Department of

Biological Chemistry

T he Sodium-potassium Pump: Prof. Steven J.D.Karlish

Dr. Daniel Tal, Dr. Adriana Katz,
structure, function, regulation Dr. Einat Kapri-Pardes,
Talya Belogus, Haim Haviv,
and pharmacology Elizabeta Dinitz

The Na, K-pump or Na/K-ATPase include sarcoplasmic reticulum

actively transports Na and K ions across Ca-ATPase, gastric cell membrane H/K-
mammalian cell membranes to establish ATPase, plasma membrane Ca-ATPase,
and maintain the characteristic trans- plant cell membrane H-ATPase, heavy
membrane gradients of Na and K ions. metal-dependent ATPases etc., with
972 8 934 2278
This function underlies essentially all of selectivity for the other cations.
mammalian cell physiology. For example, FAX 972 8 934 4118
in the kidney, the Na, K-pump controls P-type ATPases have a common [email protected]
body Na and K balance, extracellular kinetic mechanism, which involves
www.weizmann.ac.il
volume and blood pressure. In the heart covalent phosphorylation of an active
the Na, K-pump controls myocyte Ca site aspartate residue by ATP, an E1P-
balance and cardiac contractility. The E2P conformational change coupled
Na, K-pump is the receptor of digitalis to cation movement, hydrolysis of
steroids used to treat heart failure. the phosphoenzme and an E2-E1
Na/K-ATPase is a membrane protein and conformational change to complete the The β subunit plays an essential role
consists of a catalytic α subunit with ten cycle. Crystal structures of sarcoplasmic as a chaperone of α, and is known also
trans-membrane segments, and a single reticulum Ca-ATPase, and native renal to play an important role in cell-cell
trans-membrane glycosylated β subunit, Na,K-ATPase, published recently, adhesion. However, its structure has
required for stabilization. Na,K-ATPase illuminate the basic mechanism of not been well defined. We have used
is regulated by FXYD proteins which active cation transport. Neverthless Fold Recognition methods to predict
are auxiliary subunits. There are four insights into crucial features of Na,K- that the extracellular domain has an
isoforms of α(1-4) and three isoforms of pump structure, function, regulation Imunoglobulin-like fold and consists
β expressed in a tissue-specific fashion. and pharmacology are lacking. of two lobes (Fig. 2). This concept
α1 is the “housekeeping” isoform. α2 is has interesting implications for the
expressed in heart and other muscle Structure- crystalization and physiological role of the β subunit. We
and plays a key role in maintenance of modeling are now attempting to express and
blood pressure and cardiac function. We have expressed Na,K-ATPase purify these putative lobes of the protein
in the methanotrophic yeast, Pichia (Dinitz and Karlish, unpublished).
The Na, K-ATPase is a member of pastoris, and purified the protein
the P-type ATPase family of cation to homogeneity in a single step Function – E1-E2 conformational
pumps that use the free energy of (Strugatsky et. al., 2003; Cohen et. changes
hydrolysis of ATP to actively transport al, 2005; Haviv et. al., 2007). About Crystal structures have shown that
i cations against their electrochemical 1-2mg of pure, stable and functional the essence of E1-E2 conformational
gradients. Other P-type ATPases α/β complexes in a non-ionic detergent changes is a movement of cytoplasmic
can be prepared conveniently. Initially domains (N, P, and A) coupled to
Life Science Open Day ∙ 2008 ∙ Weizmann Institute of Science

