MINIREVIEW
The sodium pump
Its molecular properties and mechanics of ion transport
Georgios Scheiner-Bobis
From the Institut fu
¨
r Biochemie und Endokrinologie, Fachbereich Veterina
¨
rmedizin, Justus-Liebig-Universita
¨
t Giessen, Germany
The sodium pump (Na
+
/K
+
-ATPase; sodium- and potas-
sium-activated adenosine 5¢-triphosphatase; EC 3.6.1.37)
has been under investigation for more than four decades.
During this time, the knowledge about the structure and
properties of the enzyme has increased to such an extent that
specialized groups have formed within this field that focus on
specific aspects of the active ion transport catalyzed by this
enzyme. Taking this into account, this review, while some-
what speculative, is an attempt to summarize the informa-
tion regarding the enzymology of the sodium pump with the
hope of providing to interested readers from outside the field
a concentrated overview and to readers from related fields a
guide in their search for gathering specific information
concerning the structure, function, and enzymology of this
enzyme.
Keywords: ATPase; P-type; ouabain; palytoxin; ion

+
per electron or, in other words, 3 Na
+
per ATP) and the fact that ouabain had already been shown
to inhibit sodium fluxes on frog skin, contributed to the
overall acceptance of Skou’s conclusion from 1957, which
identified in crab nerve membrane preparations the sodium
pump as an ATPase that was activated by Na
+
and K
+
and inhibited by ouabain [1].
Undoubtedly, however, all of these findings helped to
lay the cornerstone in the research field of ion transport,
which currently includes a vast number of primarily and
secondarily active transporters or ion channels. Among
them, the Na
+
/K
+
-ATPase takes its place within the
family of the so-called P-type ATPases, enzymes that
become autophosphorylated by the gamma phosphate
group of the ATP molecule that they hydrolyze. The Na
+
/
K
+
-ATPase was the first discovered ion transporter, and
indeed the first-discovered P-type ATPase. It is still,

Abbreviations:Na
+
/K
+
-ATPase, sodium- and potassium-activated
adenosine 5¢-triphosphatase; FSBA, 5¢-p-fluorosulfonylbenzoyl-
adenosine; ClR-ATP, c-[4-(N-2-chloroethyl-N-methylamino)]benzyl-
amide ATP; FITC, 5¢-isothiocyanate.
Enzyme: sodium- and potassium-activated adenosine
5¢-triphosphatase (EC 3.6.1.37).
(Received 15 October 2001, revised 11 December 2001,
accepted 28 January 2002)
Eur. J. Biochem. 269, 2424–2433 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02909.x
The sodium pump, also known as the Na
+
/K
+
-ATPase,
is responsible for establishing and maintaining this electro-
chemical gradient in animal cells. This enzyme is a
component of the plasma membrane and transports Na
+
and K
+
using ATP hydrolysis. For every molecule of ATP
hydrolyzed, three Na
+
ions from the intracellular space and
two K
+

Within the group of the P
2
-type ATPases, the Na
+
/
K
+
-ATPase, together with the colonic or gastric H
+
/
K
+
-ATPases, constitute a subgroup of oligomeric enzymes
consisting of a and b subunits. A third peptide referred to as
the c subunit appears in some tissues to be involved in
regulating the activity of the sodium pump and its
interactions with Na
+
or K
+
ions.
A number of isoforms of the a and b subunits has been
isolated from various tissues of numerous species, and it has
been repeatedly demonstrated that the function of Na
+
/
K
+
-ATPase requires the presence of both subunits.
The a subunit, which is referred to as the catalytic

associates with the sodium pump [6], possibly via interac-
tions with the C-terminal domain of the a subunit [7]. The
c subunit belongs to type I membrane proteins and is
related to phospholemman and to the human Mat8
protein, a type I membrane protein associated with
mammary tumors. The availability of the cDNA coding
for the peptide permitted analysis of the role of the
c subunit in the function of the enzyme. Consistent with
the fact that c expression is not seen in all tissues where
a or b expression is otherwise easily identified, the presence
of the c peptide is not essential for obtaining Na
+
/
K
+
-ATPase activity in heterologous expressions systems
of the enzyme [8]. Nevertheless, c subunit expression in
HEK cells apparently modifies the affinity of the enzyme
for ATP, and its expression in different segments of the
nephron is associated with modulation of the affinity of
Na
+
/K
+
-ATPase for Na
+
or K
+
ions [9,10]. These data,
together with the fact that several peptides similar to the

