic PMCA pump
isoforms
Tissue distribution
Kd (CaM), nM
Calpain sensitivity
PMCA1b
PMCA2b
PMCA3b
PMCA4b
Ubiquitous
40–50
High
Restricted (brain)
2–4
Low
Restricted (brain)
8
Low
Ubiquitous
30–40
Low
The notation 1-4b refers to the full-length pump, without splicing inserts (see text). The information given in Table 1, for instance, calmodulin
(CaM) affinities, has been extracted from several of the articles quoted in the text.
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teresting activators are acidic phospholipids (213), which
bind to two sites in the pump molecule: one is the calmodulin binding domain (28), and the other (see above) is
a stretch of ⬃40 predominantly basic amino acids in the
cytosolic loop that connects transmembrane domains 2
and 3 (330): which of the two sites is more important as a
mediator of the response to acidic phospholipids is still
not known. The activation by acidic phospholipids is
likely to be physiologically important, as they are present
in the membrane environment of the pump: it has been
calculated that the concentration of phosphatidylserine in
the environment of the erythrocyte membrane that surrounds the pump would be sufficient to permanently stimulate the pump to ⬃50% of maximal activity (212). Since
the most effective acidic phospholipid is the doubly phosphorylated derivative of phosphatidylinositol (PIP2), the
concentration of which becomes modulated in the membrane during Ca2⫹ related signaling processes, a possible
PIP2-mediated reversible cycle of PMCA activation could
be envisaged (55). Other activatory mechanisms of the
pump are phosphorylations by protein kinase C and protein kinase A (the latter only in one of the isoforms), an
oligomerization (polymerization) process involving the
calmodulin binding sequence in the COOH-terminal tail of
the pump, and the cleavage by calpain. As no conclusive
information is available on the physiological relevance of
these mechanisms, they will not be discussed here. They
are, however, discussed in detail in a number of reviews
(e.g., Refs. 43, 112). However, the cleavage by calpain will
be briefly mentioned. It occurs immediately upstream of
the COOH-terminal calmodulin binding domain (139) and
activates the pump irreversibly, making it calmodulin insensitive. This irreversible mechanism of activation could
become significant in conditions of pathological Ca2⫹
overload that would demand increased Ca2⫹ exporting
ability (258).
The cloning of the pump was soon followed by a
number of studies that showed that in mammals the enzyme was the product of a multigene family (ATP2B1-4).
Four basic gene products were soon discovered, the transcript of each undergoing a complex alternative splicing
process that increases the total number of presently recognized isoforms to ⬃30 (see Refs. 112, 230, 280 for
comprehensive reviews of this aspect of the PMCA
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MARISA BRINI AND ERNESTO CARAFOLI
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FIG. 11. Alternative splicing of rat ATP2B1-4 genes generating
multiple PMCA isoforms. The scheme summarizes the splice variants at
sites A (A) and C (B). Exons are represented by colored boxes, introns
by the black line. Details on the different splicings are described in the
text. The numbers in the boxes represent the nucleotide number of each
rat exon. Human exons are slightly different in the number of nucleotides, but display essentially the same splice variants.
be alternatively inserted in its primary transcript leading
to variants w (three exons of 33, 60, and 42 bp), y (two
exons of 33 and 60 bp), and x (one exon of 42 bp). Variant
y, however, has so far been only identified in rats, and a
variant v, in which a fourth novel exon is transcribed at
site A, has been found in the bullfrog (76) The variant of
PMCA2 in which no extra exons are included at site A is
designated with the letter z. Variant z is not found in
PMCA1, as all mature transcripts of this isoform invariably contain a 39-bp exon, which in all other isoforms is
only alternatively included, leading to variant x. Thus all
PMCA1s known correspond to the x variant. PMCA3 and
PMCA4 exist in two variants: z (no extra exons in the
transcripts) and x (two exons of 42 and 36 bp, respectively). The splicing process at site C is characterized by
splice sites internal to exons, read-through across adjoining exons, and inclusion of full extra exons. As a result, in
the multitude of variants that are generated, changes in
reading frame occur that lead to the creation of premature
stop codons, and to the truncation of the resulting proteins. Pumps in which no insertions occur at site C are
designated as b variants. As a rule, a single exon is inserted at site C in the transcripts of PMCA1 (154 bp) and
PMCA4 (175 bp), but the insertion occurs piecemeal,
leading to variants c, d, e, and a (full exon transcribed) in
PMCA1, and to variants d and a (full exon transcribed) in
PMCA4. The insertion of the 154-bp exon, or of portions
of it, in the transcript of PMCA3 leads to variants a (full
exon included) or c and d. Variants e and f have also been
described. In the former, 88 bp of the following intron and
a poly(A) tail were added to the 154-bp exon. In the latter,
a 68-bp exon, located upstream the 154-bp exon, was
included. In the case of PMCA2, two, and not only one,
novel exons (172 and 55 bp, respectively) can be included
or excluded. The resulting variants are designated as c or
a (both exons inserted). Again, the splicing at site C
shows the peculiar complexity of PMCA2.
