Hyperpolarization-activated inward leakage currents caused by deletion or mutation of carboxy-terminal tyrosines of the Na+/K+-ATPase {alpha} subunit.

Abstract

The Na(+)/K(+)-ATPase mediates electrogenic transport by exporting three Na(+) ions in exchange for two K(+) ions across the cell membrane per adenosine triphosphate molecule. The location of two Rb(+) ions in the crystal structures of the Na(+)/K(+)-ATPase has defined two "common" cation binding sites, I and II, which accommodate Na(+) or K(+) ions during transport. The configuration of site III is still unknown, but the crystal structure has suggested a critical role of the carboxy-terminal KETYY motif for the formation of this "unique" Na(+) binding site. Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human Na(+)/K(+)-ATPase alpha(2) subunit decreases the affinity for extracellular and intracellular Na(+), in agreement with previous biochemical studies. Apparently, the DeltaYY deletion changes Na(+) affinity at site III but leaves the common sites unaffected, whereas the more extensive DeltaKETYY deletion affects the unique site and the common sites as well. In the absence of extracellular K(+), the DeltaYY construct mediated ouabain-sensitive, hyperpolarization-activated inward currents, which were Na(+) dependent and increased with acidification. Furthermore, the voltage dependence of rate constants from transient currents under Na(+)/Na(+) exchange conditions was reversed, and the amounts of charge transported upon voltage pulses from a certain holding potential to hyperpolarizing potentials and back were unequal. These findings are incompatible with a reversible and exclusively extracellular Na(+) release/binding mechanism. In analogy to the mechanism proposed for the H(+) leak currents of the wild-type Na(+)/K(+)-ATPase, we suggest that the DeltaYY deletion lowers the energy barrier for the intracellular Na(+) occlusion reaction, thus destabilizing the Na(+)-occluded state and enabling inward leak currents. The leakage currents are prevented by aromatic amino acids at the carboxy terminus. Thus, the carboxy terminus of the Na(+)/K(+)-ATPase alpha subunit represents a structural and functional relay between Na(+) binding site III and the intracellular cation occlusion gate.