+
and ATP
bind with very high affinity (K
d
values of 0.19–0.26 m
M
and
0.1–0.2 l
M
, respectively) to the E
1
conformation of the
enzyme (Fig. 1, step 1), during which phosphorylation at an
aspartate residue occurs via the transfer of the c-phosphate
of ATP (Fig. 1, step 2) [12,13]. Magnesium is very
important for this reaction. Thereafter, three Na
+
ions
are occluded while the enzyme remains in a phosphorylated
condition. After the E
2
-P3Na
+
conformation is attained,
the enzyme loses its affinity for Na
+
(K
0.5
¼ 14 m
M

Intracellular ATP increases the extent of the release of
K
+
from the E
2
(2K
+
) conformation (Fig. 1, step 6) and
thereby also the return of the E
2
(2K
+
) conformation to the
E
1
ATPNa conformation. The affinity of the E
2
(2K
+
)
conformation for ATP, with a K
0.5
value of 0.45 m
M
,is
very low [12,13].
Through the juxtapositioning of these three reaction
sequences, the full catalytic cycle of Na
+
/K

concentration curve is due to the presence of two catalytic a
subunits that work cooperatively [16]. Each catalytic
subunit goes through the same conformational changes
that are described in the single-site model but in such a way
that they are shifted 180° from each other. Thus, in this
model the high affinity and low affinity ATP binding sites
occur simultaneously, and there is also simultaneous
transportofNa
+
out of the cell and K
+
into the cell.
Several experimental results support this model.
In a third model proposed by Plesner, the cooperativity of
the a subunits described by Repke occurs only in the
presence of Na
+
and K
+
[17]. The partial reactions of the
Na
+
/K
+
-ATPase are catalyzed by the ab protomeric
enzyme, as is the case with Na
+
-ATPase or K
+
-stimulated

-ATPase. Alternative proposals
suggest the existence of (ab)
4
tetrameric enzymes [20] or
enzymes with two ATP binding sites per a subunit [21].
THE K
+
-STIMULATED PHOSPHATASE
ACTIVITY
A special characteristic of the Na
+
/K
+
-ATPase is its ability
to hydrolyze phosphoesters and phosphoanhydrides in the
presence of K
+
ions [22]. This so-called K
+
-stimulated
phosphatase activity is ouabain-sensitive. The physiological
relevance of this reaction is unknown.
Fig. 1. Reaction cycle of Na
+
/K
+
-ATPase. Na
+
/K
+

on the cytosolic side, and L1/2, L3/4, L5/6, L7/8, and L 9/10
are accessible from the extracellular side.)
First, the ATP phosphorylation site is localized within
this loop as a part of the sequence DKTGT/S that is highly
conserved among all P-type ATPases. In addition, all ATP
analogs used thus far label peptide structures within this
loop, and the recently published Ca
2+
-ATPase crystal
structure was shown to contain TNP-AMP bound within
this L4/5 peptide. Therefore, it is justified to refer to this part
of the enzyme as the ATP binding domain.
By using the protein-reactive ATP analogs 2-azido-ATP
and 8-azido-ATP, it was possible to label and identify
Gly502 and Lys480, respectively, as possible recognition
sites for the adenosine moiety of ATP [23,24]. (Hereafter,
the amino-acid sequence numbers refer to that of the a1
isoform of the sheep.) The fact that Lys480 is also labeled by
both pyridoxal 5¢-diphospho-5¢-adenosine and pyridoxal
5¢-phosphate suggests that this amino acid might be
involved additionally in the recognition of phosphate
groups, as proposed by Hinz & Kirley [25]. Thus, in this
point of view, the labeling of Lys480 by 8-azido-ATP [23]
does not necessarily indicate that this amino acid directly
interacts with the adenine moiety of the ATP molecule, but
that it is merely within reach of the highly reactive azido
group of 8-azido-ATP. In the crystal structure of the
Ca
2+
-ATPase, Lys492, the equivalent of Lys480 of