porcine and human α1/β1 were purified movement of trans-membrane

. An essential feature is that specific segments, which mediates the cation
interactions with phosphatidyl serine transport. However, it is not known what
(PS) are required to stabilize the triggers the conformational changes.
protein, probably at a site near the Previously we hypothesized that changes
α/β subunit interface. More recently in charge on active site aspartate
the human α2/β1 isoform complex (D369) upon phosphorylation, are the
has been expressed, purified and trigger (Strugatsky et.al, 2003). We
stabilized (Lifshitz et. al, 2007). α2 is have now utilized purified fluorescein-
unstable compared to α1 due to weaker labeled recombinant Na,K-ATPase
phospholipid-protein interactions and (see Karlish, 1980) to look at effects
must be stabilized by a combination of of charge of D369 on conformational
PS/cholesterol. Other isoforms α and β changes, by comparing wild-type and
subunits are now being expressed. charge neutralized mutants (D369N
and D369A). Steady-state and transient
Crystalization trials are being carried kinetics of fluorescence changes show
out. If suitable crystals are obtained that the charge on D369 is indeed a
it is hoped that these will lead to crucial feature (Belogus and Karlish,
Fig. 1 Proposed immunoglobulin-like lobes determination of structure of different unpublished) ( Fig. 2).
of the β subunit ectodomain fitted into the conformations of the protein, mutants,
electron density map of the Na,K-ATPase isoforms, and complexes with FXYD In another approach we have utilized
together with the α subunit proteins (see below). the technique of Fe-catalyzed oxidative
 FYYD1 has been expressed in Pichia Selected publications
Pastoris and reconstituted with purified Karlish, S.J. (1980) Characterization of
human α1/β1 and α2/β1 complexes conformational changes in (Na,K)
to produce α1/β1/FXYD1 and α2/ ATPase labeled with fluorescein at
β1/FXYD1 complexes (Lifshitz et. the active site. J Bioenerg Biomembr,
al., 2006;Lifshitz et. al., 2007). The 12, 111-136.
functional properties of α1/β1/FXYD1
Strugatsky, D., Gottschalk, K.E.,
(phosphorylated or not at Ser68) have
Goldshleger, R., Bibi, E. and Karlish,
been characterized. A striking feature
S.J. (2003) Expression of Na+,K+-
of the α1/β1/FXYD1 and α2/β1/FXYD1
ATPase in Pichia pastoris: analysis of
Fig. 2 Stopped-flow fluorimeter traces of complexes is that they are highly
wild type and D369N mutant proteins
the E2(Rb)-E1Na conformational transitions, thermally stabilized by comparison with
by Fe2+-catalyzed oxidative cleavage
showing a large reduction in rate for the α1/β1 and α2/β1 complexes (Lifshitz
and molecular modeling. J Biol Chem,
D369N and D369A mutants compared to et. al., 2007). FXYD1 stabilizes the
278, 46064-46073.
WT. phosphatidyl serine-α/β interaction.
More recently we have expressed Karlish, S.J. (2003) Investigating the
FXYD1 in E.Coli purified the protein and energy transduction mechanism of
cleavage , developed to analyze P-type ATPases with Fe2+-catalyzed
reconstituted α1/β1/FXYD1 complexes
spatial organization of proteins around oxidative cleavage. Ann N Y Acad Sci,
(Lifshitz and Karlish, unpublished,
specifically bound Fe (reviewed Karlish, 986, 39-49.
in collaboration with the Weizmann
2003), to investigate divalent metal sites
Institute Proteomics Center). The Cohen, E., Goldshleger, R., Shainskaya,
in the Na,K-ATPase expresed in Pichia
purified α1/β1/FXYD1 complex will A., Tal, D.M., Ebel, C., le Maire, M.
Pastoris (Strugatsky et. al.,2005). This
be used for crystalization trials and and Karlish, S.J. (2005) Purification
suggested that two Mg ions are bound
detailed functional characterization. of Na+,K+-ATPase expressed in Pichia
to the protein in the complex with
Similar experiments with FXYD2 have pastoris reveals an essential role of
ATP-Mg, one in the P-domain (D710)
been initiated. phospholipid-protein interactions. J
and a second in the N domain (D443).
Biol Chem, 280, 16610-16618.
Pharmacology- an α2-selective Strugatsky, D., Gottschalk, K.E.,
Regulation- FXYD proteins
cardiac glycoside (CG)? Goldshleger, R. and Karlish, S.J.
(with Prof. Haim Garty, Dept.
Plant-derived digitalis steroids have (2005) D443 of the N domain of
Biological Chemistry)
been used for over two hundred years Na+,K+-ATPase interacts with the
FXYD proteins are a group of seven
to increase the force of contraction of ATP-Mg2+ complex, possibly via a
short single span transmembrane ii
the heart (positive inotropy), but they second Mg2+ ion. Biochemistry, 44,
proteins termed after the invariant motif
are dangerous drugs and can induce 15961-15969.
FXYD in their extracellular domain.
fatal arrhythmias. In addition it is known