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J. Gen. Physiol. Vol. 135 No. 2 115–134
www.jgp.org/cgi/doi/10.1085/jgp.200910301
115
I N T R O D U C T I O N
The Na+/K+-ATPase is an electrogenic ion pump, which
exports three Na+ ions and imports two K+ ions at the ex-
pense of one ATP molecule. The reaction cycle of the
Na+/K+-ATPase is commonly expressed as a sequence of
reversible partial reactions known as the Post-Albers
scheme (Fig. 1 A) (Albers, 1967; Post et al., 1972). The
sequential translocation of Na+ and K+ ions requires
strict cation specificity of the phosphorylation and de-
phosphorylation reactions, and the changes in the ap-
parent affinities for the individual cation species are
accompanied by alternating exposure of ion binding
sites toward the intracellular and extracellular medium.
Electrophysiological experiments and relaxation
studies have supported the notion that the major elec-
trogenic event during the Na+/K+-ATPase’s reaction
cycle occurs during Na+ transport (Fendler et al., 1985;
Gadsby et al., 1985; Nakao and Gadsby, 1986; Gadsby
and Nakao, 1989; Rakowski et al., 1991; Wuddel and
Apell, 1995; Clarke and Kane, 2007). It has been sug-
gested that electrogenicity arises from the passage of
Correspondence to Thomas Friedrich: friedrich@­chem.tu-berlin.de
Abbreviations used in this paper: ENaC, epithelial Na+ channel; TEVC,
two-electrode voltage clamp; WT, wild-type.
Na+ ions through a narrow, high field “access channel”
to the extracellular space (Läuger, 1979; Gadsby et al.,
1993; Hilgemann, 1994; Sagar and Rakowski, 1994;
Rakowski et al., 1997; Holmgren et al., 2000). The exis-
tence of a negative slope in the stationary current–voltage
curve suggested that K+ ions also bind within an extra-
cellular ion well of smaller fractional depth (Rakowski
et al., 1991). Structural evidence for the existence of an
extracellular access channel in P-type ion pumps is miss-
ing so far, and from the viewpoint of rate theory, the
movement of energy barriers along the transmembrane
field would be equally sufficient to explain the experi-
mental observations (Läuger and Apell, 1988). Never-
theless, the access channel metaphor in combination
with the term “fractional depth” (or “equivalent charge”)
is frequently used to denote that an ion passes a certain
fraction of the transmembrane electric field to reach or
exit from its binding site.
According to Holmgren et al. (2000), the extracellu-
lar release of Na+ ions occurs in three distinct steps,
Hyperpolarization-activated inward leakage currents caused
by deletion or mutation of carboxy-terminal tyrosines
of the Na+/K+-ATPase  subunit
Susan Meier, Neslihan N. Tavraz, Katharina L. Dürr, and Thomas Friedrich
Technical University of Berlin, Institute of Chemistry, D-10623 Berlin, Germany
The Na+/K+-ATPase mediates electrogenic transport by exporting three Na+ ions in exchange for two K+ ions across
the cell membrane per adenosine triphosphate molecule. The location of two Rb+ ions in the crystal structures of
the Na+/K+-ATPase has defined two “common” cation binding sites, I and II, which accommodate Na+ or K+ ions
during transport. The configuration of site III is still unknown, but the crystal structure has suggested a critical role
of the carboxy-terminal KETYY motif for the formation of this “unique” Na+ binding site. Our two-electrode voltage
clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human
Na+/K+-ATPase 2 subunit decreases the affinity for extracellular and intracellular Na+, in agreement with previous
biochemical studies. Apparently, the YY deletion changes Na+ affinity at site III but leaves the common sites unaf-
fected, whereas the more extensive KETYY deletion affects the unique site and the common sites as well. In the
absence of extracellular K+, the YY construct mediated ouabain-sensitive, hyperpolarization-activated inward cur-
rents, which were Na+ dependent and increased with acidification. Furthermore, the voltage dependence of rate
constants from transient currents under Na+/Na+ exchange conditions was reversed, and the amounts of charge
transported upon voltage pulses from a certain holding potential to hyperpolarizing potentials and back were un-
equal. These findings are incompatible with a reversible and exclusively extracellular Na+ release/binding mecha-
nism. In analogy to the mechanism proposed for the H+ leak currents of the wild-type Na+/K+-ATPase, we suggest
that the YY deletion lowers the energy barrier for the intracellular Na+ occlusion reaction, thus destabilizing the
Na+-occluded state and enabling inward leak currents. The leakage currents are prevented by aromatic amino acids
at the carboxy terminus. Thus, the carboxy terminus of the Na+/K+-ATPase  subunit represents a structural and
functional relay between Na+ binding site III and the intracellular cation occlusion gate.
© 2010 Meier et al. This article is distributed under the terms of an Attribution–Noncom-
mercial–Share Alike–No Mirror Sites license for the first six months after the publication
date (see http://www.jgp.org/misc/terms.shtml). After six months it is available under a Cre-
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The Journal of General Physiology