in close proximity to the C8 atom of the adenine
moiety. Therefore, if ATP is assumed to retain a similar
conformation when bound within the ATP binding site, one
can imagine that the C8-azido group of 8-azido-ATP labels
Lys480, which originally interacts with the a-phosphate
group of ATP. Taking into account that the distance
between Lys501 and Lys480, as determined by labeling
experiments with dihydro-4,4¢-diisothiocyanostilbene-2,2¢-
disulfonate, is approximately 1.4 nm [31], it is conceivable
that the azido group of 8-azido-ATP labels Lys480 while the
azido group of 2-azido-ATP labels Gly502.
The recently resolved crystal structure of Ca
2+
-ATPase
demonstrates that all ATP analogs used so far label
functional areas of the a subunit. The azido derivatives of
ATP, pyridoxal 5¢-diphospho-5¢-adenosine and pyridoxal
5¢-phosphate, or FITC label near the adenosine binding
pocket, as demonstrated for the binding of TNP-AMP
within the crystal structure of Ca
2+
-ATPase. This area is
referred to as the N (nucleotide binding) domain of the L4/5
peptide. Other ATP analogs such as FSBA or ClR-ATP
label the enzyme in the vicinity of the phosphorylation site,
within a substructure of the L4/5 peptide referred to as the P
(phosphorylation) domain. This area of the protein, consti-
tuting a Rossman fold, was first identified as being
conserved among various hydrolases by comparison of
the primary sequences of P-type ATPases with the primary

2+
, it was demonstrated that the peptide
TGESE(212–216) from the A domain moves towards
the phosphorylation site in the P domain, supporting
the dephosphorylation of the enzyme during the
E
2
-P fi E
2
(K
+
)-transition [33]. Because this peptide
(TGES/A) is highly conserved among all known P-type
ATPases, transport catalyzed by these other enzymes is
likely to take place by similar mechanisms.
MEMBRANE-SPANNING DOMAINS
AND THEIR INVOLVEMENT IN THE
CATION TRANSLOCATION PROCESS
Investigations using isolated Na
+
/K
+
-ATPase have shown
that after tryptic removal of the hydrophilic part of the
enzyme, the remaining C-terminal, membrane-spanning
segment (so-called Ô19-kDa membranesÕ) is still able to
Ó FEBS 2002 Sodium pump structure and properties (Eur. J. Biochem. 269) 2427
occlude Na
+
or the K

here, it may be that Na
+
mimics the binding of K
+
at
extracellular sites. Nevertheless, mutation of this Glu779 to
Gln, Asp, or Lys leads to only moderate changes in the K
0.5
for the cation activation of Na
+
/K
+
-ATPase. For this
reason, and because the Glu779fiLys mutants have a
slightly higher affinity for Na
+
, a direct role for Glu779 in
the cation binding process is fairly unlikely. Rather, it may
be assumed that Glu779 is a part of the overall structure
that participates in the formation of an ion coordination
complex involved in cation selectivity and activation of the
sodium pump.
Of all acidic amino acids examined thus far, only
nonconservative mutation of Asp804 and Asp808 leads to
a nonfunctional enzyme. It is possible that these mutations
have a deleterious effect on K
+
recognition at the
extracellular face of the enzyme [39]. The interaction with
the conservative mutation Asp808fiGlu. The conclusion