Life Science Open Day ∙ 2008 ∙ Weizmann Institute of Science

FXYD proteins act as tissue-specific Garty, H. and Karlish, S.J. (2006) Role
that digitalis-like steroids are produced
regulatory subunits, which adjust the of FXYD proteins in ion transport.
in mammals in a manner similar to
kinetics properties of the Na+, K+-pump Annu Rev Physiol, 68, 431-459.
steroid hormones, and are intimately
to the needs of the particular cell Lifshitz, Y., Lindzen, M., Garty, H.
involved in regulation of blood pressure
type or physiological state (reviewed and Karlish, S.J. (2006) Functional
and cardiac hypertrophy. Thus, there
in Garty and Karlish, 2006). We have interactions of phospholemman
is great interest in the mechanism
investigated intensively the functional (PLM) (FXYD1) with Na+,K+-
of action of endogenous CG’s and,
effects and structural interactions ATPase. Purification of alpha1/
development of safer CG’s.
of FXYD 1, 2, 4 and 5 expressed in beta1/PLM complexes expressed in
One way to reduce digitalis toxicity
in mammalian cells and Xenopus Pichia pastoris. J Biol Chem, 281,
could be to develop an inhibitor
oocytes. Most recently we have been 15790-15799.
selective for the α2 isoform. α1,α2 α3
focussing on interactions of purified
isoforms are all expressed in humans Haviv, H., Cohen, E., Lifshitz, Y., Tal,
FXYD1 (phospholemman, PLM). FXYD1
hearts, but α1 is the predominant D.M., Goldshleger, R. and Karlish, S.J.
regulates the Na+, K+-pump in cardiac
isoform. We are utilizing the human (2007) Stabilization of Na(+),K(+)-
and skeletal muscle. PLM has PKA
α1β1 and α2β1 complexes purified ATPase purified from Pichia pastoris
and PKC phosphorylation sites and
from P.pastoris membranes to try and membranes by specific interactions
responds to -adrenergic and other
develop an α2-selective inhibitor. This with lipids. Biochemistry, 46,
hormonal signals.
involves a combination of biochemical 12855-12867.
http://www.ncbi.nlm.nih.gov/entrez/
screening, synthetic chemistry and Lifshitz, Y., Petrovich, E., Haviv, H.,
query.fcgi?cmd=Retrieve&db=pubmed&d
molecular modeling. Goldshleger, R., Tal, D.M., Garty, H.
opt=Abstract&list_uids=16148001&query_
hl=3&itool=pubmed_docsum and Karlish, S.J. (2007) Purification
 of the human alpha2 Isoform of Na,K-
ATPase expressed in Pichia pastoris.
Stabilization by lipids and FXYD1.
Biochemistry, 46, 14937-14950.

Acknowledgements
SJDK is the William Smithburg Professor
of Biochemistry. This work is supported
by the Israel Science foundation and
German-Israel foundation (GIF).

INTERNAL support
This work is supported by the Minerva
foundation (Germany), Weizmann
Institute Renal Research Fund,
Mauerberger foundation (South Africa) ,
and Johnson and Johnson/Yeda.

iii
Life Science Open Day ∙ 2008 ∙ Weizmann Institute of Science