The multitude of PMCA isoforms, especially of those
generated by alternative splicing of the transcripts, is not
easily rationalized in terms of function. The four basic
isoforms have all been purified and characterized (even if
the most important information on the isolated enzyme
has been obtained on purified PMCA4), but only scarce
information is available on the differential activity of
splice variants. It is known, however, that the insertion of
the three exons at site A (which corresponds to a 45residue insertion, the w form) directs PMCA2 to the apical
domain of polarized cells, whereas smaller inserts sort the
protein to the basolateral domain of the plasma membrane (53). It is also known that the COOH-terminal truncation determined by the C inserts (the a variants) lowers,
as should be expected, the affinity of the truncated pumps
for calmodulin (27, 242).
One interesting property of the PMCA pump that is
related to the multiplicity of its isoforms is its inhomoge-
CALCIUM PUMP DYSFUNCTIONS
Physiol Rev • VOL
oxide synthase 1 (NOS-1) in a ternary complex with ␣-syntropin in which the latter binds to a pump domain further
upstream in the sequence (320). The double interaction
thus downregulates the production of NO by decreasing
Ca2⫹ in the immediate vicinity of the synthase; therefore,
the pump could function as a modulator of signal transduction, in addition to regulating cell Ca2⫹ (46). Other
proteins containing PDZ domains that interact with
PMCAs are the following: PISP (PMCA interacting single
PDZ domain protein), which could have a role in the
sorting of the pump to the plasma membrane (102);
Ania-3, a member of the Homer family of scaffolding
proteins that couple NMDA and metabotropic glutamate
receptors (274), and could recruit PMCAs to domains
close to Ca2⫹ influx channels to control neuronal Ca2⫹
signaling and synaptic function; CLP36, a member of the
LIM family of proteins that interacts with the platelet
cytoskeleton via its constitutive association with the actin
binding protein ␣-actinin (25). The association with
PMCA 4b would recruit the pump to nonfilamentous actin
complexes in resting platelets, and then associate it with
the actin cytoskeleton during its rearrangement upon
platelet activation.
In addition to the COOH-terminal domain, other regions of the pumps also interact with protein partners.
The main intracellular loop joining transmembrane domains 4 and 5 binds RASSF1 (tumor suppressor RASassociated factor 1), inhibiting the epidermal growth factor-mediated activation of the RAS signaling pathway (7).
The main intracellular loop also interacts with ␣-syntropin (see above), and with the catalytic subunit of calcineurin. Since the pump does not interact with the consensus sequence for calcineurin anchoring proteins (32),
a novel type of interaction could operate. The interaction
would downregulate calcineurin activity, thus acting as a
negative modulator of calcineurin signaling pathways.
The NH2-terminal cytosolic region of the pump has a low
degree of sequence homology among isoforms and has
thus been exploited in a search for isoform specificity in
interacting partners. The study has identified protein
14.3.3 and has further underlined the uniqueness of
PMCA2 among isoforms, as protein 14.3.3 (the ␧-isoform)
interacted with PMCA1, -3, and -4, but not with PMCA2
(185, 250). The study has also shown that the interaction
with protein 14 –3-3 inhibited pump activity.
2. The PMCA pump in the disease process
Several mammalian genetic and nongenetic pathologies linked to dysfunctions of the PMCA pump have been
described, with those of genetic origin being the best
characterized. A number of disease conditions linked to
the ablation, or to partial disruptions, including point
mutations, of the genes for the PMCAs, have been described in mice, but the only spontaneous human disease
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neous distribution in the plasma membrane: in a number
of cell types, the pump has been shown to be associated
with the caveolae (96), a functionally convenient location,
as caveolae, at least in some cell types (e.g., intestinal
smooth muscle), are in close contact with the ER (239)
and would thus facilitate the extrusion of Ca2⫹ liberated
by it. In addition to caveolae, the cholesterol-rich lipid
rafts, which are emerging as important domains in cell
signaling, also appear to be loci for specific association
with the PMCA. Recent studies on cerebellar synaptosomal plasma membranes (273) have shown that one PMCA
isoform, PMCA4, is specifically associated with the lipid
rafts, whereas the other PMCA isoforms are not. The
specific association of PMCA with caveolae/lipid rafts has
been confirmed in a recent study on intestinal smooth
muscle (81). The results of the study have indicated that
one splice variant of isoform 4 of the pump (PMCA4b)
could specifically associate with caveolin 1, whereas another splice variant (PMCA4a) could be instead associated with lipid rafts.
One important development that may rationalize the
existence of so many PMCA pump isoforms, including the
splice variants, is the identification of protein partners
that regulate activity, targeting to membrane domains,
and/or the recruiting of pump variants to cell components
(e.g., the cytoskeleton). This area of work has now grown
significantly, but only some of the information that has
been generated has so far considered differences among
isoforms. A large number of proteins interact with a PDZbinding domain in the COOH-terminal tail of the pumps.