from studies of the ionophores valinomycin and gramicidin
[40a]. This general preference for ion/dipole instead of ion/
ion interactions has also been noted for soluble enzymes
that bind monovalent cations. Should ion translocation by
the sodium pump also occur by ion/dipole interactions, one
would assume that cations interact with carbonyl or
hydroxyl groups and not just with carboxyl groups.
In analogy to the Ca
2+
-ATPase, these amino acids
would be in the membrane-spanning domains M4, M5,
M6, and M8 of the a subunit of the sodium pump. In fact,
the crystal structure of Ca
2+
-ATPase, which was recently
reported with a resolution of 2.6 A
ˆ
, shows two binding
sites for Ca
2+
within the transmembrane region (Fig. 2).
One calcium ion is bound within a pocket formed by
Asn768 and Glu771 (M5), Thr799 and Asp800 (M6), and
Glu908 (M8) [26]. These results agree well with previous
conclusions drawn from mutation experiments [40].
A second Ca
2+
binds via interaction with the carbonyl
groups of Val304, Ala305, and Ile307 (M4) and through
the side-chain oxygen atoms of Asn796 and Asp800 (M6)

Asp800 participates in the coordination of both Ca
2+
ions. The cor-
responding amino acids of the sodium pump a1 subunit of the sheep
are given in parentheses. Atoms of interest: oxygen, red; nitrogen, blue;
calcium, green.
2428 G. Scheiner-Bobis (Eur. J. Biochem. 269) Ó FEBS 2002
COUPLING OF ATP HYDROLYSIS
TO ION TRANSPORT
Despite the appreciable amount of knowledge about the
ATP-recognition area of the protein or its ion coordination
sites, the molecular mechanisms that couple ATP hydrolysis
to the opening of the ionophore for the translocation of ions
against their electrochemical gradient are not well under-
stood. Comparison with some other known ion transporters
might be helpful in understanding the translocation process,
or at least in gaining some room for speculation.
The Kdp-ATPase of bacteria is a particularly interesting
K
+
-transporting ATPase made up of three protein
components: KdpA, KdpB, and KdpC. KdpA is inserted
into the membrane and is similar in sequence to the
hydrophobic portion of other P-type ATPases. KdpB is
hydrophilic and analogous to the hydrophilic, ATP-binding
L4/5 domains of other P-type ATPases. Finally, the KdpC
protein is equivalent to the b subunit of K
+
-transporting
P-type ATPases [41]. Furthermore, the KdpA component

is bound
by ion/dipole interactions to carbonyl groups of the M4,
M5, M6, and M8 domains. This applies for a sodium ion in
an aqueous milieu. Because K
+
is larger (r ¼ 1.33 A
˚
)than
Na
+
(r ¼ 0.95 A
˚
), K
+
would not fit into the Na
+
binding
site. Phosphorylation of Na
+
/K
+
-ATPase causes a
conformational change that brings about an alteration in
the Na
+
binding site, allowing Na
+
to exit toward the
extracellular side. One can assume that this conformational
change occurs concomitantly with an expansion of the

would be required to remove two water
molecules 0.38 A
˚
(difference in ionic radii between Na
+
and
K
+
)fromNa
+
. This results in a preference for selecting K
+
over Na
+
of 10
6
: 1. The mechanism of ion selectivity
proposed by Armstrong guarantees that despite an
enormous excess of Na
+
in the extracellular medium, the
binding of K
+
is preferred. Thus, the Eisenman hypothesis,
which dictates that smaller ions pass more easily through a
pore than larger ones, does not apply for all ion channels
or pores. It is conceivable that after the release of Na
+
,
the selectivity for K