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116C-terminal truncations of the Na+ pump’s  subunit
It has been suggested from the crystal structure
(Morth et al., 2007) that the conserved (K/R)E(T/S)YY
motif at the carboxy-terminal end of the Na+/K+-
ATPase’s  subunit is important for the formation of
the unique Na+ binding site (see Fig. 1 B). In accor-
dance, mutations or deletions of one or both carboxy-
terminal tyrosines, but also larger deletions or C-terminal
extensions, decrease the affinity for extracellular as well
as intracellular Na+ (Morth et al., 2007; Tavraz et al.,
2008; Blanco-Arias et al., 2009; Toustrup-Jensen et al.,
2009). Changes in Na+ affinity have also been reported
in a recent electrophysiological study of a KESYY-trun-
cated Xenopus 1 isoform (Yaragatupalli et al., 2009).
Remarkably, these affinity changes were assigned to the
effects of the KESYY deletion on the two common
binding sites rather than on the proximal unique site.
Furthermore, the deletion led to hyperpolarization-in-
duced inward currents, which were attributed to low af-
finity passive flow of Na+ ions through the KESYY-deleted
pump (Yaragatupalli et al., 2009). Here, we specifically
investigate the consequences of deletion or mutation of
only the two carboxy-terminal tyrosines on Na+/K+-
ATPase pump function. By analyzing stationary and
presteady-state currents in two-electrode voltage clamp
(TEVC) experiments on Xenopus oocytes, we show that
the less extensive YY deletion dominantly affects the
apparent Na+ affinity at the unique binding site. Fur-
thermore, we characterize the hyperpolarization-acti-
vated inward leakage currents in more detail to provide
a framework for understanding this potentially energy-
dissipating operation mode of carboxy-terminally trun-
cated Na+ pumps.
M AT E R I A L S A N D M E T H O D S
cDNA constructs
Human Na+/K+-ATPase 2 and 1 subunit cDNAs were subcloned
into a modified pCDNA3.1 vector, which had been optimized for
expression in Xenopus oocytes (Koenderink et al., 2005). For mu-
tagenesis, the QuikChange site-directed mutagenesis kit (Agilent
Technologies) was used. All PCR-derived fragments were verified
by sequencing (Eurofins MWG Operon). Mutations Q116R and
N127D were introduced to obtain an ouabain-resistant protein
with an IC50 in the millimolar range (Price and Lingrel, 1988),
which is referred to as ATP1A2 WT in this paper. Amino acid
numbering refers to the human Na+/K+-ATPase 2 subunit.
cRNA synthesis and oocyte treatment
cRNA synthesis was performed with the T7 mMessage mMachine
kit (Applied Biosystems). Oocytes were obtained by partial ovari-
ectomy from anesthetized Xenopus laevis females, followed by col-
lagenase 1A treatment (Sigma-Aldrich). Oocytes were injected
with 25 ng 2 subunit and 2.5 ng 1 subunit cRNA and stored in
ORI buffer (contents in mM: 110 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2,
and 5 HEPES, pH 7.4) containing 50 mg/L gentamycin at 18°C
for 3–4 d. In preceding experiments, [Na+]int was elevated by incu-
bating the oocytes for 45 min in Na+ loading solution (contents in
mM: 110 NaCl, 2.5 Na-citrate, 2.5 3-(N-Morpholino)-propanesul-
fonic acid [MOPS], and 2.5 Tris, pH 7.4), followed by an incubation
from which only deocclusion and release of the first Na+
ion is associated with a large charge movement. This
highly electrogenic release of the first Na+ ion is most
probably rate limited by the major E1P-E2P conforma-
tional change. After release of the first Na+ ion, the high
field access path to the other Na+ occlusion sites is re-
structured such that the exit of the remaining two Na+
ions contributes only little to the overall electrogenicity
(Wuddel and Apell, 1995; Holmgren et al., 2000). In
terms of the access channel model, positive voltage
pulses drive Na+ ions extracellularly out of the access
channel and result in positive transient currents. Con-
versely, negative voltage pulses promote the movement
of Na+ ions through the extracellular access channel to
enhance binding to sites in E2P and induce negative
transient currents. Due to the electrogenicity of reverse
binding of extracellular Na+, the occupancy of the Na+
binding sites is controlled by [Na+]ext and voltage. The
amount of charge moved in response to certain voltage
steps follows a Boltzmann-type function, which is cen-
tered at a half-maximal voltage (V0.5), at which 50% of
the pump molecules are in E2P and 50% are in E1P
(Holmgren et al., 2000). Thus, changes in V0.5 at a given
[Na+]ext indicate changes in the affinity for Na+ext
(Holmgren et al., 2000; Holmgren and Rakowski, 2006).
Since 2000, the understanding of P-type ATPase func-
tion has been advanced by several crystal structures of
the Ca2+-ATPase from sarcoplasmic/endoplasmic retic-
ulum (Toyoshima et al., 2000; Toyoshima and Nomura,
2002; Olesen et al., 2004, 2007; Sørensen et al., 2004;
Toyoshima and Mizutani, 2004; Jensen et al., 2006),
which cover the reaction cycle rather completely. The
wealth of information has recently been expanded by
the structures of an H+-ATPase from plants (Pedersen
et al., 2007) and the Na+/K+-ATPase (Morth et al., 2007;
Shinoda et al., 2009).
The K+ binding sites I and II of the Na+/K+-ATPase are
formed by residues homologous to those coordinating
the two Ca2+ ions in the sarcoplasmic/endoplasmic re-
ticulum pump’s E1 conformation (Morth et al., 2007;
Shinoda et al., 2009). Because this pocket most likely
accommodates two Na+ ions in the E1 form of the Na+
pump as well, sites I and II will be referred to as “com-
mon” sites in this study. The third binding site, which
exclusively coordinates Na+ ions and is therefore termed
“unique,” has been located between transmembrane
domains M5, M6, and M9 according to structural
modeling (Ogawa and Toyoshima, 2002) and mutagen-
esis work (Van Huysse et al., 1993; Li et al., 2005, 2006).
The unique site has been correlated to a noncanonic
transport mode (Li et al., 2006), which only occurs in
the absence of extracellular Na+ and K+ (Rakowski et al.,
1991; Efthymiadis et al., 1993; Wang and Horisberger,
1995; Rettinger, 1996) and is characterized by hyperpo-
larization-activated, mainly H+-driven inward currents
(Vasilyev et al., 2004).

