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
Trang 1M I N I R E V I E W
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 underinvestigation formore than fourdecades
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 transport
T H E S O D I U M P U M P : A B R I E F
R E T R O S P E C T I V E
Today there is a vast amount of information concerning ion
transport through biological membranes and primary
structures, crystals, mutants, and chimeras of ion
trans-porters It is difficult to imagine that the impressive progress
achieved thus far was originally generated by a few
researchers who had the ability to observe simple
phenom-ena connected with ion distribution, to question their origin,
and to assemble experimental evidence in ways that did not
allow any otherconclusion but that there must a mechanism
that enables ions to be actively transported against their
electrochemical gradients This mechanism, termed a
sodium pump by Dean in 1941, originates from the
observation that sodium ions within muscle fibers can
exchange with radioactive sodium added to their
environ-ment Nevertheless, although a large amount of data and
interpretation of it followed Dean’s proposal, it was not
until 1954 that Gardos discovered that ion pumping in red
blood cell ghosts was supported by ATP, which in turn
became hydrolyzed (Due to space limitations, some of the
early, seminal work is not included in the reference list;
instead, an up-to-date selection of papers from a variety of groups from which both the current progress in the field can
be assessed and in which earlier, landmark discoveries are fully referenced is provided.)
These observations, together with the finding that 18 sodium ions were transported for each molecule of oxygen consumed (4.5 Na+per electron or, in other words, 3 Na+ perATP) 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, however, not well understood; after many years of investigation, the sodium pump is still at the center
of researchers’ attention
N a+/ K+- A T P A S E : S U B U N I T
C O M P O S I T I O N
Every living cell is negatively charged in comparison with its environment Thus, in principle, the cell/environment pair constitutes a battery Just as a battery can be used to perform work, a cell uses this electrochemical gradient to obtain nutrients, ionic or nonionic, from its environment and to extrude metabolites and ions from its interior In this fashion, the composition of the intracellular milieu remains constant while allowing foradaptation to a changing environment to occur
Correspondence to G Scheiner-Bobis, Institut fu¨rBiochemie und
Endokrinologie, Fachbereich Veterina¨rmedizin,
Justus-Liebig-Universita¨t Giessen, Frankfurter Str 100,
D-35392 Giessen, Germany.
Fax: + 49 641 9938189, Tel.: + 49 641 9938180,
E-mail: Scheiner-Bobis@vetmed.uni-giessen.de
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 October2001, revised 11 December2001,
accepted 28 January 2002)
Trang 2The 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+ ions from the external medium are exchanged
Thus, the sodium pump contributes substantially to the
maintenance of the membrane potential of the cell, provides
the basis forneuronal communication, and contributes to
the osmotic regulation of the cell volume In addition, the
electrochemical Na+gradient is the driving force behind
secondary transport systems
The Na+/K+-ATPase belongs to the P-type ATPases, a
family of enzymes that become phosphorylated during
transport by the c-phosphate group of ATP at an aspartic
acid localized within the highly conserved sequence
DKTGS/T [2] This family, which contains more than 50
members, includes membrane-bound enzymes responsible
for the transport of heavy metal ions (P1-type ATPases),
othermetal ions (P2-type ATPases), and the K+-selective
Kdp-ATPase of Escherichia coli (P3-type ATPase)
Within the group of the P2-type ATPases, the Na+/
K+-ATPase, togetherwith the colonic orgastric 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+orK+ions
A numberof 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
subunit, has a relative molecular mass of 100–113 kDa,
depending on the presence of different isoforms: a1, a2, a3,
or a4 It crosses the membrane 10 times, forming