+
-ATPase, Na
+
-independent specific ouabain binding
can still be measured in the presence of Mg
2+
and ATP [44].
Apparently, the b subunit of the H
+
/K
+
-ATPase confers a
conformational change on the a subunit that enhances the
binding of ouabain.
Besides verifying that the interaction between a and
b subunits involves the L7/8 region, our own investigations
using an NGH26 chimera have additionally shown that the
binding of specific inhibitors is mediated through this
interaction. Thus, an NGH26/HKb heterodimer recognizes
not only palytoxin and ouabain but also the gastric
H
+
/K
+
-ATPase-specific inhibitor SCH 28080 [45].
Taken together, these results point to the function of the
b subunit as being more than just a vehicle for the transport
of the a subunit from the ER to the plasma membrane [46].
This hypothesis is supported by the fact that there are three
or possibly even four isoforms of the b subunit. Besides the

suggested by the fact that it is expressed in tissues that
contain no b1 isoform, including pineal gland, photorecep-
tor cells, and astrocytes, and also in tissues in the CNS
Ó FEBS 2002 Sodium pump structure and properties (Eur. J. Biochem. 269) 2429
(glia, choroid plexus, arachnoid membrane) that have
specialized ion-translocating characteristics. Nevertheless,
although these observations suggest that the b2 subunit
influences ion transport via the sodium pump, data that
confirm this function are still lacking.
An extracellularly localized peptide composed of 34
amino acids of the b1 subunit (Val93-Asp126) interacts with
the 26 amino-acid peptide of the a1 subunit already
mentioned [48]. The corresponding fragment of the b2
subunit (Val96-Arg129) has only 29% identity with the
Val93-Asp126 fragment of the b1, and 47% homology.
Whether these differences in the primary structure of these
two regions are responsible for any differences in enzyme
characteristics has yet to be investigated.
Nevertheless, the overall impression is that the 26 amino-
acid peptide and possibly the entire L7/8 region are
somehow involved in ion conduction by the pump. Our
own results show that mutations of Asp884 and Asp885
from within the L7/8 peptide to Arg considerably affect the
interactions of the enzyme with Na
+
, while, if anything, the
affinity for K
+
increases [49]. Notably, an SYG motif is
present within the 26-amino-acid peptide that somewhat

+
/K
+
-ATPase in ion transport, Cu
2+
-catalyzed
cleavage of the L7/8 loop (possibly near His875) results in
the loss of Rb
+
occlusion [51] usually obtained with the
19-kDa-membrane preparations of the a subunit. This,
together with the likelihood that the b subunit may play a
roleincationocclusion[52],makestheL7/8areaandthe
26 amino-acid peptide within this region attractive for
further investigation.
Besides this peptide, aromatic amino acids from the
transmembrane domain of the b subunit might be import-
ant for a/b subunit interactions and might influence the
properties of the enzyme. In the membrane-spanning
domains of the b1, b2, and b3 subunits of the sodium
pump, there is a relatively high number of amino acids with
aromatic side chains (phenylalanine, tyrosine, tryptophan)
whose position is conserved in almost all isoforms. In a
more recent study it was confirmed that Tyr40 and Tyr44 of
the membrane-spanning domain of the b1 subunit influence
the transport kinetics of the Na
+
/K
+
-ATPase and its

and thus ion transport. The Na
+
/K
+
-ATPase is the only
enzyme known to interact with this class of substances.
Cardioactive steroids, especially water-soluble ouabain
(g-strophanthine), have often been used to identify
Na
+
/K
+
-ATPase and to study ion transport mechanisms
involved in this system. Under optimal conditions, 1 mole of
Na
+
/K
+
-ATPase binds 1 mol of ouabain. Optimal binding
occurs when the incubation medium contains one of the
following groups of ligands: (a) Mg
2+
,Na
+
,andATPor
(b) Mg
2+
and P
i
. Because both conditions can induce the

