Sodium-Potassium
Pump
Monica Kriete '11 and Mary Clare Higgins-Luthman '11
Contents:
I.
Introduction
The
transmembrane sodium-potassium ATPase is of key importance in living
systems.
Three sodium ions are pumped out in exchange for the uptake of two
potassium
ions by utilizing the Gibbs free energy of ATP hydrolysis. This enables
cells
to establish and maintain an electrochemical gradient used to maintain
cell
volume and pH. This also provides the energy for secondary active
transport of
other ions and nutrients, such as protons.
The
sodium-potassium ATPase belongs to the P-type ATPase family, so named
for the
reversible autophosphorylation of an
aspartate residue as part of ATP hydrolysis (Brampkamp et. al. 2007).
P-type
ATPases transport cations, including heavy metals (transported by
P1-ATPases)
and non-heavy metals, such as sodium, potassium, and calcium
(transported by
P2-ATPases) (Lutsenko and Kaplan, 1993).
Although most P-type
ATPases are single-subunit proteins (Geering), the Na+
,K+ -ATPase
is
comprised of
three subunits, named alpha, beta, and gamma, all of which are
necessary for
correct functioning (Morth et. al.).
The α-subunit
is
homologous
to single-subunit P-type ATPases, such as the sarco(endo)plasmic
reticulum Ca2+-ATPase
(SERCA). The β-subunit
is
necessary for routing
of the
alpha-subunit to the plasma membrane, and also plays a role in ion
occlusion
during exchange. The γ-subunit
regulates
pumping activity
tissue-specifically.
The Na+ /K+ pump
is shown crystallized
in the E2 state
(see below) bound
to MgF42-, a
phosphate
analogue
,
and
two Rb+
ions,
which are K+
analogues
.
II.
Mechanism
The
P-type ATPases contain three highly-conserved domains: the actuator (A)
domain
;
nucleotide-binding
(N) domain
;
the
and the phosphorylation
(P) domain
.
These three domains effect
conformational
changes in response to ligand binding that result in ion exchange
across the cell membrane. In the Na+
/K+
pump, all three domains
are
contained in the alpha subunit, along with the Na+
and K+
binding
sites. Two sites are present between helices 4, 5 and 6 that
serve to bind two K+
or two of the three Na+
ions, which in this model
are shown bound to two Rb+ ions. The
third Na+
binding site is
found on the carboxy-terminal.
In
the Na+/K+ pump reaction cycle,three Na+
ions bind to the enzyme in
the E1 state, open to the lumen of the
cell, with ATP already bound to the N domain. Sodium-ion binding causes
movement of the A domain, which results in the occlusion of two of the
three sodium ions and the subsequent hydrolysis of ATP at the P domain.
The resulting ADP and carboxy-terminal Na+
ion are released to the
exterior of the cell, and the enzyme adopts the E2 conformation, open
to the exterior of the cell. The remaining two Na+
ions are exchanged
for two K+
ions. The enzyme occludes
the K+
ions with inorganic
phosphate still bound. The phosphate is coordinated by loop of Thr 212,
Gly 213, Glu 214, and Ser 215.
Although
one of the carboxyl
oxygens in Glu 213 is at a hydrogen bond distance from a
fluorine in MgF42-(the phosphate analog), the
high electronegativity of fluorine makes this conformation
unstable. Therefore, Glu 213 may be forced into this configuration by
surrounding residues (Toyoshima). A
water molecule replaces
the inorganic
phosphate, after which ATP-binding prompts conversion back to the E1
state. The K+
ions are released to the
cell lumen and the process
begins again.
A clearer depiction
of the conformational changes between states is given for another
P-type ATPase,
SERCA.
III. Alpha-Subunit:
Potassium Binding
In
addition to the A, N
and P domains
discussed earlier,
the α-subunit
also contains a transmembrane domain consisting
of ten alpha-helices designated αM1-10
αM4
and αM6
are
partially unwound to make
room for the K+ ions, as in SERCA.
