Advances in Biological Chemistry, 2011, 1, 74-92
doi:10.4236/abc.2011.13010 Published Online November 2011 (http://www.SciRP.org/journal/abc/
ABC
).
Published Online November 2011 in SciRes. http://www.scirp.org/journal/ABC
Endogenous modulators in the regulation of ion transporting
enzymes: structure, function, interactions, recent
advancements and future perspectives
Parimal C. Sen
Division of Molecular Medicine, Bose Institute, Kolkata, India.
Received 23 August 2011; revised 29 September 2011; accepted 6 October 2011.
ABSTRACT
A prerequisite for life is the ability to uphold electro-
chemical imbalance across biomembranes. Ion trans-
porting enzymes, known as specific pumps, are re-
sponsible for the transport of various ions across cell
membranes to sustain the same. In all eukaryotes, the
plasma membrane potential and secondary transport
systems are maintained by the activity of P-type ion
transporting enzymes, commonly known as ATPase
membrane pumps. Malfunction of pumps leads to
various cell disorders and subsequently diseases like
cardiac problems, renal malfunctionings, diabetes,
cataract, even cancer. Activities/functions of these
pumps are regulated either by exogenous agents or
by endogenous substances like proteins, peptides,
hormones, etc., which are collectively known as mo-
dulators. Some of these endogenous modulators may
be useful for developing drugs depending on the na-
ture of regulation. For more than last two decades,
researchers across the globe are exploring the me-
chanism of action of different endogenous modula-
tors on these ion transporting enzymes with the aim
of developing target-specific drugs. In this review, we
have discussed recent advances in our understanding
of ATPase pumps, e.g., Ca2+-, Na+, K+-, Ca2+, Mg2+-,
H+, K+-ATPases, with the emphasis on their func-
tional regulation by a number of endogenous modu-
lators, and the implications of development of some
of these modulators as potential dru gs.
Keywords: ATPases; Endogenous Protein/Peptides; FX-
YD; Phospholamban; Sarcolipin; Interleukin; Regula-
tions
1. INTRODUCTION
Transport enzymes are those responsible for the tran-
sport of ions across the cell membranes. The transport
takes place against ion gradient, energy required for the
process is provided by ATP, which is hydrolyzed to ADP
and Pi during these transport phenomena. These are a
large group of evolutionary conserved ion pumps that
are found in bacteria, archaea and higher eukaryotes and
belong to P-type ATPases (includes Na+, K+, Ca2+, H+,
K+-ATPases) and are involved in performing different
fundamental processes in biology and medicine, ranging
from the generation of membrane potential to muscle
contraction, the removal of toxic ions from cells, main-
taining proper acidity inside cells etc. [1]. Mutation or
dysfunction of these ATPases leads to several diseases.
Malfunction of Ca2+-ATPase may lead to defect in car-
diac function, infertility, diabetes and even cancer [2].
Impairment of sodium pumps, on the other hand, cause
diseases including osteoporosis, hypertension, familiar
hemiplegic migraine. P-type ATPases, that transport mo-
novalent and divalent cations such as Na+, K+, Ca 2+, Cu+,
Ag+, Zn2+, Cd2+, Ni2+ etc., are divided into five subfami-
lies on the basis of the conserved sequences [3].
It is now well established that all these P-type ATP-
ases are regulated by endogenous modualtors like, pep-
tides, proteins and other small molecules.
1.1. Na+, K+-ATPase
The Na+, K+-ATPase commonly known as sodium
pump, is responsible for coupled extrusion and uptake
of Na+ and K+ ions across the plasma membranes of
most eukaryotic organisms. Na+, K+-ATPase is a mem-
ber of the P2c family of P-type ATPases superfamily [4].
The pump drives three sodium ions out of the cell and
two potassium ions into the cell against substantial
concentration gradient. The activity of this enzyme is
required for diverse functions like maintenance of cel-
lular osmotic balance, generation of neuronal mem-
brane potentials and renal as well as intestinal handling
of solutes. This is a (  /  )2 dimeric integral membrane

P. C. Sen / Advances in Biological Chemistry 1 (2011) 74-92 75
protein and is composed of a 100-kDa -subunit and a
heavily glycosylated
 -subunit of molecular weight
about 55 kDa [5]. In addition, Na+, K+-ATPase is asso-
ciated with a third subunit belonging to the FXYD pro-
tein family [6]. Isoforms exist for both the α (α1, α2, α3)
and β (β1, β2, β3) subunits. These isoforms are ex-
pressed variedly in different mammalian tissues [7-10]
with various αβ combinations. The Na+, K+-ATPase can
function as an αβ protomer, but it is postulated that the
Na+, K+-ATPase is composed of (α/β)2 dimer in vivo. α
subunit contains ATP binding site, phosphorylation site,
and amino acids essential for the binding of cations and
cardiac glycosides which suggests that this subunit
plays a major role in the catalytic function of the en-
zyme. The β subunit appears to be involved in matura-
tion of the enzyme, localization of ATPase to the pla-
sma membrane, and stabilization of a K+-bound inter-
mediate form of the protein [11]. The other low mo-
lecular mass transmembrane proteins were named after
the invariant extracellular motif FXYD [6]. One central
role of the FXYD proteins is to interact with the Na+,
K+-ATPase and modulate its properties. There are
seven FXYD proteins. Because each of these proteins
has a different tissue distribution and functional effects,
the current hypothesis is that FXYD proteins act as
tissue-specific modulators of Na+, K+-ATPase that ad-
just or fine-tune its kinetic properties to the specific
needs of a given tissue, cell type, or physiological state,
without affecting it elsewhere [12,13].
1.2. Ca2+-ATPase
Ca2+-ATPase or the Ca2+-pump on the other hand is
responsible for the transport of Ca2+ across cell mem-
branes and thus maintains intracellular calcium concen-
tration. Because of its peculiar flexibility as a ligand,
calcium regulates all important aspects of cellular ac-
tiveity, beginning with the creation of new life at fer-
tilization and ending with the dramatic event of apop-
totic suicide at the end of the life cycle. Ca2+ may also
function as a bonafide first messenger since it interacts
with the exterior of cellular membrane as if it were a
hormone or growth factor [14]. Ca2+ is distinctly am-
bivalent molecule that is essential for life. Cells have
an absolute dependence on the messenger function of
Ca2+. In order to function properly, its homeostasis
must be controlled with absolute precision, failing of
which, there can be a sustained cellular Ca2+ overload
leading to apoptotic and/or necrotic cell death [15]. The
cytoplasmic free Ca2+ concentration in all cell types at
rest is very low (50 nM - 150 nM) which is 103 - 104
times lower than the free Ca2+ concentration in the ex-
tracellular space (usually 1 mM) or in the lumen of
sarco(endo)plasmic reticulum (SR/ER) (0.1 mM - 2.0
mM). Such large Ca2+ gradients across cellular bounda-
ries are established and maintained by the powerful
calcium pumps located in the plasma membranes and in
sarco (endo) plasmic reticulum [16] with contributions
from other cellular organelles.
The Ca2+ transporting systems can be classified into
four basic transporting modes i.e. ATPases, exchangers,
channels and uniporters. In general, whenever the situa-
tion demands the fine regulation of Ca2+ in submicro-
molar concentrations, the ATPase mode are chosen,
since this appears to be the only transport system with
the ability to interact with high Ca2+ affinity and is there-
fore used by plasma membrane and sarcoendoplasmic
reticulum.