presence of Mg
2+
and P
i
, low concentrations of Na
+
have
the effect of lowering the affinity of Na
+
/K
+
-ATPase for
cardioactive steroids when K
+
is present.
Inhibition of the sodium pump by cardiac steroids is
clinically relevant. Application of these substances, especi-
ally of digitalis and its congeners, helps to increase muscular
contractility of the failing heart, possibly by indirectly
inducing an elevation in the Ca
2+
concentration in the
myocardium. The wide use of digitalis for many centuries in
medicine, the great therapeutic impact of these substances,
and the need for a regulatory substance that increases heart
tonus without influencing its beating frequency led more
than 50 years ago to the proposal that endogenous factors
must exist that either have a similar structure or act in a
similar way to the cardiac steroids currently in use for clinical
purposes. The discovery of various isoforms of the sodium

experiments demonstrating mitogen-activated protein kin-
ase activation in rat cardiomyocytes by low concentrations
of ouabain [55,56], however, indicate that investigating
signal cascades induced by the glycoside might be helpful in
understanding its potential physiological relevance and its
possible involvement in vascular tone regulation or in the
pathogenesis of hypertension.
The Na
+
/K
+
-ATPase is a target of other substances
besides the cardiac glycosides. Palytoxin, produced by
corals of the genus Palythoa, is the most potent toxin of
animal origin. The LD
50
for rodents is 10–250 ngÆkg
)1
[57].
Previous investigations demonstrated that palytoxin opens
ion channels in vertebrate cells with a conductance of
approximately 10 pS. These channels remain open for some
time and allow K
+
ions to flow out of the cytosol. This is
probably the reason for the high toxicity of palytoxin, as the
outflow of K
+
and the resulting collapse of the membrane
potential lead to a general loss of basic cell functions.

+
/K
+
-ATPase hetero-
logously in yeast [58]. Untransformed yeast cells are
insensitive to palytoxin, whereas cells transformed with
both subunits of the Na
+
/K
+
-ATPase show a marked
efflux of K
+
in response to the toxin. This fact, and the
observation that this palytoxin-induced K
+
efflux is inhib-
ited by ouabain and other cardiotonic steroids, confirmed
that the sodium pump is the target of palytoxin. In vitro
expression experiments have lent further support to this
theory by showing that the palytoxin-induced channel is
directly associated with the presence of the Na
+
/K
+
-
ATPase [59]. Through its binding to the Na
+
/K
+

channel because palytoxin produces K
+
efflux in yeast cells
expressing an Asp369Ala mutant of the a1 subunit that is
enzymatically inactive.
Palytoxin is apparently not the only molecule that
converts the sodium pump into an ion channel. Sanguin-
arine, one of a number of alkaloids developed by the
plant Sanguinaria canadensis in the course of evolution to
protect itself from herbivores, was described about
25 years ago as an inhibitor of the sodium pump.
Nevertheless, the interactions between sanguinarine and
the pump were not pursued because at that time
experiments that would yield conclusive results were not
possible. Using the yeast expression system for the sodium
pump, we recently showed that sanguinarine induces the
formation of a ouabain- or proscillaridin A-sensitive
channel in the sodium pump that allows K
+
ions to
flow out of the cell cytosol [60]. Sanguinarine also appears
to bind primarily to the E
1
-P conformation of the enzyme
and to inhibit the binding of [
3
H]ouabain, although,
as with palytoxin, phosphorylation is not absolutely
required.
The experiments with palytoxin and sanguinarine show

PROSPECTS FOR FUTURE RESEARCH
Although much has been learned about the mechanics of
the transport of ions against their electrochemical gradients
by ATPases or the role of these enzymes as targets of either
endogenous or foreign toxins, the picture is still not
complete. The resolution of the crystal structure of Ca
2+
-
ATPase has appeared at a time when it was being suggested
that additional efforts might only result in semantic
refinements rather than the gain of new information. This
structure has provided new hope that the mechanisms of
this enzyme can be unveiled by addressing new questions in
new projects, and with the expectation of gaining new
perspectives. Thus, although they are long-known enzymes,
ATPases remain a fresh target for researchers and may soon
be discovered anew.
Ó FEBS 2002 Sodium pump structure and properties (Eur. J. Biochem. 269) 2431
ACKNOWLEDGEMENTS
The author has been supported through DFG, grants Sche 307/5-1 and
307/5-2. He wishes to thank Drs W. Schoner and R. A. Farley for many
constructive discussions.
REFERENCES
1. Skou, J.C. (1957) The influence of some cations on adenosine-
triphosphatase from peripheral nerves. Biochim. Biophys. Acta
23, 394–401.
2. Lutsenko, S. & Kaplan, J.H. (1995) Organization of P-type
ATPases: significance of structural diversity. Biochemistry 34,
15607–15613.
3. Antolovic,R.,Bruller,H.J.,Bunk,S.,Linder,D.&Schoner,W.