Among them are members of the MAGUK (membrane
associated guanylate kinases, or SAP) family of protein
kinases which contain PDZ domains and are associated
with the cortical actin cytoskeleton (63). The pump variants studied have been the 2b and 4b: it has been suggested that the interaction could maintain the pumps in
specific membrane domains to control local Ca2⫹ homeostasis. Along similar lines of work, PMCA 4b has been
shown to redistribute to newly formed platelet filopodia
via the PDZ binding domain (61). CASK (Ca2⫹-calmodulin
dependent serine protein kinase) is another MAGUK protein that interacts with PMCA4b (266). It is a coactivator
of the transcription of T-element containing promoters
and has been suggested to downregulate the T-elementdependent reporter activity by depleting Ca2⫹ in the immediate environment of the enzyme: the suggestion has
been made that the interaction of the pump with CASK
could have a role in the regulation of synaptic activity, as
CASK is located at brain synapses. Another PDZ domaincontaining protein, NHERF2 (Na⫹/H⫹ exchanger regulatory factor 2), has been shown to interact with PMCA2b,
but not with PMCA4b (62): it could provide a scaffolding
mechanism to bring together membrane proteins, signaling molecules, and the cytoskeleton. The PDZ binding
domain also directs the interaction of PMCA 4b with nitric
1363
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MARISA BRINI AND ERNESTO CARAFOLI
FIG. 12. A: immunolocalization of the wild-type PMCA2 pump in the
stereocilia of the mouse cochlear hair cells (B). Staining of the stereocilia with an anti-actin antibody is shown. Bars, 25 ␮m.
Physiol Rev • VOL
PMCA2 pump was described in the deafwaddler mouse
(279), which carried either a point mutation in the first
cytosolic loop of the pump (G283S), or a frame shift
mutation in codon 471 of the gene, generating a null
mutant. The mutants had equilibrium problems and severe hearing impairment. Soon afterwards, another spontaneous PMCA2 mutation that produced hearing loss and
equilibrium imbalance was described in the Wriggle
Sagami mouse (287). In this case a replacement (K412E)
occurred in the fourth transmembrane domain of the
pump, preventing its targeting to the stereocilia. The mistargeting of the pump in the outer hair cells of the Wriggle
Sagami mouse was to be expected, as it is generally
assumed that mutations in the transmembrane domains of
the pump prevent its correct delivery to the plasma membrane (115). Thus the recent finding that another deafness-inducing point mutation (S877F) in the sixth transmembrane domain of mice PMCA2 (278) still permitted its
correct delivery to the plasma membrane was surprising.
Two deafness-inducing PMCA2 mutations described
in humans (91, 269) have extended the information provided by the mouse models. In both cases, the mutation of
PMCA2 required a concurrent mutation in cadherin 23 to
generate, or to exacerbate, the hearing loss phenotype.
Cadherins are a family of plasma membrane receptors
that mediate Ca2⫹-dependent cell-cell adhesion and play a
key role in the regulation of organ and tissue development
during embryogenesis. Cadherin 23 is a component of the
tip-links in the stereocilia of the hair cells and participates
in the mechanoelectrical transduction process, i.e., in the
conversion of mechanical force into electrochemical signals. In the family described by Schultz et al. (269), a
substitution (V586M) in the sequence of the pump that
defines the ATP binding site enhanced the auditory defect
in three out of five siblings who also had an accompanying
mutation in the gene of cadherin 23. The PMCA2 mutation
(G293S) in the family described by Ficarella et al. (91) had
a number of additional aspects of interest. It clearly defined a digenic mechanism for the hearing loss phenotype,
as the affected child carried both the mutation in the
PMCA2 gene and a concomitant mutation in cadherin 23
(different from that described by Schultz et al.). However,
the father of the child had the cadherin 23 mutation and
had no hearing loss phenotype, and the mother, who had
the PMCA2 mutation, was equally healthy. The G293S
mutation, which still permitted the regular targeting of the
pump to the stereocilia, was located only 10 residues
downstream of that of the deafwaddler mice and involved
the same two amino acids. Evidently, the cytosolic loop
where the deafwaddler and the human mutations occurred, even if distant from the active site and apparently
not involved in any of the known important functions of
the pump, is a functionally sensitive region. The study by
Ficarella et al. (91) also analyzed molecularly the defect
of the pump; as mentioned above, the PMCA2 pump has
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related to a PMCA pump defect so far identified is a form
of hereditary deafness (91, 269). The defect concerns the
PMCA2 pump, which is abundantly expressed in the
brain, particularly in the Purkinje cell of the cerebellum
and in the hair cells of the Corti organ of the inner ear,
where it locates specifically to the stereocilia (Fig. 12).