The ATP dependent Ca2+ pumps of sarco (endo) pla-
smic reticulm (SERCA) constitute a large family of pro-
teins of 100 kDa - 138 kDa [17-21] and a proteolipid of
molecular mass 6 kDa - 12 kDa [22], belonging to P2
subfamily (subtype 2A) of P-type ATPases. They are
structurally distinct from the Ca2+ pump of the plasma
membrane, but share similarities in the mechanism of
calcium translocation. The intracellular location of SER-
CA exclusively in SR/ER membranes is maintained by
the presence of specific retention/retrieval motifs in their
primary sequences. The Ca2+ transport is reversible and
under favourable condition results in the formation of
ATP molecule for two Ca2+ ions released from the lumen
of SR [23]. Counter-transport of H+ and fluxes of ions
through the anion and cation channels of SR prevent
large changes in membrane potential during Ca2+ trans-
port. The SERCA pumps have high affinity for Ca2+ (Km
about 0.1 µM), and are capable of maintaining a resting
cytoplasmic [Ca2+] of 10 nM - 20 nM.
The plasma membrane Ca2+-ATPase (PMCA) is the
only high affinity Ca2+ transporting system present in the
plasma membrane and belongs to the P2 (subtype 2B)
subfamily of P-type ATPases [24]. The molecular mass
is 130 kDa - 140 kDa and are characterized by the for-
mation of an aspartyl phosphate intermediate as part of
their reaction cycle [25]. At variance with the closely
allied SERCA, PMCA contains only one Ca2+ binding
site, and indeed transports one Ca2+ as one ATP molecule
is hydrolyzed.
In addition to Mg2+-dependent Ca2+-ATPase, another
Ca2+-ATPase which can be activated without any Mg2+
has also been reported from a number of tissues and
sources with varying sensitivity to calcium and insensi-
tivity to magnesium [26-34]. Both these ATPases are
having similar properties [35-37]. They may either be
the two forms of the same enzyme having separate cata-
lytic sites or same catalytic site with different sensitivi-
ties to Mg2+ [36,38,39].
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P. C. Sen / Advances in Biological Chemistry 1 (2011) 74-92 76
1.3. H+, K+-ATPase
The gastric H+, K+-ATPase is an alpha beta (αβ) het-
erodimeric member of the eukaryotic alkali-cation P2-
type ion-motive ATPase family which undergoes a cycle
of phosphorylation and dephosphorylation coupled to the
outward and inward transport of hydrogen and potassi-
um ions, respectively. The secretory canaliculus of the
parietal cell present in the gastric glands of the stomach
perform secretion of hydrochloric acid upon hormonal
stimulation [40-43]. The ATPase sustains a 10-fold in-
ward potassium gradient (150 mM K+ in, 15 mM K+ out)
and a transmembrane outward hydrogen ion gradient of
greater than 1 million fold to generate a luminal pH of
0.8. This is the largest ion gradient generated by a P2
type ATPase.
ABC
The molecular weight of α subunit is ~114 kDa and
that of the glycosylated β subunit is ~65 kDa. The α
subunit of hog, rat and sheep is predicted to span the
membrane 10 times and a β subunit only once [40,41].
The primary structure of the α subunit of gastric H+, K+
-ATPase (HK α1) shows significant homology to the Na+,
K+-ATPase (62%) and SR Ca2+-ATPase (29%) while the
β subunits of H+, K+-ATPase and Na+, K+-ATPase are
35% identical [41].
All these above mentioned ATPases have been re-
ported to be regulated by endogenous proteins, peptides,
hormones and/or other small molecules to different ex-
tent [44,45], collectively known as ‘modulators’. For
more than two decades scientists across the globe have
been exploring different aspects of the regulation of
these ion transporting enzymes by endogenous modul-
tors.
In the present review, structure-functions of different
ATPases, how they are regulated by endogenous modu-
lators like peptides, proteins and other compounds, as
well as the mechanism of their regulation and the impli-
cations/importance have been described.
2. STRUCTURE AND FUNCTION OF
ATPASES
Jens Skou in 1957 first examined the effect of different
cations, e.g. Na+ and K+ (later named as Na+, K+-ATPase)
[46] in leg nerve homogenates of crabs. He was finally
awarded the Nobel Prize in Chemistry in 2001, i.e. , 40
years after the discovery. It is specifically and character-
istically identified in its inhibition by extra cellular
binding of cardiac glycosides, the most widely used and
well known one is ouabain [47].
Subsequent to his discovery in 1961, other ion pumps,
like Ca2+ -pump [48] with comparable characteristics but
different properties were identified from different tissues
and organisms. Most important and well characterized of
them are sarcoplasmic-reticulm (SR) Ca2+-ATPase, whi-
ch controls the contraction of skeletal muscl [17].
H+, K+-ATPase, another ion transporting enzyme ma-
inly present in the gastric cells, is well known for its
vigorous role during acid secretion process. It is ho-
mologous in sequence to the Na+, K+-ATPase and the
Ca2+-ATPase and has a similar pattern of transmem-
brane helices.
The biochemical properties that are common among
these ATPases are: 1) formation of an acid-stable, pho-
sphorylated aspartic acid residue during the pumping
cycle (that is phosphorylated intermediate) and 2) inhi-
bition by orthovanadate.
The mechanism of the Ca2+ ATPase is usually dis-
cussed in terms of the E1-E2 model developed from the
Post-Albers scheme for Na+, K+-ATPase [49]. All these
ATPases follow a similar catalytic cycle as described by
Post-Albers [49] for Na+, K+-ATPase. The reaction cycle
of the Ca2+ ATPase is shown in Fi gure 1.
Either ATP or Ca+2 can bind first to the E1 conforma-
tion of the Ca2+-ATPase. A series of conformational
changes lead to the intermediate E1”Ca2.ATP, which un-
dergoes phosphorylation to give E2PCa2. This leads to
the release of Ca2+ into the lumen followed by dephos-
phorylation to form E2, which then returns to E1.
The above model proposes that Ca2+-ATPase (SERCA)
can exist in one of the two distinct forms, E1 or E2. In the
E1 conformation, the ATPase can bind two Ca2+ ions
from the cytoplasmic site of the membrane with high
affinity whereas in the E2 conformation these two sites
are closed. Following binding of ATP, the enzyme is
phosphorylated on Asp-351 to give a phosphorylated
intermediate E2PCa2, a state in which two Ca2+ binding
sites are of low affinity and face inwards, the lumen of
the SR. Following the loss of Ca2+ to the lumen of the
SR, the ATPase dephosphorylates to E2 and then recycles
to E1. The phosphorylation events on the Ca2+-ATPase
are reversible.
Putative structure of the plasma membrane Ca2+-AT-
Pase indicating the key functional sites are shown in
Figure 1.
Putative structure of the plasma membrane Ca2+-ATP-
ase indicating the key functional sites [50] are shown in
Figure 2.
Figure 1. E1/E2 scheme for Ca2+-ATPase.

P. C. Sen / Advances in Biological Chemistry 1 (2011) 74-92 77
Figure 2. Putative structure of the plasma membrane Ca2+-ATPase indicating the key functional sites [50].