trans-membrane domains M1 to M10; both N- and C-termini are
localized on the cytosolic side [3] Various studies have
shown that both ATP binding and ion occlusion occurs in
this subunit
The b subunit is highly glycosylated and has a relative
molecularmass of about 60 kDa The mass of the protein
moiety of this subunit is 36–38 kDa, depending on the
isoforms b1, b2, or b3 The b subunit crosses the membrane
only once, and the N-terminus is localized on the
intracel-lular side of the membrane The respective roles of these
proteins is still not entirely clear More recent results have
shown that the b subunit makes direct contact with the
a subunit [4], thereby stabilizing the a subunit and assisting
in its transport from the endoplasmic reticulum to the
plasma membrane [5] In addition, numerous experiments
have shown that the b subunit is important for ATP
hydrolysis, ion transport, and the binding of inhibitors such
as ouabain
The third subunit of Na+/K+-ATPase, the c subunit of
7–11 kDa, was first identified as a component involved in
the binding of [3H]ouabain The c subunit specifically
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 forthe 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 forobtaining 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 forNa+orK+ions [9,10] These data, togetherwith the fact that several peptides similarto the
c subunit have already been determined to interact with and influence the sodium pump [11] confirm that the ion pumping activity can be finely modulated by type I membrane peptides and also offers the possibility
of addressing physiologically relevant questions in connec-tion with the regulaconnec-tion of the expression of this type
of protein
T H E C A T A L Y T I C M E C H A N I S M
O F T H E N a+/ K+- A T P A S E
The Na+/K+-ATPase has two conformational states, E1 and E2 These states are not only characterized by differ-ences in theirinteractions with Na+, K+, ATP, orouabain, they also have been clearly defined by tryptic cleavage experiments
In the first step of the reaction sequence, Na+and ATP bind with very high affinity (Kdvalues of 0.19–0.26 mMand 0.1–0.2 lM, respectively) to the E1 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 Afterthe E2-P3Na+ conformation is attained, the enzyme loses its affinity forNa+(K0.5¼ 14 mM) and the affinity forK+is increased (Kd 0.1 mM) Thus, three
Na+ions are released to the extracellular medium (Fig 1, step 3) and K+ ions are taken up (Fig 1, step 4) The binding of K+ to the enzyme induces a spontaneous dephosphorylation of the E2-P conformation The dephosphorylation of E2-P leads to the occlusion of two
K+ions, leading to E2(2K+) (Fig 1, step 5) [12,13] Intracellular ATP increases the extent of the release of
K+from the E2(2K+) conformation (Fig 1, step 6) and thereby also the return of the E2(2K+) conformation to the
E1ATPNa conformation The affinity of the E2(2K+) conformation for ATP, with a K0.5value of 0.45 mM, is very low [12,13]
Through the juxtapositioning of these three reaction sequences, the full catalytic cycle of Na+/K+-ATPase is obtained (Fig 1)
All P-type ATPases function in a similarway: they all hydrolyze ATP and occlude ions during the translocation process within the membrane-inserted segment of the protein Through this process, the ionophore of every ion-transporting ATPase is accessible from only one side of the membr ane at any given time
The sequential model presented above, however, often referred to as the Albers–Post scheme [13] does not take into
Trang 3consideration that the sodium pump might exist as a
diprotomer of cooperating (ab)2subunits and thus contain
two binding sites forATP
The concentration–effect curve for ATP hydrolysis is
biphasic, which can be explained by an extrapolation of the
single-site model shown in Fig 1 Each ab protomer has a
single ATP binding site that changes from high affinity to
low affinity with changes in conformation This model is
strongly supported by experiments showing that the
stoichiometry of binding for either ATP, phosphate, or
ouabain is 1 per a subunit, and that solubilized enzyme
retains its catalytic activity [14] Results obtained with highly
purified enzyme from duck salt gland lend credence to this
hypothesis [15]
A second model, which was originally put forward by