That the PMCA2 pump was involved in the hearing process had become clear from the analysis of the phenotype
of the PMCA2 knockout mice (162), which was characterized by balance impairment and hearing loss. The study
of the vestibular inner ear revealed the absence of the
otoconia and the progressive degeneration of the hair
cells beginning 10 days after birth. The PMCA2 pump is
probably also essential in neuronal Ca2⫹ homeostasis,
since PMCA2 null mice and a defective mutant mouse
(deafwaddler mouse, see below) showed a significant
reduction in the number of spinal cord motor neurons
(168) and abnormalities in Purkinje neurons (167). However, the balance defect was apparently not due, or was
only partially due, as could have been expected, to a
cerebellar dysfunction, but was apparently related to the
absence of the calcium carbonate crystals of the otoconia
embedded in the otolithic membrane overlying the sensory epithelium that sense gravity and linear acceleration
in the inner ear.
At about the time of the generation of the PMCA2
knockout mice, the first spontaneous mutation of the
CALCIUM PUMP DYSFUNCTIONS
Physiol Rev • VOL
13. A: monitoring of cytosolic Ca2⫹ concentration ([Ca2⫹]c) in
CHO cells transfected with the recombinant Ca2⫹-sensitive photoprotein
aequorin (cytAEQ) or cotransfected with cytAEQ and splice variants z/b
and w/a of the PMCA2 pump isoform. B: monitoring of [Ca2⫹]c in CHO
cells cotransfected with cytAEQ and wild-type or mutated (G283SS or
G293S) splice variant w/a of the PMCA2 pump isoform. The cells were
challenged with ATP, a purinergic agonist that generates InsP3.
FIG.
opening probability and unitary conductance of the
mechanoelectrotransduction (MET) channels, eventually
depolarizing the hair cells, and compromising the generation of the hearing signals (Fig. 14).
In addition to the inner ear, PMCA2 is also prominently expressed in other brain regions, and its dysfunction may have a role in some typical neurodegenerative
diseases: a transcriptional downregulation of PMCA2 has
for instance been described in the brains of mouse models
of Huntington’s disease and in the brains of Huntington’s
disease patients (164). The dysfunction may contribute to
the alteration of the homeostasis of Ca2⫹ which is widely
considered to have a role in the etiology of the disease.
PMCA2 is also prominently expressed in the epithelial
cells of the mammary gland and has an important role in
the regulation of the concentration of Ca2⫹ in the milk.
Mammary gland epithelial cells need to continuously and
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uniquely high activity in the absence of the activator
calmodulin and reacts poorly to it, i.e., it appears to be
programmed to continuously pump Ca2⫹ out of the cells
where it resides with reasonable efficiency irrespective of
whether calmodulin and presumably other putative activators act on it. This is at variance with the other three
basic isoforms of the pump, which only pump Ca2⫹ efficiently when activated by calmodulin (or, possibly, by
other factors). Importantly, the variant of the PMCA2
pump present in the stereocilia is not the full-length z/b,
but the w/a variant (107) which contains the A site insert
specified by the three extra exons that direct it to the
apical pole of the cell. It is also COOH-terminally truncated and would thus be expected to have even lower
calmodulin reactivity than the conventional z/b variant of
the PMCA2 pump that reacts poorly to calmodulin, as it
has lost about half of the calmodulin binding domain.
Thus it would eject Ca2⫹ continuously but would be expected to respond even more poorly than the full-length
PMCA2 pump to the sudden arrival of a Ca2⫹ bolus. The
comparison of the Ca2⫹ ejecting properties of the z/b and
w/a PMCA2 pump variants shown in Figure 13A indeed
shows that the two pump types expressed in model cells
behaved as expected, i.e., the variant resident in the stereocilia was much less able to control the Ca2⫹ transient
induced by the stimulation of the cells with a suitable
agonist than the full-length z/b variant. Figure 13A is
particularly informative as it describes an experiment
performed in the native intracellular environment. Evidently, the special properties of the PMCA2 pump satisfy
the peculiar Ca2⫹ homeostasis demands of hair cells, in
which the role of the pump is to eject Ca2⫹ from the
stereocilia to the endolymph with an efficiency poised to
maintain the free Ca2⫹ concentration in it which is much
lower than in all other conventional extracellular spaces
(⬃20 ␮M, Ref. 321). The reasons for the unusually low
free Ca2⫹ concentration of the endolymph are likely to be
related to the physiology of the tip links and of the gating
of the mechanoelectrical transduction channels that are
opened by the displacement of the stereocilia bundle to
permit influx of K⫹ (and Ca2⫹) into the stereocilia. They
may also be related to other special regulatory demands
of the stereocilia/endolymph system. Figure 13B shows
that the mutant G293S pump, and the deafwaddler G283S
mutant shown for comparison, did not differ from the
wild-type w/a pump in the poor ability to control the Ca2⫹
pulse, but were peculiarly inefficient in the long-term,
nonactivated pumping of Ca2⫹ out of the model cell used
for the analysis. It is plausible to assume that they would
act in the same way in the real inner ear situation, in
which they are requested to pump Ca2⫹ out of the stereocilia to the endolymph. Experiments on organotypic cochlea and utricula cultures have indeed shown that this is
the case (91). The lowering of Ca2⫹ near the tip links
linked to the dysfunction of the pump could increase the
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MARISA BRINI AND ERNESTO CARAFOLI
the effect of activators like calmodulin. To respond to the
demands of lactation, the w/b variant becomes highly
expressed during lactation (246).