In the plasma membrane, the enzyme is organized
with ten transmembrane domains, and the NH2 and
COOH termini are both located on the cytosolic side of
the membrane. The bulk of the protein mass is facing the
cytosol and consists of three major parts: the intracellu-
lar loop between transmembrane segments 2 and 3, the
large unit between membrane-spanning domains 4 and 5,
and the extended “tail” following the last transmembrane
domain [51,52]. The first intracellular loop region be-
tween membrane-spanning domains 2 and 3 corresponds
to the “transduction domain” is believed to play an im-
portant role in the long-range transmission of conforma-
tional changes occurring during the transport cycle. The
large cytosolic region of ~400 residues between mem-
brane spanning segments 4 and 5 contains the major
catalytic domain including the ATP binding site and the
invariate aspartate residue that forms the acyl phosphate
intermediate during ATP hydrolysis. Finally, the ex-
tended COOH-terminal tail corresponds to the major
regulatory domain (calmodulin-binding domain) of the
PMCAs [53,54].
The PMCAs closely resemble that of the SERCA
[sister pump of sarco (endo) plasmic reticulum] [55].
Indeed, the major global difference between the two
types of calcium pumps is confined to the C-terminal tail,
which is generally much smaller in the SERCAs (rang-
ing from <20 to ~50 residues) than in the PMCAs (70 to
200 residues). Unlike SERCA pump, it only contains one
Ca2+-binding site, and indeed transports one Ca2+ as one
ATP molecule is hydrolyzed.
All P-type ATPases have an architectural commonality,
with cytoplasmic domains which are linked to a trans-
membrane module. The three cytoplasmic domains [k-
nown as the phosphorylation (P), nucleotide binding (N)
and actuator (A) domains], as revealed from the first
high resolution X-ray structure of the sarco (endo) plas-
matic reticulum Ca2+-ATPase (SERCA) which exchan-
ges Ca2+ for protons) [17] are responsible for ATP hy-
drolysis. The P-type ATPases have six transmembrane
helices (M1 - M6) that make up the core of the mem-
brane transport domains. Both these subclasses have ten
trans-membrane helices (M1 - M10): the core segment
(M1 - M6) and an additional carboxyterminal transmem-
brane segment (M7 - M10). Many P-type ATPases also
have regulatory (R) domains, which typically inhibit
their function [24]. Crystallographic studies have pro-
vided detail information on the pumping mechanism of
rabbit SERCA1a [17-19,56-58].
The functional cycle of P-type ATPases is typically
denoted by E1 and E2 states which in case of SERCA
relate to the binding and active transport of cytoplasmic
Ca2+ and the countertransport of luminal H+ to the
cytoplasm, respectively. In Ca2+ translocation, ATP is
involved as the key substrate in the formation of the Ca2+
occluded E1-P state [56]. However, ATP at physio-lo-
gical concentrations also exhibits a general, stimulatory
effect on the functional cycle of SERCA relating to a
noncatalytic, modulatory mode of binding to the various
ATPase intermediates. A key question as to the mecha-
nism of SERCA is then to address the structural and
functional properties of the modulatory ATP binding site
in comparison to the catalytic site and to make possible
extrapolations to other P-type ATPases [59].
There is evidence from mutational studies [60,61],
and X-ray crystallography [19,56] which strongly su-
ggests for a direct involvement of the conserved residues
in the N-domain in nucleotide binding region of rabbit
SERCA1a. Another puzzling observation relates to the
structure of SERCA in the Ca2E1 state, which exhibits an
open conformation with the N- and A-domains detached
from the P-domain [17].
The complete structural shape of the Na+, K+-ATPase
and identification of the individual domains based on a
docking model derived from SERCA1a have been re-
ported [62]. Recently, new information on the structure
and function of the Na+, K+-ATPase and the H+-ATPase
has emerged from X-ray crystal structures of the auto-
inhibited plasma membrane H+-ATPase from Arabidop-
sis thaliana (AHA2) [63] and of the pig renal α1β1γ Na+,
K+-ATPase complex (pNKA) [64,65]. A structure of
shark Na+, K+-ATPase (sNKA) was determined at 2.4 A
resolution [66] and was found to be similar to that of
pNKA. This structure more accurately resolved the
atomic interactions within the Na+, K+-ATPase, com-
pleted the β subunit ectodomain structure cholesterol
[64,65]. The mechanism by which P-type ATPases func-
tion and how it is coupled to ATP hydrolysis, general and
specific transport mechanisms have been proposed on
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P. C. Sen / Advances in Biological Chemistry 1 (2011) 74-92 78
the basis of protein chemistry and mutagenesis experi-
ments using various P-type ATPases [15,67-71] together
with structural studies of the Ca2+-ATPase from rabbit
SERCA [19,69,72,73].
When the transported cations (three Na+ for the Na+,
K+-ATPase; one proton for H+-ATPases) are bound at the
membrane transport domain of the ATPase, a confor-
mational change occurs and transmits to the P domain,
predominantly by the M4 helix [17,18,58,59,74]. Con-
formational changes associated with the functional cycle
can be examined from studying the Ca2+-ATPase. The
bound cations are occluded in the E1P state. Occlusion
occurs during phosphorylation owing to the formation of
an ATP mediated cross link of the N and P domains,
which moves the A domain to the side [19,73]. As the K+
ions of the Na+ pump bind (or when the H+-ATPase has
formed a proposed intramolecular salt bridge), the in-
duced closure of the E2P state stimulates dephosphory-
lation, and this is mediated by the TGE motif of the A
domain which activates a water molecule at the phos-
phorylation site. Dephosphorylation leads to an occluded
E2 state, with two K+ ions present in the occlusion cav-
ity of Na+, K+-ATPase [19,64,75]. In the E2 state, ATP is
bound at the N domain and stimulates an E2 to E1 tran-
sition by interfering with residues involved in tertiary
interactions between domains [59].
For the Na+, K+-TPase and SERCA, the M7 - M10
segments provide additional coordinating groups fo ions
bound at the core region of the M domain and thus con-
tribute to selectivity [17,64]. For Na+, K+-ATPase, the
M7 - M10 helices were shown to have strong influence
on the Na+ affinity, as determined by the α subunit C
terminus and its interaction network [64,65,76-79]. An
overview of the structural aspects of plasma membrane
Na+, K+-ATPase and H+-ATPase ion pumps has been
published recently [80].
3. DIVERSITY IN UNITY IN THE
FUNCTION OF ATPASES
The mechanism of ion coordination and transport of
P-type ATPase was first revealed after determination of
the molecular structure of the sarcoplasmic reticulum
Ca2+-ATPase in an E1 conformation at 2.6 A resolution
[17]. Furthermore, the structure of the Ca2+-ATPase in E2
conformations bound with phosphate analogues has also
been identified [19,57,73,81]. These structures show that
the three cytoplasmic domains rearrange to move six out
of ten transmembrane helices, thereby changing the af-
finity of the Ca2+-binding sites and the gating of the ion
pathway. These structural data have allowed construction
of homology models that address the central questions of
mechanism of active cation transport by all P-type cation
pumps. However, for Na+, K+-ATPase and H+, K+-ATPase,
which consist of both α and β-subunits, there may be
some specific differences in regions of subunit interac-
tions. Mutagenesis, proteolytic cleavage, and transition
metal-catalyzed oxidative cleavages have provided evi-
dence about residues involved in binding of Na+, K+,
ATP, and Mg2+ ions and changes accompanying E1-E2 or
E1-P-E2-P conformational transitions [67]. Recently Ogawa
et al. [75] described the crystal structure of Na+, K+-
ATPase with bound ouabain, a representative cardiac
glycoside, at 2.8 A resolution in a state analogous to E2.