Repke, postulates that the biphasic nature of the ATP
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
occursimultaneously, and there is also simultaneous
tr anspor t of Na+ 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 orK+-stimulated phosphatase
The models of Repke and Plesnerdifferfrom the single-site model in that they predict the presence of two binding sites on each functional enzyme entity The results of many investigations support the existence of two binding sites on one (ab)2diprotomer Kinetic studies have shown that the single-site model is not sufficient to explain the coupling of ATP hydrolysis to ion transport [18] Moreover, crystallo-graphic studies have demonstrated that Na+/K+-ATPase crystallizes in a way that allows ab protomers to be in close contact with each other[19] Finally, radiation inactivation has shown in several cases that the target size is consistent with that of an (ab)2diprotomeric structure These data, however, are not compelling proof of the simultaneous existence of two ATP binding sites and therefore do not definitively establish the (ab)2 diprotomer as the basic functional unit of Na+/K+-ATPase Alternative proposals suggest the existence of (ab)4 tetrameric enzymes [20] or enzymes with two ATP binding sites per a subunit [21]
T H E K+- S T I M U L A T E D P H O S P H A T A S E
A C T I V I T Y
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+-ATPase binds Na+and ATP in the E 1 conformational state (step 1) and is phosphorylated at
an aspartate residue by the c-phosphate of ATP This leads to the occlusion of three Na + ions (step 2) and then to their release to the extracellular side (step 3) This new conformational state (E 2 -P) binds K + with high affinity (step 4) Binding of K + leads to dephosphorylation of the enzyme and to the occlusion of two K+cations (step 5) K+is then released to the cytosol after ATP binds to the enzyme with low affinity (step 6) The dashed box highlights the electrogenic steps of the catalytic cycle.
Trang 4T H E A T P B I N D I N G D O M A I N
The cytosolic protein structure between membrane domains
M4 and M5 (L4/5) is of great importance for the function of
the enzyme, because a series of amino acids within this
region have been identified to be either essential for or
highly involved in ATP hydrolysis and enzyme function
(The prefix L stands for loop, a transmembrane
domain-connecting peptide L2/3, L4/5, L6/7, and L8/9 are localized
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 Ca2+-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
Ca2+-ATPase, Lys492, the equivalent of Lys480 of
the sodium pump a1 subunit, seems to interact with the
phosphate group of TNP-AMP [26] Site-directed
muta-genesis experiments have confirmed the importance of
Lys480 forATP hydrolysis and enzyme function [27]
Various other ATP analogs such as
5¢-p-fluoro-sulfonylbenzoyl-adenosine (FSBA) or
c-[4-(N-2-chloro-ethyl-N-methylamino)]benzylamide ATP (ClR-ATP) were
successfully used foridentifying amino acids within the
L4/5 peptide Nevertheless, although these substances
resemble nucleotide triphosphates and their interaction with
the enzyme can be prevented by ATP, they are not
substrates of the sodium pump Thus, it was still uncertain
whetherCys656 and Lys719, the FSBA labeling sites [28],
and Asp710, the ClR-ATP labeling site [29], were truly
constituents of the ATP binding site In contrast to these
ATP-like substances, fluorescein 5¢-isothiocyanate (FITC),
a protein-reactive probe, was shown to modify Lys501 of
the sodium pump a1 subunit [30] Although there is no
apparent similarity between FITC and ATP, the fact that
ATP prevents modification of Lys501 by FITC led to the
conclusion that Lys501 is localized within the
adenosine-recognizing moiety of the a1 subunit This proposal has
been supported by findings concerning the conformation of
Mg2+-complexed ATP analyzed by 1H-NMR and
ultra-violet spectrophotometric methods According to these
reports, the a-phosphate group of the ATP molecule is