The
binding sites for K+ are
contained within the transmembrane domain,
between helices
αM4, αM5,
and αM6,
and are
designated 1
and 2
.
Several residues are involved in ion binding
and stabilization. Asp 804
donates a
side-chain oxygen ligand to each site
.Glu
327 donates a
side-chain
oxygen to site 2
.
The side chains of residues Ser
775, Asn 776, and Glu
779
could
all potentially donate ligands
for binding, either directly or through an intervening water molecule,
and have been assigned roles in liganding by mutagenesis. Asp 808
and Gln
923 may also be
involved
.
Identical
or closely-related residues are found at corresponding positions in
SERCA. Therefore,
cation selectivity must be determined by
subtle differences in the side chains of the cation-binding residues
between SERCA and the Na+/K+ pump.
IV. Alpha-Subunit:
Sodium Binding
Two
of the Na+ ions involved in
exchange bind to the same sites as K+;
the third is proposed to bind to a site comprised of the C-terminal end
of the alpha-subunit and several nearby residues. The C-terminal end of
the alpha-subunit is extended eight residues relative to SERCA. The
first six residues of this extension form an α-helix
which
is accommodated between the
transmembrane helix of
the beta-subunit, αM7,
and αM10
.
The final two residues of the extension, Tyr 1015
and Tyr
1016
are
recognized by a binding pocket
between αM5, αM7
and αM8
.
Tyr
1016
seems
to
interact with Lys 766
and
Arg
933 in
the loop connecting αM8
and αM9.
Tyr 771, Thr
807, and Glu 954
are
the other residues proposed to be involved
this binding site,
based on mutagenesis studies. Due to their distance from the C-terminal
end, it is likely that these residues stabilize interactions between
the helices rather than coordinating the ligands.
The
region surrounding the
C-terminal extension is highly
electropositive, containing six
arginine
residues
.
In other types of ion channels, clusters of arginine
residues act as voltage sensors that move in response to membrane
depolarization; thus, Morth et. al
propose that this cluster could function similarly to control the
voltage-sensitivity of the Na+/K+ pump.
V.
Beta and Gamma Subunits
The
Beta
Subunit:
The
β subunit directs the α subunit to the
plasma membrane. Only the transmembrane segment of the β
subunit, an α
helix, was able to be crystallized. This helix makes direct
contact
only with αM7and αM10
helix of the alpha subunit.
Three
amino acid
residues, Tyr 39, Phe
42, and Tyr 43,
interact with αM7 around Gly
848.
The
extracellular domain of
the β subunit contains a large glycosylated
region and is
thought to
cover
the
alpha subunit,
preventing K+
escape.
The
Gamma subunit:
The
transmembrane segment of the ɣ subunit
is also an alpha helix that interacts
minimally with the alpha subunit. Gly
41 of the ɣ
subunit interacts with Glu 953
of αM9 of the alpha subunit.
Several
other
residues on αM9, Phe 949,
Leu
957, and Phe 960,
are thought
to interact with
the ɣ subunit
based on a
mutagenesis study
.
The
extracellular domain of the ɣ
subunit contains a conserved FXYD motif and moves between the alpha and
beta
subunits to regulate enzyme activity.
VI.
References
Bramkamp, Marc,
Karlheinz Altendorf, and Jorg-Christian Greie, 2007.
"Common patterns and unique features of P-type ATPases: a comparative
view on the KdpFABC complex from Escherichia coli (Review)." Molecular Membrane Biology
24(5-6): 375-386.
Geering, Kathi, 2001. "The Functional Role of Beta-Subunits in
Oligomeric P-type ATPases." Journal
of Bioenergetics
and Biomembranes 33: 425-438.
Lutsenko, Svetlana and Jack H. Kaplan, 1995."Organization of P-Type
ATPases: Significance of Structural Diversity" Biochemistry
34, No. 48: 15607-15613.
Morth, J. Preben, et al, 2007. "Crystal structure of the
sodium-potassium pump." Nature
450: 1043-1049.
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