2K+. Pi. Ouabain is deeply inserted into the transmem-
brane domain with the lactone ring very close to the
bound K+, in marked contrast to previous models. Due to
antagonism between ouabain and K+, the structure
represents a low-affinity ouabain-bound state. Yet, most
of the mutagenesis data obtained with the high-affinity
state are readily explained by the present crystal struc-
ture, indicating that the binding site for ouabain is essen-
tially the same [75]. The crystal structure also shows that
the beta-subunit has a critical role in K+ binding and
explains, at least partially, why the homologous Ca2+-
ATPase counter-transports H+ rather than K+, despite the
coordinating residues being almost identical [66]. Morth
et al. [64] has shown that the beta- and gamma-subunits
specific to the Na+, K+-ATPase are associated with
transmembrane helices alpha-M7/alphaM10 and alphaM9,
respectively. Electron microscopy has revealed the over-
all shape of proton pumps. The structure of a P-type pro-
ton pump determined by X-ray crystallography shows
the ten transmembrane helices and three cytoplasmic
domains define the functional unit of ATP-coupled pro-
ton transport across the plasma membrane, and the struc-
ture is locked in a functional state not previously obser-
ved in P-type ATPases [80].
4. ATP ASES AND ENDOGENOUS
MODULATORS
4.1. Regulation of Na+, K+-ATPase
Na+, K+-ATPase is found to be regulated by the endoge-
nous factors , e.g. peptides, proteins, hormones or other
small molecules, known as modulators [45]. There are
two types of factors: 1) peptidic and 2) nonpeptidic
[82-84]. Physiologically, ATPase actions are controlled
by endogenous regulator proteins. It has been reported
that an endogenous inhibitor protein inhibits porcine
spem motility and a competitive inhibitor of Na+, K+-
ATPase and is identical to  -microseminoprotein [85].
The regulators of Na+, K+-ATPase can be divided into
two groups: (a) direct modulators e.g., ouabain, some
proteins and peptides and (b) indirect modulators which
include many peptides, nonpeptideic hormones and neu-
rotransmitters etc. [82,83]. The former group can bind
directly with enzyme protein but the latter group of com-
pounds affect via binding to membranes of specific re-
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P. C. Sen / Advances in Biological Chemistry 1 (2011) 74-92 79
ceptors. Na+, K+-ATPase is also inhibited by some en-
dogenous glycosides [86], insulin [87], aldosterone [88],
endothelin-1 (ET-1) [89], thyroxin [90], bradykinin [91]
and the N-terminal fragment of substance P (SPI-5) [92]
most likely via receptor-mediated mechanism.
A group of proteins known as FXYD proteins, a fam-
ily of seven homologous single transmem-brane segment
proteins (FXYD1-7), has been observed to be expressed
in a tissue-specific fashion and regulate Na+, K+-ATPase
activity. Most of them are short chain single proteins (>
100 amino acids) except FXYD5 which is an atypically
long N-terminal sequence [44]. It has been suggested
that seven members of this family, FXYD1 (phospho-
lemman) [93], FXYD2 (-subunit of Na+, K+-ATPase)
[94,95], FXYD3 (Mat-8) [96], FXYD4 (CHIF) [97,98],
FXYD5 (RIC, dysadherin) [99], FXYD6 (Phosphohip-
polin) (7) and FXYD7 [100], are auxiliary subunits of
Na+, K+-ATPase. FXYD6 is unique in its expression in
the inner ear, suggesting a role in endolymph compo-
stion [101]. Another FXYD protein called FXYD10 with
74 amino acids has been reported to regulate the activity
of shark Na+, K+-ATPase [102]. Some endogenous pep-
tides of varying molecular weights regulate Na+, K+-
ATPase [103,104]. Recent work of our laboratory has
suggested that endogenous proteins of varying molecular
masses isolated from different sources can either inhibit
or stimulate ATPase activities [37,105-107]. The inhibit-
tors were found to inhibit partly the H+, K+-ATPase also.
The findings suggested that these proteins inhibit spe-
cifically monovalent ion transporting enzymes. Partial
amino acid sequence of the 70 kDa inhibitor protein of
Na+, K+-ATPase from goat spermatozoa cytosol [108]
showed about 80% - 100% homology with mice [109],
pig [110] and human [111] testicular aryl sulphatase.
Another novel protein, MONaKA was reported to inter-
act with plasma membrane Na+, K+-ATPase and modu-
lates its activity [112].
Purification of non-peptidic endogenous Na+, K+-AT-
Pase inhibitors to its high purity has been reported [113]
and the structures of three peptidic inhibitors (SPAI-1,
-2,-3) have been revealed [114]. A small peptide of mo-
lecular mass approximately 600 dalton isolated from
human cerebrospinal fluid has been found to specifically
inhibit Na+, K+-pump [115].
The involvement of cAMP-dependent protein kinase
(PKA) in acute sodium pump regulation has been docu-
mented in 20 different mammalian tissues and in lower
vertebrates [116]. The result can be either stimulation or
inhibition of the pump, however, and in no case is the
pathway completely understood. These structure of the
Ca2+-ATPase loop homologous to the Na+, K+-ATPase, it
was suggested that PKA site should be inaccessible to
the kinase [117]. Regulation occurs on several levels:
biosynthesis and degradation; reversible recruitment to
and internalization from the plasma membrane; altera-
tion of affinity for Na+; and either stimulation or inhibit-
tion of activity.
4.2. Regulation of Ca2+-ATPase
Fewer reports are available on endogenous protein sti-
mulators/inhibitors of Ca2+-ATPases. Calmodulin is the
naturally occurring activator of plasma membrane Ca2+-
ATPase. A protein of molecular weight 63 kDa from
human erythrocyte membrane [118], and another ~56
kDa - 60 kDa from dog and beef heart sarcolemma have
been reported to stimulate Mg2+, Ca2+-ATPase. Regu-
calcin, a calcium binding protein plays a pivotal role in
maintaining intracellular Ca2+ homeostasis due to active-
tion of Ca2+ pump enzymes in plasma membrane (baso-
lateral membrane), microsomes (endoplasmic reticulum)
and mitochondria of many cell types [119]. Other acti-
vators are acidic phospholipids and long chain polyun-
saturated fatty acids [120]. Phosphorylation by protein
kinase A and protein kinase A inhibitor (PKI) purified
from bovine heart stimulates Ca2+, Mg2+-ATPase activity
in human erythrocytes [121]. Recently, PDC-109, the
major secretory protein from bull seminal vesicles has
been described to stimulate bovine sperm membrane
Ca2+-ATPase. An analysis of the enzyme kinetics data
suggested irreversible, cooperative interaction of PDC -
109 with the enzyme, the stimulation being organ spe-
cific, but not species specific [33]. A 12 kDa protein
from rat brain cytosol reported from the author’s labora-
tory has been found to inhibit Mg2+-independent Ca2+-
ATPase while stimulating the Mg2+-dependent form of
the enzyme [37], another 14 kDa protein either from
goat spermatozoa [122,123] or bovine brain [124,125] is
found to stimulate Mg2+-independent Ca2+-ATPase with-
out any significant effect on Mg2+-dependent form of the
ezyme. Narayanan et al. [126] reported a cytosolic pro-
tein fraction, termed CPF-I, obtained by (NH4)2SO4 frac-
tionation of rabbit heart cytosol which caused marked
inhibition (up to 95%) of ATP-dependent Ca2+ uptake by
cardiac sarcoplasm reticulumn. An endogenous inhibitor
of SR Ca+2-ATPase has been reported from human pla-
centa, distributed in cytosol and microsomal fractions
[127].