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 Ca2+-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, orFITC label nearthe adenosine binding pocket, as demonstrated for the binding of TNP-AMP within the crystal structure of Ca2+-ATPase This area is referred to as the N (nucleotide binding) domain of the L4/5 peptide OtherATP analogs such as FSBA orClR-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 sequence of the L-2-haloacid dehalogenase from Pseudo-monas sp and was thought to directly participate in the phosphorylation/dephosphorylation of Asp369 via the terminal phosphate of ATP More recent studies, however, have suggested that this area of the protein is a Mg2+ binding site [32]
The distance between the adenosine binding area of the
N domain and the phosphorylation site in the P domain is rather large (2.5 nm) to be bridged by the ATP molecule Thus, some conformational transition must occur prior to ATP hydrolysis, which results in the two domains approaching each other A third subdomain formed by the L2/3 peptide might be involved in these conforma-tional changes This area of the protein is referred to as the actuatordomain (A domain) No functional analysis has yet been published, however, that supports this proposal Nevertheless, the A domain undoubtedly contributes to the conformational transitions associated with ATP hydrolysis, ion transport, and dephosphorylation of the phosphoenzyme formed by the transfer of the c phosphate group of ATP In experiments involving ascorbate/
H2O2-catalyzed peptide cleavage in the presence of ATP-Fe2+, 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
E2-Pfi E2(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 similarmechanisms
M E M B R A N E - S P A N N I N G D O M A I N S
A N D T H E I R I N V O L V E M E N T I N T H E
C A T I O N T R A N S L O C A T I O N P R O C E S S
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 19-kDa membranes) is still able to
Trang 5occlude Na+orthe K+analog Rb+[34], indicating that
the ionophore, as expected, must consist of
membrane-spanning domains Negatively charged amino acids within
this structure are viewed as possible interfaces between the
protein and ions being transported However, analysis of
mutants has not always demonstrated that substitution of
acidic amino acids within the membrane-spanning domains
has a marked effect on enzyme activity Substitution at
Glu327 (within the fourth membrane-spanning domain,
denoted M4), Asp926 (M8), Glu953, orGlu954 (both M9)
does not lead to significant changes in the affinity of the
mutant enzyme forNa+ orK+ oraffect its electrical
properties [35–37] Mutation of Glu953 or Glu954 also has
no effect on the interaction of the enzyme with palytoxin (G
Scheiner-Bobis, unpublished observations)
Mutation of Glu779 from the sixth membrane-spanning
domain has a numberof effects, depending on the nature of
the substitution A Glu779Ala mutant has an ATPase
activity that is independent of K+(a Na+-ATPase) [38];
here, it may be that Na+ mimics the binding of K+at
extracellular sites Nevertheless, mutation of this Glu779 to
Gln, Asp, orLys leads to only moderate changes in the K0.5
forthe cation activation of Na+/K+-ATPase Forthis
reason, and because the Glu779fiLys mutants have a
slightly higheraffinity forNa+, 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
drawn from these studies is that Asp804 and Asp808 from
the sixth membrane-spanning domain of the a1 subunit
are involved in cation coordination [39] The data
reported thus far, however, give the impression that the
mutations have an effect only on K+ and not on Na+
recognition
The examination of various acidic residues from the
transmembrane domains of the sodium pump has not
brought us closer to the goal of identifying amino acids that
are essential for ion transport In general, it would appear as
if it weren’t the individual negatively charged amino acids of
the membrane-spanning domains that were directly
involved in ion transfer, but larger peptide structures that
contain these amino acids This conclusion, as
unsatisfac-tory as it may be, agrees well with investigations of a
considerable number of mutants of the Ca2+-ATPase that
clearly demonstrate that numerous amino acids within the
transmembrane domains M4, M5, M6, and M8 are
important for the function of the enzyme, independent of
whether they are charged or not [40]
If no single acidic residue from the transmembrane
domains is essential for ion transport, then which structures
are important?