Very recently, a missense mutation of PMCA3 gene
which causes a T543M substitution was identified in human pancreatic cancer cells (145). Interestingly, the mutation was located between the catalytic D residue and
the K which is a component of the ATP-binding site, and
affected a residue located in one of the two docking site
for the calmodulin binding domain. No indications have
been offered for a role of the PMCA3 in pancreatic tumorigenesis, but it is interesting that the PMCA3 pump has
been identified in pancreatic islet cells (147).
FIG. 14. Mechanotransduction (MET) in hair cells of the Corti organ
of the inner ear. A: scanning electron micrograph of hair bundle (outer
hair cells in adult mice; K. S. Steel, Cambridge, UK, at http://www.
neuroscience.cam.ac.uk/directory/profile.php?kps1). The view shows
the stereocilia arranged in order of increasing height. B: model for
mechanotransduction. Deflection of a hair cell’s bundle bends the stereocilia and the tip links (the thick black lines connecting the individual
stereocilia) between them. MET channels open in response to the tension on the tip link and permit influx of K⫹ (and Ca2⫹) into the stereocilium. The PMCA2wa pump reexports the Ca2⫹ that has entered
through the MET channels. Mutations in its gene reduce the ability of
PMCA2wa to remove Ca2⫹ from the intracellular ambient, thus decreasing the endolymphatic Ca2⫹ concentration and increasing the open
probability of the MET channels (91).
efficiently eject Ca2⫹ to supply it to the milk during lactation, and this is probably why they express the w/b
splice variant, which contains the A splice insert that
directs it to the apical pole of the cell, where it would
continuously eject Ca2⫹ to the milk. In keeping with the
special properties of PMCA2, it would do it irrespective of
Physiol Rev • VOL
The PMCA2 knockout mice have already been mentioned. The ablation of the PMCA2 gene, in addition to
generating the hearing loss phenotype discussed above,
also strongly reduced the concentration of Ca2⫹ in the
milk (248). Knockout mice have also been developed and
the phenotypes studied for PMCA1 and PMCA4, but not
yet for PMCA3. This isoform is widely expressed in developing embryos and may thus be essential for normal
gestation.
The ablation of the PMCA1 gene has resulted in
early embryonic lethality in homozygotes, underlying
the essential role of PMCA1 in the early stages of
development and in organogenesis, in line with the
suggested housekeeping function of this isoform (226).
Heterozygotes, in contrast, appeared healthy; however,
loss of one copy of the PMCA1 gene in mice with a
PMCA4 null background led to increased propensity to
apoptosis in the smooth muscles of blood vessels, possibly due to Ca2⫹ overload (226).
The ablation of the PMCA4 gene did not cause a very
evident general pathological phenotype (264), but local
defects were present. This is interesting, as PMCA4 is also
very widely expressed and has also been proposed to play
a housekeeping role. However, it has now become evident
that PMCA4 plays more specialized roles as well and is
not essential for the general function of controlling Ca2⫹
homeostasis in all cells, although it contributes to it. One
prominent observed defect caused by PMCA4 dysfunction
was male infertility, which reflected the dominance of the
PMCA4 pump in the testis, where it represents ⬎90% of
the total PMCA protein (241). Sperms of PMCA4 null mice
were still able to fertilize eggs (264) but were incapable of
achieving hyperactivated motility, and thus could not
reach them to perform the fertilization. Ultrastructural
analyses of the sperm tail failed to reveal abnormalities in
the motility apparatus but showed increased condensation of mitochondria, indicating that they had become
overloaded with Ca2⫹ and had thus become dysfunctional. The ablation of the PMCA4 gene produced other
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3. Genetic manipulations of the PMCA pumps
CALCIUM PUMP DYSFUNCTIONS
Physiol Rev • VOL
are related to the regulation of the Foxo3a protein degradation pathway by the PMCA4-nNOS complex (218).
4. Nongenetic alterations of PMCA pumps
Nongenetic alterations of PMCA pumps have also
been claimed to contribute to pathological processes (reviewed in Ref. 176). As a rule, these alterations decrease
the content and/or the activity of the pump (however, see
below for the case of some cancers), predictably generating situations of cytosolic Ca2⫹ overload that would
disturb normal cell life. In general, these alterations and
their effects are less precisely defined, molecularly, than
those linked to genetic defects, and it is frequently difficult to decide whether the pump deficiency has a primary
causative role in a given disease or is a consequence of it.