Sarcolipin (SLN) in vitro inhibits SERCA1 or SER-
CA2 pump activity. However the exact nature of regula-
tion by sarcolipin is not fully understood [128].
Phopholamban (PLN), an oligomeric proteolipid has
been reported to inhibit cardiac Ca2+-ATPase [129]. It is
composed of 52 amino acids and is organized in three
domains: (a) cytosolic domain 1a (amino acids 1 - 20);
cytosolic domain 1b (amino acids 21 - 30) and domain II
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P. C. Sen / Advances in Biological Chemistry 1 (2011) 74-92 80
(amino acids 31 - 52) which tranverse the domains [130].
Various attempts have been made to therapeutically
target of SERCA2a and PLN in animal and human heart
failure models [131]. In vitro mutagenesis studies evalu-
ating the interaction between PLN and SERCA2a have
been directed toward the cytoplasmic domains (aa resi-
due, 1 - 30), which include the phosphorylation sites in
PLN. These studies demonstrate the importance of PLN
interaction with SERCA2a and suggest that interferance
with this interaction may provide a novel therapeutic
approach for prevention of dilated cardiomyopathy. Con-
vincing evidence suggested that the impaired function of
the SR to cycle Ca2+ during diastole and systole is a
critical defect in cardiomyocytes from failing hearts.
Strategies to interfere with the PLN/SERCA2a interact-
tion have been proposed as therapy to improve Ca2+ cy-
cling, contractility, and relaxation in failing and nonfail-
ing animal and human heart models [132]. The recent
mutation identified in a human genetic study has sug-
gested the need for further refinement of the new thera-
peutic concept of PLN inhibition [133]. A class of in-
hibitors has emerged, named “caloxins” defined as sub-
stances that bind to the PEDs (putative extracellular do-
mains) in PMCA to inhibit any conformational changes
in them during the reaction cycle and hence modulate
PMCA activity. The first caloxin (caloxin 2A1) was dis-
covered as a peptide that would bind a synthetic peptide
corresponding to the sequence of PED2 of PMCA [134].
Caloxin 2A1 does not affect the activities of Mg2+-AT-
Pase or Na+, K+ ATPase in the erythrocyte ghosts,
SERCA1 Ca2+, Mg2+-ATPase in fast twitch skeletal mu-
scle. Caloxin 3A1, based on PED3, inhibits the PMCA
pump but not the sarcoplasmic reticulum Ca2+ pump
[135].
Interleukin-2 (IL-2), one of the most important cyto-
kines, generally produced by activated helper T-lympho-
cytes and stimulates proliferation and effector functions
of various cells of immune system, has been shown to
increase the activity of SR Ca2+-ATPase in rat cardio-
myocytes [136].
However, it also decreases the sensitivity of SR Ca2+-
ATPase to calcium. It has been demonstrated that 3,5,3’-
tri-iodo-L-thyronine (T3), in rat thymocytes, increases
plasma membrane Ca2+-ATPase activity [137]. The ef-
fect of T3 is rapid, concentration related, evident at a
physiological concentration as low as 1 pM. The amphi-
philic peptide mastoparan, isolated from wasp venom,
has been shown to be a potent inhibitor of the sarco-
plasmic reticulum Ca2+-ATPase. The peptide also de-
creases the affinity of the enzyme for Ca2+ and abolishes
the cooperativity of Ca2+ binding [138]. Myotoxin a, a
polypeptide of 43 amino acids from the prairie rattle
snake Crotalus viridis viridis [139] and mellitin, a basic
peptide isolated from bee venom inhibit the activity of
the Ca2+-ATPase of skeletal muscle sarcoplasmic reticu-
lum [140]. Palytoxin, a coral toxin significantly reduces
Ca2+ pumping of isolated SR vesicles [141]. A variety of
polyamines, including spermine, spermidine and polyar-
ginine inhibit the Ca2+-ATPase of skeletal muscle [142].
The skeletal muscle SR Ca2+-ATPase is stimulated by
jasmine and jasmonate and increase the rate of depho-
sphorylation of the ATPase [143]. Reports show that
ceramide stimulates the plasma membrane Ca2+-ATPase
activity in a dose dependent manner and an additive ef-
fect in activation was observed in presence of calmo-
dulin and ethanol [144]. Ceramide affects the affinity for
Ca2+ and Vmax of the enzyme, and also stimulates Ca2+
transport in inside-out plasma membrane vesicles from
erythrocytes. Sphingosine, on the other hand, inhibits the
calmodulin stimulated enzyme [141]. Ivermectin, a ma-
crocyclic lactone (IC50 = 7 µM), and cyclosporin A (a
cyclic oligotide) have been shown to be potent inhibit-
tors of SERCA1. Ivermectin inhibition has been shown
to be competitive with respect to high concentrations of
ATP, increase of Km at the regulatory binding site [145].
5. MECHANISM OF REGULATION
It is evident from the above discussion that different
endogenous regulators modulate various ion transporting
enzymes. Questions come, what are the mechanisms of
regulation?
Na+, K+-ATPase has been reported to be inhibited by a
number of proteins of varying molecular weights. A 12
kDa - 13 kDa molecular mass protein from rat brain was
found not to compete with ouabain (a specific inhibitor
of Na+, K+-ATPase) in inhibiting the enzyme, whereas,
an additive effect was observed in combination with
ouabain, suggesting their different binding sites on AT-
Pase. The inhibitor is responsible for controlling the
phosphorylation of the ATPase and, thus, its activity and
this is due to the binding to E2 state of the enzyme. The
inhibition was found to be due to conformational change
of the ATPase on binding to the inhibitor [105].
Another group of high molecular mass (70 kDa - 75
kDa) proteins isolated from rat brain and goat sper-
matozoa cytosol was found to inhibit Na+, K+-ATPase
without having any appreciable effect on Ca2+-ATPase.
In both the cases, inhibition was found due to blocking
of the phosphorylation step of the overall reaction se-
quences [106,107]. The one isolated from rat brain
(75kDa) inhibits Na+, K+-ATPase activity reversibly and
binds to the E2 state of the enzyme. CD spectra indicates
a slight loss of α -helix and random coil in the enzyme-
inhibitor complex [107]. While the inhibition of the Na+,
K+-ATPase by 70 kDa protein isolated from goat sper-
matozoa was found to be competitive in nature with re-
spect to its substrate, the binding is reversible and inhi-
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P. C. Sen / Advances in Biological Chemistry 1 (2011) 74-92 81
bition was found to be due to the change in conformation
of the enzyme [106]. The fact that both these inhibitors
bind reversibly with the enzyme may act as control ele-
ment of the transport enzyme and suggests their impor-
tant role as endogenous regulators.
The SPAIs isolated from porcine duodenum, has been
shown to inhibit Na+, K+-ATPase by the competitive
mode against Na+ and non-competitive to K+ [146]. An-
other regulator, PE-60 has an activating influence on Na+,
K+-ATPase from rat brain frontal cortex, the peptide
stimulates the enzyme apparently due to Na+-dependent
steps of the Na+, K+-ATPase system. The activating ef-
fect was enhanced by preincubation at low concentra-
tions of ATP that transforms the enzyme into the Na+-
form [103]. It is obvious that the interactions of PE-60,
as well as SPIs, with Na+, K+-ATPase are relatively
complex, both of them exerted their effect by binding to
two different sites of the enzyme with different affinities.