It is known that cations are transferred along the
backbone of carbonyl groups by ion/dipole interactions
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 occurby ion/dipole interactions, one would assume that cations interact with carbonyl or hydroxyl groups and not just with carboxyl groups
In analogy to the Ca2+-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 Ca2+-ATPase, which was recently reported with a resolution of 2.6 Aˆ, shows two binding sites forCa2+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 Ca2+ 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) and Glu309 (M4) [26]
A similarsituation could be assumed forthe coordination
of cations within the membrane-spanning domains of the
Na+/K+-ATPase, because several structures are, as dem-onstrated in extensive and thorough theoretical work, very similarto those of the Ca2+-ATPase This constellation would also explain why single mutations within this region
do not lead to a complete loss of transport, because the cations are coordinated simultaneously by several amino acids If this is the case, then only the mutation of several amino acids concomitantly would lead to a marked change
in ion transport properties
Fig 2 Cation coordination sites of Ca2+-ATPase The view is a cross-section of the protein from the lumen of the sarcoplasmic reticulum Areas of the protein not involved in Ca2+coordination have been eliminated Two Ca 2+ ions shown in green are coordinated by Val304, Ala305, Ile307 and Glu309 (M4), Asn768 and Glu771 (M5), Thr799 and Asp800 (M6), and Glu908 (M8) The side chain carboxyl group of 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.
Trang 6C O U P L I N G O F A T P H Y D R O L Y S I S
T O I O N T R A N S P O R T
Despite the appreciable amount of knowledge about the
ATP-recognition area of the protein or its ion coordination
sites, the molecularmechanisms 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,
orat least in gaining some room forspeculation
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 otherP-type ATPases Finally, the KdpC
protein is equivalent to the b subunit of K+-transporting
P-type ATPases [41] Furthermore, the KdpA component
has similarities to K+channels [42] Taking into account
these observations, one could speculate that during
evolu-tion an ion channel has, togetherwith the help of an ATP
hydrolase, been selected to move ions against their
electro-chemical gradients In the further development of P-type
ATPases, ATP hydrolases and ion channels became
phys-ically fused
In the case of Na+/K+-ATPase, by taking into
consid-eration Armstrong’s proposal regarding the selectivity of
ion channel ionophores for Na+orK+[43], such
trans-formations in the ion binding structure could explain how
one single structure could coordinate Na+in one instance
and K+in another In the E1conformation, Na+is bound
by ion/dipole interactions to carbonyl groups of the M4,
M5, M6, and M8 domains This applies fora 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
cation binding site (E2conformation of Na+/K+-ATPase),
so that now the larger K+can be accommodated The ion/
dipole interactions in this case are also those of K+in an
aqueous environment This newly expanded binding site
does not bind Na+well because Na+cannot be adequately
coordinated by the carbonyl groups In this state, an
exchange of the water molecules surrounding Na+ for
carbonyl groups would be thermodynamically unfavorable
For the rigid pore opening of K+channels, Armstrong [43]
calculated that an energy expenditure of approximately
10 kcalÆmol)1 would be required to remove two water
molecules 0.38 A˚ (difference in ionic radii between Na+and
K+) fr om Na+ This results in a preference for selecting K+
overNa+ of 106: 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 largerones, does not apply forall ion channels
orpores It is conceivable that afterthe release of Na+,
the selectivity forK+ at the extracellular side of
Na+/K+-ATPase is maintained by such a rigid pore opening, which may be formed by the L7/8 peptide of the
a subunit as well as the b subunit
T H E R O L E O F T H E a / b S U B U N I T
I N T E R A C T I O N S F O R I O N T R A N S P O R T
The a and b subunits of the sodium pump must interact with each other in order to accomplish ion transport In several reports from the laboratory of Fambrough and colleagues,
it was shown that 26 amino acids from within the L7/8 peptide loop of the a subunit interact with extracellular parts of the b subunit [4] Such interactions appear not only
to stabilize the a/b heterodimer but also to have functional relevance, as ATP hydrolysis, ouabain binding, and paly-toxin-induced K+efflux occuronly in the presence of both subunits and are markedly influenced by mutations in this region of the enzyme
Moreover, the b subunit appears to influence the confor-mation and ion sensitivity of the sodium pump If the b subunit of the sodium pump is replaced by that of the H+/