The problem is exacerbated by the existence of concurrent membrane defects that could in turn affect the function of the PMCAs. However, in a number of pathologies,
the dysfunction of the pump appears to be at least one
of the causative etiological factors. A selection of the
cases that seem to have particular interest will be presented here. It will be necessarily succinct, as the complete coverage of all conditions that have been described
would place an unacceptable burden on the space of this
contribution. In addition, a large number of the reported
findings at the moment are still essentially phenomenological.
PMCA defects have been described in a large number
of disease conditions, among them the oxidative stress in
several tissues, particularly brain, brain ischemia, diabetes, atherosclerosis, aging, neurodegenerative diseases,
and various disturbances of calcium metabolism. Numerous studies have also reported PMCA defects in various
cancer types (reviewed in Ref. 206) in line with the accumulating evidence on the involvement of Ca2⫹ homeostasis dysfunction in the process of malignant transformation. The findings of overexpression of the mRNA of
PMCA2 and, albeit to a lower degree, of PMCA4 in some
breast cancer lines (173) is thus particularly interesting. A
previous study by the same authors had found that the
mRNA of PMCA1 was also overexpressed in breast cancer
lines. They also found that the antisense-mediated inhibition of PMCA pumps in the tumorigenic breast cancer cell
line MCF-7 dynamically inhibited cell proliferation when
PMCAs were inhibited to a level that did not completely
prevent Ca2⫹ efflux and did not induce cell death (174).
Similar studies have been performed on intestinal colon
cancer cells (12), showing that the expression of the
PMCA4 pump was specifically augmented during the differentiation of the HT-29 cancer line (PMCA1 pump was
unaffected), suggesting that less differentiated stages
could be associated with compromised Ca2⫹ efflux. Indeed, the PMCA4 pump transcripts were downregulated
in the early progression of some colon cancers (G. Mon-
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localized dysfunctions, for instance, in the modulation of
the Ca2⫹ signals in B-lymphocytes (52), and in the contractility of vascular smooth muscles (226). The absence
of phasic contractions, however, was due to the large
number of smooth muscle cells dying in the isolated
tissue, not to the dysfunction of the PMCA4 pump. Interestingly, when PMCA4 knockout mice were backcrossed
with the original strain, the smooth muscle phenotype
disappeared, to be restored by superimposing a heterozygous PMCA1 null mutation: thus both PMCA1 and PMCA4
pumps appear to be essential for the control of the homeostasis of Ca2⫹ in vascular smooth muscle cells. Further studies on PMCA4 knockout mice and PMCA1 heterozygous mice (with only one copy of the PMCA1 gene)
had shown that these two isoforms may have different
roles in the regulation of bladder smooth muscle contractility (186): the PMCA1 isoform appears to be important
for the generalized Ca2⫹ extrusion function and the
PMCA4 isoform for the modulation of acetylcoline receptor signaling. Again, this suggests that the PMCA4 isoform
may be a signaling enzyme rather than a housekeeping
Ca2⫹ controlling pump. Important physiological functions
of the PMCA4 isoform were also revealed by studies on
mice overexpressing it rather than by ablating it. Early
studies in which the PMCA4 pump had been expressed
specifically in the myocardium of the rat, and in vascular
smooth muscle cells in mice, had indicated a role in the
modulation of myocardial growth and hypertrophy (118).
They also indicated a role in the regulation of peripheral
vascular tone, as the mice displayed increased peripheral
blood pressure (111, 265).
Transgenic mice overexpressing the PMCA4 pump
or a COOH-terminally truncated version lacking 120
residues, and thus also the PDZ-binding domain, have
extended the work that had shown that the pump interacts with the neuronal nitric oxide synthase (nNOS)
via its PDZ domain and inhibits its activity by decreasing local Ca2⫹ concentration (267). nNOS is important
in heart physiology, as it regulates cardiac contractility
(11), Ca2⫹ cycling (154, 271), and redox equilibrium
(121) and is needed to sustain the ␤-adrenergic response (217). Overexpression of the full-length pump,
but not of the COOH-terminally truncated version, reduced the ␤-adrenergic contractility response. nNOS
inhibitors reduced the contractility to the levels observed
by overexpressing PMCA4, confirming the role of PMCA4 as
a regulator of the activity of nNOS in heart (218). PMCA4,
however, also has other roles in cardiac physiology. The
ablation of its gene, in addition to the effects described
above, affected the process of cardiac hypertrophy. The
hypertrophic response to tranversal aortic restriction was
abolished in PMCA4 knockout mice. In contrast, the hypertrophic response to physical exercise was preserved.
The hypothesis has been put forward that these effects
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MARISA BRINI AND ERNESTO CARAFOLI
Physiol Rev • VOL
PMCA3 pump were unchanged. The decrease was
found to precede the demise of the neurons. The isoform specificity of the transcript changes emphasizes
the differential activity, and thus the differential role, of
the PMCA pumps , and underlines the complex interplay of the PMCA pump isoforms in the control of
neuronal Ca2⫹ homeostasis under changing physiological conditions and under the stress of pathology. This
point has been repeatedly touched on above, but deserves to be mentioned again.