At low concentration, PEC-60 acts as an activator of the
enzyme and at higher concentrations the activating effect
is reversed by binding another molecule of PEC-60
[103]. Although SPIs have been reported to inhibit Na+,
K+-ATPase, the effect was found to be biphasic, at sub-
micro-molar concentration about 20% stimulation was
observed from intestine whereas at higher peptide con-
centration inhibition was found [146]. The porcine
sperm motility inhibitor (molecular mass, 15 kDa) is
identical to β-microseminoprotein and is a competitive
inhibitor of Na+,K+-ATPase [85]. With MONaKA, it has
been revealed that it binds tightly to the β1 and β3 sub-
units of the Na+, K+-ATPase. The association between
MONaKA and Na+, K+-ATPase β-subunit was confirmed
by coimmunoprecipitation from transfected cells, mouse
brain, and cultured mouse astrocytes [112]. Hormones
like aldosterone has been reported to increase the Na+,
K+-ATPase function in cultured AT2 cells. This was as-
sociated with an increase in the β1-subunit mRNA levels
and β1-subunit protein abundance in AT2 cell plasma
membranes. It has been demonstrated that the human
Na+, K+-ATPase is transcriptionally regulated by aldos-
terone and may involve a direct interaction with poten-
tial hormone response elements present in the promoter
region of these genes. Endothelin-1 (ET-1), a hormone,
increases the Na+, K+-ATPase activity in epithelial cells
by enhancing the mRNA and protein levels of the α1
subunit suggesting ET-1 could play a homeostatic role in
modulating aqueous humor formation by having differ-
ential effects on the activity and expression of Na+, K+-
ATPase by the ciliary epithelium in the eye [89].
FXYD proteins modulate the function of Na+, K+-
ATPase by affecting its kinetic properties in specific way.
Although effects of FXYD proteins on parameters such
as K+
1/2, Na+ 1/2, KmATP and Vmax are modest, usually two
folds, however these effects may have important long-
term consequences for homeostasis of cation balance.
Functional modulators are likely to affect the ATPase
activity possibly by altering rate limiting steps, particu-
larly the E1P-E2P and E2(K)-E1 conformational transi-
tions, or binding of the transported cations, particularly
cytoplasmic Na+ ions (or competing K+ ions), which
may limit the rate of active Na+ pumping in vivo. Al-
though some conclusions on the effects of FXYD on
cation binding and conformational transitions have been
drawn, direct observations with purified α/β/FXYD com-
plexes would be more conclusive [44].
Evidence suggests multiple sites of interactions be-
tween FXYD and α/β subunits. Data supporting this
came from the fact that the anti-γC terminus neutralizes
the effect of γ on the apparent ATP affinity in renal Na+,
K+-ATPase or HeLa cells transfected with γ, but not with
K+: Na+ antagonism [16,44]. Expression in HeLa cells of
γ with either 4 or 10 C-terminal residues or 7-deleted N-
terminal residues removes the effect of γ on ATP affinity
but does not affect the K+: Na+ antagonism. Replacement
of the deleted 7 N-terminal residues with 7 alanines re-
stores the effect on ATP affinity [93].
Mutational analysis combined with expression in
Xenopus oocytes reveals that Phe956, Glu960, Leu964, and
Phe967 in TM9 of the Na+, K+-ATPase α subunit repre-
sent one face interacting with the three FXYD proteins
i.e. FXYD2, FXYD4, and FXYD7. Leu964 and Phe967
contribute to the efficient association of FXYD proteins
with the Na+, K+-ATPase α subunit, whereas Phe956 and
Glu960 are essential for the transmission of the functional
effect of FXYD proteins on the apparent K+ affinity of
Na+, K+-ATPase. The relative contribution of Phe956 and
Glu960 to the K+ effect differs for different FXYD pro-
teins, due to the intrinsic differences of FXYD proteins
on the apparent K+ affinity of Na+, K+-ATPase. In con-
trast to the effect on the apparent K+ affinity, Phe956 and
Glu960 are not involved in the effect of FXYD2 and
FXYD4 on the apparent Na+ affinity of Na+, K+-ATPase.
The mutational analysis followed by a docking model of
the Na+,K+-ATPase/FXYD7 complex, predicted the im-
portance of Phe956, Glu960, Leu964, and Phe967 in subunit
interaction [147]. It has been suggested that FXYD pro-
teins modulate Na+, K+-ATPase activity in close coop-
eration with post-translational modifications such as
phosphorylation [148].
Recent study shows FXYD1 associated with the Na+,
K+-ATPase α and β subunits, and that the effects of
phosphorylation by PKA on the Na+, K+-ATPase regula-
tory activity of FXYD1 could be due primarily to chan-
ges in electrostatic potential near the membrane surface
and near the Na+/K+ ion binding site of the Na+, K+-ATP-
ase α subunit [149]. Furthermore, protein kinase phos-
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P. C. Sen / Advances in Biological Chemistry 1 (2011) 74-92 82
ABC
phorylation also seems to involve direct modification of
Na+, K+-ATPase catalytic activity both in vitro and in
vivo, at least in some systems [149]. Interestingly, FXYD
proteins are found to be involved in the regulation of
some diseases. Recently Okudela et al. [150] has re-
ported that FXYD3 levels were also lower in a consid-
erable proportion in primary lung cancers than in non-
tumoral airway epithelia; its expression levels decreased
in parallel with the dedifferentiation process. Therefore,
it was suggested that inactivation of FXYD3 through a
gene mutation or unknown mechanism could be one of
the causes of the atypical shapes of cancer cells and play
a potential role in the progression of lung cancer.. The
latest addition of FXYD11 gene (zFXYD11) regulating
the transport ability of NaK-MRCs (mitochondrion-rich
cells) by modulating Na+, K+-ATPase activity may be
involved in the controlling of body fluid and electrolyte
homeostasis [151].
Anionic phospholipids increase the intermolecular
cross-linking between the FXYD10 C-terminus (Cys74)
and the Cys254 in the Na+, K+-ATPase (in shark) A-
domain. However it has been suggested that phosphory-
lation involves only modest structural rearrangements
between the cytoplasmic domain of FXYD10 and the
Na+, K+-ATPase A-domain [147]. The salinity-dependent
expression of pFXYD (pufferfish FXYD gene) protein
and Na+, K+, as well as the evidence for their co-loca-
lization and interaction in pufferfish gills suggested that
pFXYD regulates Na+, K+-ATPase activity in gills of
euryhaline teleosts upon salinity challenge [152].
The ATPase described here gets phophorylated by
different protein kinases for their function. The protein
kinase A/protein kinase C (PKA/PKC) phosphorylation
profile of H+, K+-ATPase is very similar to the one found
in the Na+, K+-ATPase. PKC phosphorylation is taking
place in the N-terminal part of the α-subunit with a
stoichiometry of 0.6 mol Pi/mole α-subunit. PKA phos-
phorylation is in the C-terminal part and requires deter-
gent, as is also found for the Na+, K+-ATPase. The stoi-
chiometry of PKA-induced phosphorylation was 0.7 mol
Pi/mole α-subunit. The Na+, K+-ATPase is also known to
be regulated by membrane trafficking controlled by N-
terminal PKC phosphorylation [63] through a mecha-
nism that involves binding of phosphoinositide 3-kinase
to a polyproline motif in the N-terminus [153]. Con-
trolled proteolysis of the N-terminus abolished PK C
phosphorylation of native H+, K+-ATPase [102].