K+-ATPase, Na+-independent specific ouabain binding can still be measured in the presence of Mg2+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 inhibitorSCH 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 orpossibly even fourisoforms of the b subunit Besides the b1 isoform, which is the most widely distributed isoform, there is the b2 isoform that is found in excitable tissues (muscle and nervous tissue), the b3 in testes, adr enal, and brain, and the bm in skeletal and heart muscle In view of the variety of isoforms that have been identified, it is not unreasonable to speculate that this multiplicity has a physiological relevance
Interestingly, the b2 isoform was known for some time in glial cells as adhesion molecule on glia [47] This lends further support to the idea that the b2 isoform has a function besides that of stabilizing the a subunit For example, in tissue sections from cerebellum, Fab fragments
of monoclonal antibodies against adhesion molecule on glia inhibit the migration of granulocytes In the cochlea, the expression of b2 is specifically associated with the striata vulgaris, a tissue that forms the barrier between endolymph and extracellular fluid The endolymph contains a high concentration of K+and almost no Na+ It is also strongly electropositive, and K+must be transported against this potential (+80 mV) Thus, it appears that b2 expression is associated with structures that have a high K+-transporting capability Finally, a dual function forthe b2 isoform is also suggested by the fact that it is expressed in tissues that contain no b1 isoform, including pineal gland, photorecep-torcells, and astrocytes, and also in tissues in the CNS
Trang 7(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 forK+increases [49] Notably, an SYG motif is
present within the 26-amino-acid peptide that somewhat
resembles the GYG motif of the P-loop of K+channels
There, this tyrosine is essential for ion translocation
Although it is not clear yet whether the corresponding
tyrosine of the a subunit is also involved in K+conduction,
it is certainly interesting to note that all but one of the
K+-transporting P-type ATPases, which always have a and
b subunits, have this tyrosine residue conserved (in Hydra, it
is a phenylalanine) A further point worth mentioning is that
naturally occurring mutation of the highly conserved GYG
sequence of the pore opening of K+ channels to SYG
(which is the sequence in the Na+/K+-ATPase) leads to a
reduction in K+ selectivity and an increase in Na+
permeability [50] Although there are currently no data
directly indicating a role for the SYG(894–896) sequence of
the Na+/K+-ATPase in ion transport, Cu2+-catalyzed
cleavage of the L7/8 loop (possibly nearHis875) results in
the loss of Rb+occlusion [51] usually obtained with the
19-kDa-membrane preparations of the a subunit This,
togetherwith the likelihood that the b subunit may play a
r ole in cation occlusion [52], makes the L7/8 ar ea and the
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
affinity towards K+ [53] However, the mechanism by
which the tyrosine residues might influence interactions of
the enzyme with K+are not yet understood
S P E C I F I C I N H I B I T O R S
Possibly due to its key function in cellularphysiology and
indeed the entire organism, the sodium pump has been a
target of a vast number of toxins produced by both plants and animals Thus, its ion pumping activity is specifically inhibited by a series of naturally occurring steroids, termed cardiac steroids or cardiac glycosides, such as ouabain and digitalis Other substances, like palytoxin from marine corals of the genus Palythoa or sanguinarine from the plant Sanguinaria canadensis,are also specific inhibitors of the sodium pump Unlike the cardioactive steroids, which inhibit ion flow through the pump, palytoxin and possibly also sanguinarine convert the enzyme into an open channel that allows ions to flow down their concentration gradient
In all cases, however, the toxin/receptor interactions result
in loss of the membrane potential, a fatal situation for the cell ororganism
Cardioactive steroids bind reversibly to the extracellular side of the Na+/K+-ATPase and inhibit ATP hydrolysis 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 Underoptimal 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) Mg2+, Na+, and ATP or (b) Mg2+and Pi Because both conditions can induce the
E2-P conformation of the enzyme, this is the conformation
to which the cardioactive steroids bind, resulting in the formation of a stable phosphoenzyme/cardioactive steroid complex, termed [E2–P*Æouabain] The presence of the ions