Diabetes has also seen a large number of contributions on the possible involvement of PMCA pumps.
Ca2⫹ is known to have an important role in the process
of pancreatic insulin secretion: glucose induces Ca2⫹
entry and oscillations in the ␤-cells whose duration
depends on its extracellular concentration. ␤-Cells express a unique combination of PMCA pump isoforms
(1b, 2b, 3a, 3c, and 4a) (147, 307), a finding that suggests an important, probably isoform-specific, role of
the PMCA pump in the regulation of ␤-cell Ca2⫹ homeostasis (147, 307). The PMCA pump is inhibited by
glucose, cogently suggesting that its modulation indeed
participates in the homeostasis of Ca2⫹ in ␤-cells (127,
181). The PMCA pump activity of ␤-cells of non-insulindependent diabetic mellitus rats is lower than in the
controls, suggesting that a PMCA-mediated defect in
Ca2⫹ homeostasis may contribute to the diabetic pathology. The PMCA pump activity defect is actually not
limited to ␤-cells: erythrocytes of diabetic patients have
also been shown to have increased Ca2⫹ content and
decreased PMCA pump activity (104, 105), although the
PMCA pump defect was only detected in patients with
diabetic neuropathy (202). Decreases of PMCA pump
activity were also found in renal cortical cells (180) and
in brain (228). Somewhat paradoxically, however,
PMCA pump activity was instead found to increase in
the sarcolemmal membranes of skeletal muscles of rat
in which diabetes was induced by the treatment with
streptozotocin (286).
Finally, defects in the PMCA pump have been described in disease conditions in which the general organismic Ca2⫹ homeostasis is altered. Considering the
essential role of the PMCA pump in the homeostasis of
Ca2⫹ in cells, these defects are not surprising. One
could mention the downregulation of the expression of
the PMCA2 and PMCA3 pumps in the renal tubules of
hypercalciuric rats (44) and the general PMCA pump
dysfunction in familial hypercalcemia (72).
IV. CONCLUDING REMARKS
Ca2⫹ pumps have been known for more than 40
years as essential actors in the maintenance of the
cellular homeostasis of Ca2⫹. Of the three pumps that
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teith and S. J. Roberts-Thomson, personal communication).
The case of cancer cachexia, the tissue wasting
that frequently complicates malignancies, is of interest
from another angle as well, as it links the PMCA pump
defect to the abnormal activation of calpain induced by
the specific decline of the natural calpain inhibitor
calpastatin. The total level of the PMCA protein was
found to be decreased in skeletal muscles in cachexia
(58), and it is of interest that a reduction was also found
in the hearts of tumor bearers. The degradation of
PMCA pumps by calpain in vivo may be a more general
phenomenon in pathology and has indeed been observed in the brain of rats subjected to global brain
ischemia followed by 48 h of reperfusion (95, 210). A
decrease of the PMCA pump protein has also been
observed in several regions of the brain of gerbils subjected to forebrain ischemia followed by prolonged
reperfusion (177, 178). The study did not establish that
the decrease was due to the degradation of the PMCA
pumps, but it is interesting that the decrease only concerned PMCA1, the content of which in the forebrain
membranes decreased by ⬃40%. Since a study on the
purified pump isoforms has established that PMCA1
pump has peculiar calpain sensitivity (114), it appears
very likely that the decrease found in the gerbil forebrain was due to the degradation of PMCA1 by calpain.
A large number of studies have detected damage to
the PMCA pumps by reactive oxygen (and nitrogen)
species. The case of 4-OH-2,3-trans-nonenal (HNE), an
aldehyde product of fatty acid oxidation, deserves to be
mentioned. HNE has been found to inhibit the PMCA
pump in erythrocytes (244) and is present in oxidized
low-density lipoproteins (LDLs), which are found in
atherosclerotic lesions. Exposure of platelets to oxidized LDLs (not to native LDLs) inhibited the platelet
PMCA pump and massively increased platelet Ca2⫹
content (329), suggesting that a pump defect induced
by oxidized LDLs could contribute to pathologies like
atherosclerosis and hypertension. Reactive nitrogen
species (RNS) such as the NO radical and peroxynitrite
(ONOO⫺) have also been found to inactivate the pump
(277), degrading it when applied to it in the purified
state (331). The ONOO⫺-induced degradation pattern of
the erythrocyte PMCA has also been observed in newborn asphyxia, a major cause of childhood neurological
defects (331).
The brain damage caused by seizures, complex
partial seizures, and status epilepticus has also been
related to PMCA pump alterations. An interesting study
on kainate-induced seizures has revealed a changed
expression pattern of the PMCA isoforms (97). The
transcript levels of the PMCA1 and PMCA2 pumps were
decreased in the pyramidal cells of the CA1 and CA3
regions of the hippocampus, whereas those of the
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CALCIUM PUMP DYSFUNCTIONS
TABLE
3.