A number of low molecular mass proteins reported
from the author’s laboratory has been found to affect
Mg2+, Ca2+-ATPase and Ca2+-ATPase differentially. One,
molecular mass of 12 kDa from rat brain has been re-
ported to stimulate Ca2+, Mg2+-ATPase and inhibit Ca2+
-ATPase (SERCA family), the binding between enzy-
mes and the protein was found to be reversible and non-
competitive in nature. The modulator was found to be
negatively charged protein and could be a good tool in
distinguishing the regulation of these two ATPases [37].
On the other hand, a protein with molecular mass 13,961
from goat testes cytosol [122] has been found to stimu-
late Mg2+-independent Ca2+-ATPase without having any
appreciable effect on Mg2+-dependent one. The stimul-
torbinds to a single site of the enzyme. The effect was due
to enhancement of the dephosphorylation rate of the overall
reaction steps and acceleration of the calcium uptake [123].
Another 14 kDa mass stimulator of Mg2+-independent
Ca2+-ATPase (belongs to SERCA family) has been reported
by Ghoshal et al. [124,125] from bovine brain and was
found to enhance the Ca2+-uptake. It also stimulates sperm
motility suggesting its role in fertility. Proteomic analysis
suggests its similarity with thyroid hormone-responsive
protein [154]. The effects in both the above cases were
found to be non-species specific against SERCA.
Myotoxin a, a polypeptide from the prairie rattle sna-
ke was found to inhibit Ca2+-ATPase by decreasing the
rate of dephosphorylation, with no effect on the Ca2+-
transport step ]139].
Based on the findings of the author’s laboratory, fo-
llowing schemes (Figures 3(a) and (b)) have been pro-
posed for the regulation of Mg2+-independent and Mg2+-
dependent Ca2+-ATPases by various endogenous modu-
lator proteins.
Each of the modulator reported from the author’s la-
boratory [37,105-107,122,124] has been found to bind to
the respective ATPase in a reversible manner. Thus it has
been hypothesized that they can act as endogenous
regulators of the ATPases. Moreover, an “on” and “off”
type of mechanism has been proposed, whereby the mo-
dulator remains inactive under normal condition (off). It
gets activated (on) when a malfunction of the ATPase/
pump occurs, binds to the enzyme, brings it to the nor-
mal condition then dissociates. A cartoon as shown be-
low in Figur e 4 has been proposed.
The most important among the regulators of Ca2+-
ATPase is calmodulin. The PMCA pump has high affin-
ity for Ca 2+ (Km < 0.5 µM) when complexed with cal-
modulin, the physiological activator of the pump [155].
Calmodulin stimulates the Vmax of the enzyme, but espe-
cially decreases the Km (Ca2+) by one order of magnitude
i.e. from about 10 µM - 20 µM to about 0.5 µM. The
pump interacts with calmodulin in a Ca2+-dependent man-
ner and has high affinity for it: a Kd of about 1 nM has
been observed [53]. The regulation of the pump by cal-
modulin binding in its C-terminal tail provides a striking
example of the autoregulation of the Ca2+. Ca2+-
calmodulin binds to a region in the COOH-terminal por-
tion of the PMCAs, located ~40 residues downstream of
the last transmembrane domain[156]. In the absence of

P. C. Sen / Advances in Biological Chemistry 1 (2011) 74-92 83
E+ATP2Ca
2+
(in) E.MgATP.Ca
2
A
DP
E
2
P
Mg
2+
+2Ca
2+
P
i
E
S
t
i
m
u
l
a
t
o
r
E
1
P.Mg.Ca
2
(a)
E2Ca2+(low,in)+ATP E.Ca.AT P
C
a
2
+
.
A
D
P
E1P
E2P
Ca2+ (high,out)
E
Mo
d
u
l
a
tor
P
i
(b)
Figure 3. (a) Mg2+-dependent Ca2+-ATPase. Phosphorylation is followed by a complex
formed in presence of Mg2+ and ATP followed by dephosphorylation. Endogenous protein
was found to stimulate (↑) the enzyme activity. (b) Mg2+-independent Ca2+-ATPase. Phos-
phorylation and dephosphorylation are controlled by low (high affinity) and high (low affin-
ity) concentration of Ca2+. The modulator proteins can either stimulate (↑) or inhibit (↓) the
enzyme activities.
Figure 4. A sc hematic model showing binding of the modulator with the ATPase and its dissociation.
Ca2+-calmodulin, this sequence acts as an “autoinhibi-
tory domain”; cross-linking studies using labeled pep-
tides demonstrated that the calmodulin binding domain
interacts intramolecularly with two separate regions of
the pump, one located in the first cytosolic loop and the
other in the major catalytic unit between the phosphory-
lation and the ATP binding site [155]. This intramolecu-
lar interaction probably hinders the access of Ca2+ and/or
ATP to the active site, preventing catalytic turnover, keep-
ing the pump in an inhibited state. An elevation in the cyto-
plasmic Ca2+ results in an increase in Ca2+-calmodulin,
which then binds with high affinity to the autoinhibitory
domain of the PMCA, thereby releasing the inhibition and
stimulating pump activity to near-maximal potential.
The peptide, caloxin inhibits Ca2+, Mg2+-ATPase ac-
tivity in leaky erythrocyte ghosts with a Ki value of 0.4
mmol/L - 0.8 mmol/L. The inhibition is noncompetitive
with respect to the substrates and calmodulin [157]. Ca-
loxin1A1 inhibits PMCA by binding to the first ex-
tracellular domain [135] while caloxin1B1 is isoform
selective with a higher affinity for PMCA4 than PMCA1
[158]. The effect of spermine, particularly, is highly spe-
cific; inhibition resulting from the decrease in the rate of
dissociation of Ca2+ from the phosphorylated ATPase
(Ca2E1P → E2P) [142]. It has been suggested that regu-
calcin (maintains intracellular Ca2+-homeostasis) may
act on the SH groups of Ca2+-ATPase by binding to mi-
crosomal membranes [119]. Effects of different endoge-
nous modulators on affecting activities and/or affinities
of the enzymes to the substrate and/or co-factors are
shown in Tables 1 and 2.
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Ta ble 1. Effects of different modulators on the activities and kinetic pameters of Na+, K+-ATPase. ↑ stimulation and ↓ inhibition of
the enzyme activities.
Modulators Enzymes Kinetic parameters change Activity (↑↓) Refs.
Na+, K+-ATPase K1/2 Na+ - [93]
(rat kidney, bovine and rat cardiac sarcolema); K1/2 K+
FXYD1
rat cardiac Na+, K+-ATPase - ↑ [159]
Na+, K+-ATPase (renal) KmATP - [94]
knockout mouse K1/2 Na+ - [94] FXYD2
(kidney membrane) KmATP
Na+, K+-ATPase K1/2 Na+
FXYD3
(X.oocytes) K1/2 K+
FXYD4 Na+, K+-ATPase (mammalian) K1/2 Na+ - [160]
FXYD7 Na+, K+-ATPase (brain) K1/2 K+ - [100]
Na+, K+-ATPase K1/2 Na+ ↓ [114]
SPAI-1
(mammalian kidney K1/2 K+
12 kDa - 13 kDa Na+, K+-ATPase K1/2 Na+ ↓ [105]
Protein (rat brain) (rat brain) K1/2 K+
75 kDa protein Na+, K+-ATPase K1/2 Na+ ↓ [107]
(rat brain) (rat brain) K1/2 K+
70 kDa protein Na+, K+-ATPase K1/2 Na+ ↓ [106]
(goat testis) (rat brain) K1/2 K+
Table 2. Effects of different modulators on the activities and kinetic pameters of Mg2+-independent and Mg2+-dependent
Ca2+-ATPases. ↑ stimulation and ↓ inhibition of the enzyme activities.