9. Therien, A.G., Karlish, S.J. & Blostein, R. (1999) Expression and
functional role of the c subunit of the Na,K-ATPase in mam-
malian cells. J. Biol. Chem. 274, 12252–12256.
10. Arystarkhova, E., Wetzel, R.K., Asinovski, N.K. & Sweadner,
K.J. (1999) The c subunit modulates Na
+
and K
+
affinity of the
renal Na,K-ATPase. J. Biol. Chem. 274, 33183–33185.
11. Mahmmoud,Y.A.,Vorum,H.&Cornelius,F.(2000)Identifi-
cation of a phospholemman-like protein from shark rectal
glands. Evidence for indirect regulation of Na,K-ATPase by
protein kinase C via a novel member of the FXYDY family.
J. Biol. Chem. 275, 35969–35977.
12. Skou, J.C. (1988) Overview: the Na,K-pump. Methods Enzymol.
156, 1–25.
13. Glynn, I.M. (1993) Annual review prize lecture. ÔAll hands to the
sodium pumpÕ. J. Physiol. 462, 1–30.
14. Vilsen, B., Andersen, J.P., Petersen, J. & Jorgensen, P.L. (1987)
Occlusion of
22
Na
+
and
86
Rb
+
in membrane-bound and soluble
protomeric ab-subunits of Na,K-ATPase. J. Biol. Chem. 262,

rammine-ATP. J. Ultrastruct. Mol. Struct. Res. 102, 189–195.
20. Taniguchi, K., Kaya, S., Abe, K. & Mardh, S. (2001) The oli-
gomeric nature of Na/K-transport ATPase. J. Biochem. 129,
335–342.
21. Ward, D.G. & Cavieres, J.D. (1993) Solubilized ab Na,K-
ATPase remains protomeric during turnover yet shows apparent
negative cooperativity towards ATP. Proc. Natl Acad. Sci. USA
90, 5332–5336.
22. Bader, H. & Sen, A.K. (1966) (K
+
)-Dependent acyl phosphatase
aspartofthe(Na
+
+K
+
)-dependent ATPase of cell mem-
branes. Biochim. Biophys. Acta 118, 116–123.
23. Tran, C.M., Scheiner-Bobis, G., Schoner, W. & Farley, R.A.
(1994) Identification of an amino acid in the ATP binding site of
Na
+
/K
+
-ATPase after photochemical labeling with 8-azido-
ATP. Biochemistry 33, 4140–4147.
24. Tran, C.M., Huston, E.E. & Farley, R.A. (1994) Photochemical
labeling and inhibition of Na,K-ATPase by 2-azido-ATP.
Identification of an amino acid located within the ATP binding
site. J. Biol. Chem. 269, 6558–6565.
25. Hinz, H.R. & Kirley, T.L. (1990) Lysine 480 is an essential