Genes, isoforms, tissue expression, and genetic diseases of Ca2⫹ pumps
Pump and Isoform
Tissue Distribution
TP2A1
SERCA1a (adult)
SERCA1b (neonatal)
Fast-twitch skeletal muscle fibers
ATP2A2
SERCA2a
Heart, slow-twitch skeletal muscle, brain
ATP2A3
SERCA2b
SERCA2c
SERCA3a, b, c, e
All tissues
Epithelial, mesenchymal, and hematopoietic cells
Most tissues
Abundant in the hematopoietic cells, salivary
glands, trachea, lung, pancreas, kidney, and
colon
All tissues
All tissues
Highly expressed in human epidermal
keratinocytes
Abundant in the gastrointestinal tract. Also
present in trachea, salivary glands, thyroid,
keratinocytes, prostate, mammary gland, brain,
and testis
All tissues
ATP2C1
SERCA3d, f
SPCA1a
SPCA1b
ATP2C2
SPCA2
ATP2B1
ATP2B2
PMCA1a
PMCA1b
PMCA2z/b, z/a, w/b, w/a
Brain, mammary gland (lactating)
ATP2B3
PMCA3a, b
Brain, spinal cord, and adrenal gland
ATP2B4
PMCA4a, b
All tissues
operate in mammalian cells, one (the SPCA pump) has
only become known recently, adding new complexity to
the already crowded panorama of cellular transporters
of Ca2⫹. Important distinctive features of the reaction
mechanism, of the regulation, and of the specific roles
played by the three pumps in the process of Ca2⫹
homeostasis have gradually become known. The recent
solution of the 3D structure of the SERCA pump has
greatly advanced knowledge in the area, providing important new information on the catalytic mechanism of
the pump and on the path used by Ca2⫹ to traverse its
molecule. In parallel with the spectacular advances in
the understanding of the molecular structure of the
pumps, the area of pump malfunction has also emerged
recently. It has rapidly acquired prominence, in keeping
with the growing awareness that one of the distinctive
features of the Ca2⫹ signal is ambivalence. Ca2⫹ pump
dysfunctions are generally not linked to the extreme
situations of cell catastrophe induced by the uncontrolled, massive, and protracted Ca2⫹ overload. They
generate instead subtler disease phenotypes that are
generally cell specific, and could possibly even only
prevail in defined subcellular domains. Table 3 offers a
summary of all genetic or related defects of the three
pumps that are known today. As the table shows, only
a handful of genetic Ca2⫹ pump diseases have become
known in humans, but the larger number of spontanePhysiol Rev • VOL
Disease
Brody’s myopathy
Congenital pseudomyotonia (Chianina cattle)
Congenital muscle dystonia (Belgian Blue cattle)
Darier’s disease
Prostate cancer?
Heart failure
Colon, lung, liver cancer?
Head and neck squamous cell carcinoma?
Type II diabetes?
Head and neck squamous cell carcinoma?
Hailey-Hailey’s disease
Breast cancer?
Hereditary deafness (digenic)
Hearing loss
Breast, colon cancer?
Familial hypercalcemia?
Pancreatic cancer?
Breast cancer?
ous and induced conditions which have been detected
and studied in model animals have considerably enlarged the spectrum of possible pump dysfunctions.
The genetic phenotypes have so far attracted most of
the attention, but nongenetic pump alterations are also
being increasingly investigated. The topic of Ca2⫹ pump
dysfunction is expanding rapidly and will certainly generate important dividends, including a more complete
understanding of the significance of the Ca2⫹ signaling
process in healthy cells.
NOTE ADDED IN PROOF
Very recently a correlation has been found between
human systolic blood pressure and the locus of the
PMCA1 gene (ATP2B1) on chromosome 12q24 in a vast
number of Korean individuals (53a). The authors have
noticed the correlation of their findings with the vascular
phenotypes (loss of vein contraction) in mice with no
copies of the murine gene for PMCA4 (Ref. 226 in text)
and have suggested that PMCA1 could function as a modified locus for phasic contraction.
ACKNOWLEDGMENTS
We are grateful to Dr. C. Toyoshima (Tokyo, Japan) for
providing the image of the SERCA pump structure in Figure 3
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Gene
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MARISA BRINI AND ERNESTO CARAFOLI
and the cartoon in Figure 4, Dr. L. Raeymaekers (Leuven, Belgium) for providing the structural model of the SPCA pump in
Figure 7, and Dr. S. Pantano (Montevideo, Uruguay) from providing the structural model of PMCA in Figure 10.
Address for reprint requests and other correspondence: E.
Carafoli, Dept. of Biochemistry, Viale G. Colombo 3, 35131
Padova, Italy (e-mail: [email protected]).
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