Modulators Enzymes Kinetic parameters change Activity (↑↓) Refs.
PDC-109 Mg2+-independent KmAT P ↑ [33]
(bovine seminal and dependent vesicles) Ca2+-ATPase
(bovine spermatozoa)
12 kDa protein Mg2+-independent K1/2 Ca2 ↓ [37]
(rat brain) Ca2+-ATPase KmATP
Mg2+-dependent
Ca2+-ATPase K1/2 Ca2+ ↑ [37]
(goat spermatozoa) KmATP
4 kDa protein Mg2+-independent K1/2 Ca2+ ↑ [124]
(bovine brain) Ca2+-ATPase
4 kDa protein Mg2+-independent K1/2 Ca2+ ↑ [122]
(goat testis) Ca2+-ATPase
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ABC
Two other low molecular weight proteins which can
modulate SERCA pump activities are phospholamban
and sarcolipin [161,162]. In vitro, phospholamban in-
hbits SERCA1 and SERCA2, but not SERCA3. The p-
hosphorylation of phospholamban by the catalytic su-
bunit of the cAMP-dependent protrein kinase reverses
the inhibitory effect of Ca2+-pump. This suggests that
phosphorylation of an inhibitor of the Ca2+-ATPase in
cardiac SR and dephopshorylation relieves its inhibition.
The direct interaction between hydrophilic portion of
PLN may be one of the mechanisms of the regulation. It
is clear that SERCA2a and PLN have a critical role in
SR Ca2+ cycling and contractility. Thus, elucidating mo-
re specific roles that these proteins play in the deve-
lopment of cardiomyopathy may aid in development and
improvement of drugs for the treatment of cardiac dis-
ease, and ultimately lead to the generation of novel ge-
netic therapy for human heart failure [129]. PLN binding
inhibits the Ca2+ pumps by lowering their Ca2+ affinity
[161]. It can exist as a monomer or as a pentamer and it
is the latter which appears to be responsible for the inhi-
bition [163]. The transmembrane interaction sites (a re-
sidue, 31 - 52) were also shown to mediate the regula-
tory effects of PLN on SERCA2a affinity, with some
mutations yielding increased inhibition while others abo-
lished the PLN inhibitory effects on SERCA2a in vitro
[164]. The total expression levels of one phospholamban
per Ca2+-ATPase result in full inhibition of the enzyme
activity. The excess PLN expressed in the heart over that
required for inhibition suggests a capability for graded
responses of the Ca2+-ATPase activity to endogenous ki-
nases and phosphatases that modulate the level of phos-
phorylation necessary to relieve inhibition of the Ca2+-
ATPase by PLN [165]. Mutagenesis studies show the
SERCA sequence Lys397-Asp Acid-Asp Acid-Lys-Pro-
Val402 are essential for regulation by phospholamban
[133]. This sequence interacts with the cytoplasmic do-
main of phospholamban. Since this sequence is missing
in SERCA3, it is not inhibited by PLN. It interacts with
the SERCA pump both at the cytosolic nucleotide bind-
ing domain (Lys400 in the N domain) [166] and with the
transmembrane sector, maintaining the pump in an in-
hibited state. The inhibition is removed by phosphoryla-
tion of residues Ser16, Thr17 and Ser10 in the hydrophilic
portion by protein kinase A, a Ca2+/calmodulin-dependent
kinase-II and protein kinase C respectively. The phos-
phorylation is assumed to induce a conformational chan-
ge of PLN that forces its detachment from the pump both
at the cytosolic and the transmembrane interacting sites.
PLN can be again dephosphorylated by protein phos-
phatases.
Sarcolipin is a 31 amino acid peptide expressed pre-
dominantly in the fast twitch skeletal muscle [167], al-
though in vitro it inhibits SERCA1 or SERCA2 pump
activity. Amino acid sequence comparison and modeling
studies have shown that the transmembrane helices of
SLN and PLN share considerable homology, suggesting
both proteins interact in a similar way with SERCA [128,
168-170]. A recent study suggests that the lumenal do-
main could be involved in the retention of SLN in the
ER, although it might have a different function too [171].
The flexible nature of the C-terminus also leaves Tyr-29
and Tyr-31 residues available for interactions with vari-
ous aromatic residues in the transmembrane helices of
SERCA and suggests that lumenal domain could be in-
volved in the regulation of SERCA-SLN interaction
[128,171]. Recently Morita et al has proposed the inter-
actions among PLN, NF-SLN and SERCA1a and found
that mutation of amino acids Ile40, Ile47, and Ile48 in PLN
and mutation of Val19, Ile22 and Trp23 in NF-SLN dimin-
ished either the super-inhibition imposed on SERCA1a
function by the PLN-NF-SLN binary complex or the
physical interactions between PLN and NF-SLN or both
[172].
6. CONCLUSIONS
The foregoing discussion along with the supporting in-
formation suggest that endogenous modulators of vary-
ing molecular masses regulate ATPase activities and are
believed to act as physiological regulators of different
ATPases. The question may be raised whether the modu-
lator(s) particularly when they are proteins would reach
the target site in their native form when use from outside
since they may be degraded by proteolysis. The other
possibility is to develope antibody against these modu-
lators due to prolong use of them.
However, inspite of these short comings, protein re-
gulators may be useful tool for develping drugs with be-
tter acceptability since they are endogenous in nature.
We have found that one of the modulators of Ca2+-ATP-
ase [37] can act as a female contraceptive agent when
tested on rat and rabbit. An Indian patent has been a-
warded on this work. Simuilarly, it is clear that SERCA-
2a and PLN have a critical role in SR Ca2+ cycling and
contractility. Thus, elucidating more specific roles that
these proteins play in inducing cardiomyopathy may aid
in development and improvement of drugs for the treat-
ment of cardiac disease, and ultimately lead to the gen-
eration of novel genetic therapy for human heart failure
[131]. SERCA is responsible for the reuptake of cytop-
lasmic Ca2+ in muscle, where it ensures the efficient re-
laxation at the end of a contraction event. Recently it has
been reported that FXYD3 levels were decreased in a
considerable proportion of primary lung cancers than in
nontumoral airway epithelia. Its expression levels de-
creased in parallel with the dedifferentiation process.

P. C. Sen / Advances in Biological Chemistry 1 (2011) 74-92 86
Therefore, it has been suggested that inactivation of
FXYD3 through a gene mutation or unknown mecha-
nism could be one of the reasons of the atypical shapes
of cancer cells and play a potential role in the progress-
sion of lung cancer [150]. Therefore activation of FXYD3
by any out side agent may provide a lead of developing
an anticancer drug.
Hence, it may be suggested that some of these modu-
lators may be utilized as drug against a specific disease
which is considered to be quite important and interesting
from physiological, biochemical and medicinal point of
view.
7. ACKNOWLEGDEMENTS
The work in the author’s laboratory was supported by Bose Institute
and a part by a project from Council of Scientific and Industrial Re-
search [EMR-37 (1299)/07)]. The author is thankful to Dr. Atin K.
Mandal, Ms Pinki Nandi and Ms Swatilekha Ghosh, Division of Mo-
lecular Medicine, for comments, criticism and meaningful discussion
during preparation of the article.
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