lytic site. FEBS Lett. 217, 111–116.
30. Farley, R.A., Tran, C.M., Carilli, C.T., Hawke, D. & Shively,
J.E. (1984) The amino acid sequence of a fluorescein-labeled
peptidefromtheactivesiteof(Na,K)-ATPase.J. Biol. Chem.
259, 9532–9535.
31. Gatto, C., Lutsenko, S. & Kaplan, J.H. (1997) Chemical
modification with dihydro-4,4¢-diisothiocyanostilbene-2,2¢-
disulfonate reveals the distance between K480 and K501 in the
ATP-binding domain of the Na,K-ATPase. Arch. Biochem.
Biophys. 340, 90–100.
32. Jorgensen, P.L. & Pedersen, P.A. (2001) Structure–function
relationships of Na
+
,K
+
,ATP,orMg
2+
binding and energy
transduction in Na,K-ATPase. Biochim. Biophys. Acta 1505,
57–74.
33. Patchornik, G., Goldshleger, R. & Karlish, S.J. (2000) The
complex ATP-Fe
2+
serves as a specific affinity cleavage reagent
in ATP-Mg
2+
sites of Na,K-ATPase: altered ligation of Fe
2+
(Mg
2+

39. Kuntzweiler,T.A.,Arguello,J.M.&Lingrel,J.B.(1996)Asp804
and Asp808 in the transmembrane domain of the Na,K-ATPase
alpha subunit are cation coordinating residues. J. Biol. Chem.
271, 29682–29687.
40. Rice, W.J. & MacLennan, D.H. (1996) Scanning mutagenesis
reveals a similar pattern of mutation sensitivity in transmem-
brane sequences M4, M5, and M6, but not in M8, of the Ca
2+
-
ATPase of sarcoplasmic reticulum (SERCA1a). J. Biol. Chem.
271, 31412–31419.
40a. Eisenmann, G. & Dani, J.A. (1987) An introduction to molecular
architecture and permeability of ion channels. Annu. Rev. Bio-
phys. Biomol. Struct. 16, 247–263.
41.Altendorf,K.,Siebers,A.&Epstein,W.(1992)TheKDP
ATPase of Escherichia coli. Ann. NY Acad. Sci. 671, 228–243.
42. Durell, S.R., Bakker, E.P. & Guy, H.R. (2000) Does the KdpA
subunit from the high affinity K
+
-translocating P-type KDP-
ATPase have a structure similar to that of K
+
channels? Biophys.
J. 78, 188–199.
43. Armstrong, C. (1998) The vision of the pore. Science 280, 56–57.
44. Eakle, K.A., Lyu, R M. & Farley, R.A. (1995) The influence of
b subunit structure on the interaction of Na
+
/K
+

M7/M8 extracellular loop of the sodium pump a subunit in ion
transport. Structural and functional homology to P-loops of ion
channels. J. Biol. Chem. 272, 16158–16165.
50. Silverman,S.K.,Kofuji,P.,Dougherty,D.A.,Davidson,N.&
Lester, H.A. (1996) A regenerative link in the ionic fluxes
through the weaver potassium channel underlies the pathophy-
siology of the mutation. Proc. Natl Acad. Sci. USA 93, 15429–
15434.
51. Shimon, M.B., Goldshleger, R. & Karlish, S.J. (1998) Specific
Cu
2+
-catalyzed oxidative cleavage of Na,K-ATPase at the
extracellular surface. J. Biol. Chem. 273, 34190–34195.
52. Lutsenko, S. & Kaplan, J.H. (1993) An essential role for the
extracellular domain of the Na,K-ATPase b-subunit in cation
occlusion. Biochemistry 32, 6737–6743.
53. Hasler, U., Crambert, G., Horisberger, J.D. & Geering, K.
(2001) Structural and functional features of the transmembrane
domain of the Na,K-ATPase b subunit revealed by tryptophan
scanning. J. Biol. Chem. 276, 16356–16364.
54. Schoner, W. (2002) Endogenous cardiac glycosides, a new class
of steroid hormones. Eur. J. Biochem. 269, 2440–2448.
55. Haas, M., Askari, A. & Xie, Z. (2000) Involvement of Src and
epidermal growth factor receptor in the signal-transducing
function of Na
+
/K
+
-ATPase. J. Biol. Chem. 275, 27832–27837.
56. Xie, Z. & Askari, A. (2002) Na