to be transported influences the dissociation constant of the enzyme–ouabain complex of the Na+/K+-ATPase: K+ lowers the affinity of the enzyme for cardioactive steroids at theirhigh affinity, extracellularbinding site The presence of extracellular Na+competitively inhibits this effect of K+, and high concentrations of Na+ enhance cardioactive steroid binding This probably occurs via interaction with sites from which Na+ is released to the extracellular medium On the otherhand, with purified enzyme in the presence of Mg2+and Pi, 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 Ca2+ 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 eitherhave a similarstructure oract in a similar way to the cardiac steroids currently in use for clinical purposes The discovery of various isoforms of the sodium pump that are specifically expressed in discrete tissues indirectly supports this concept of an endogenous digitalis-like factor, especially because in some cases distinctive differences were found in the interaction of the various pump isoforms with cardiac steroids and transported cations Recently, various research groups have succeeded in both isolating endogenous circulating factors that interact with the sodium pump and inhibit 86Rb+ uptake (Rb+ is a
Trang 8surrogate for K+) and also in identifying several of them as
ouabain orits congeners [54] In addition, evidence was
provided in several investigations that the concentration of
so-called endogenous ouabain increases in plasma upon
excessive work and is present at higher levels in the serum of
hypertensive patients [54]
All these data indicate that ouabain might be directly or
indirectly involved in the regulation of vascular tone and
possibly also in the pathogenesis of hypertension
Never-theless, the mechanisms that might be relevant have not yet
been elucidated, and ouabain orcardiac glycosides do not
appearin the list of vasoactive endogenous substances that
includes such agents as endothelin and nitric oxide Recent
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 vasculartone regulation orin 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 LD50forrodents 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
Furthermore, depolarization is a key event that affects
numerous secondary systems Thus, the concentration of
Ca2+ becomes elevated in several organs through the
opening of Ca2+channels and leads to the production of
inositol trisphosphate [57], the activation of
phospholi-pase A2and metabolism of arachidonic acid, and numerous
otherphysiological responses that all stem from the
increased Na+ influx and the ensuing increase in the
concentration of cytosolic Ca2+that accompany the initial
K+outflow [57]
The actual binding site forpalytoxin has been the subject
of controversy for some time, despite the fact that the Na+/
K+-ATPase was known to be inhibited by the toxin This
issue was resolved by expressing Na+/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+-ATPase,
the toxin appears to convert the enzyme into a permanently
open conformation that allows K+ to flow down its
concentration gradient out of the cell This channel is
possibly the permanently open state of the natural
iono-phore of the sodium pump
Palytoxin binds predominantly to the E1-P conformation
of the pump This observation results from experiments
demonstrating that ATP and Na+, which first induce the
E1-P conformation, enhance the binding of 125I-labeled palytoxin Mg2+and Pi, which support the direct formation
of the E2-P conformation, decrease binding [57] ATP hydrolysis or enzyme autophosphorylation, however, are not necessary for the formation of the palytoxin-induced 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 E1-P conformation of the enzyme and to inhibit the binding of [3H]ouabain, although,
as with palytoxin, phosphorylation is not absolutely required
The experiments with palytoxin and sanguinarine show that under the appropriate conditions an ion channel can be created within an ion pump This ion channel, which is possibly the ionophore of the pump arrested into a permanently open state, is regulated under normal, physio-logical conditions so that at any given time it is open to only one side of the membrane Interestingly, the electrogenic step in the catalytic cycle of the sodium pump is associated with the E1-P conformation of the enzyme
Viewed from this standpoint, the reaction cycle of the sodium pump (Fig 1) takes on a new aspect: in the first part
of the reaction up to the occlusion of Na+, the pump can be seen as a ligand-inactivated ion channel where Pi is the ligand that blocks the backflow of Na+out of the occlusion pocket In the last part of the reaction sequence, the release
of K+ into the intracellular medium, the enzyme can be viewed as a ligand-activated ion channel where ATP is the ligand whose binding opens the occlusion pocket and allows the release of K+to the cytosol
P R O S P E C T S F O R F U T U R E R E S E A R C H
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 Ca2+ -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
Trang 9A C K N O W L E D G E M E N T S
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.
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