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Meier et al.117
buffer (100 NMDG × Cl, 1 CaCl2, 5 BaCl2, 5 NiCl2, 2.5 MOPS, and
2.5 Tris, pH 7.4), and Li+ buffer (100 LiCl, 1 CaCl2, 5 BaCl2,
5 NiCl2, 2.5 MOPS, and 2.5 Tris, pH 7.4). All buffers contained
10 µM ouabain to inhibit the endogenous Xenopus Na+/K+-ATPase.
The heterologously expressed Na+/K+-ATPase could be inhibited
by 10 mM ouabain. To determine the voltage dependence of the
currents, cells were subjected to voltage pulse protocols as follows:
starting from 30-mV holding potential, cells were clamped for
200 ms to test potentials between +60 and 140 mV (in 20-mV
decrements), followed by a step back to 30 mV. Unless stated
differently, all currents in one experiment were normalized to the
amplitude at [Na+]ext = 100 mM and [K+]ext = 10 mM and 0 mV.
K+-induced pump currents
Solutions containing distinct K+ concentrations were prepared by
adding the appropriate amounts of KCl to Na+ buffer, NMDG+ buf-
fer, or Li+ buffer. The [K+]ext-dependent currents were calculated
by subtracting currents measured in K+-free Na+, NMDG+, or Li+
buffer from the currents measured in the presence of a distinct K+
concentration (added to Na+, NMDG+, or Li+ buffer). These K+-in-
duced difference currents are denoted as NaIxK, NMDGIxK, and LiIxK,
respectively, whereby the top left index symbolizes the major mon-
ovalent cation present (concentration: 100 mM) and the bottom
right index “xK” denotes [K+]ext (with [x] in millimolars). Alterna-
tively, Na+/K+-ATPase pump currents were determined as ouabain-
sensitive currents by calculating the difference between currents
measured in Na+, NMDG+, or Li+ buffer containing a certain K+
concentration and in Na+, NMDG+, or Li+ buffer containing 10 mM
ouabain. These ouabain-sensitive, K+-induced difference currents
are denoted as NaIxK(ouab), NMDGIxK(ouab), and LiIxK(ouab), respectively.
K0.5 values for the stimulation of stationary pump currents by ex-
tracellular K+ were determined using fits of a Hill equation

I
I
K
K[
nH
=
+



+
max
.
]
1
0 5

to the normalized currents at a given membrane potential (K0.5 is
the half-maximal concentration and nH the Hill coefficient). Hill
parameters from the fits were between 1 and 1.5.
Extracellular [Na+] dependence of ouabain-sensitive currents
in the absence of K+ext
Experiments to determine the dependence on [Na+]ext used
Na+[150] buffer (contents in mM: 150 NaCl, 1 CaCl2, 5 BaCl2, 5 NiCl2,
2.5 MOPS, and 2.5 Tris, pH 7.4), Na+[100] buffer (Na+ buffer),
Na+[50] buffer (1:1 mixture of Na+ buffer with NMDG+ buffer),
and Na+[0] buffer (NMDG+ buffer). Currents were determined by
calculating the difference between currents measured in one of
these buffers in the absence and presence of 10 mM ouabain.
pH dependence of ouabain-sensitive inward currents
in the absence of K+ext
Na+/K+-ATPase currents at different pHext were measured in Na+8.1
buffer (contents in mM: 100 NaCl, 1 CaCl2, 5 BaCl2, 5 NiCl2, and
5 Tris, pH 8.1), Na+5.5 buffer (contents in mM: 100 NaCl, 1 CaCl2,
5 BaCl2, 5 NiCl2, and 5 MES, pH 5.5), or NMDG+5.5 buffer (contents
in mM: 100 NMDG × Cl, 1 CaCl2, 5 BaCl2, 5 NiCl2, and 5 MES,
pH 5.5) by calculating the difference between currents mea-
sured in one of these buffers in the absence and presence of
10 mM ouabain.
Analysis of ouabain-sensitive presteady-state currents
Transient currents were measured at [Na+]ext = 100 mM by the ap-
plication of voltage step protocols and calculating the difference
between corresponding current traces, which had been measured
of at least 30 min in Na+ buffer (contents in mM: 100 NaCl,
1 CaCl2, 5 BaCl2, 5 NiCl2, 2.5 MOPS, and 2.5 Tris, pH 7.4), as
described previously (Rakowski et al., 1991).
Electrophysiology
Currents were recorded using the TEVC technique with an ampli-
fier (Turbotec 10CX; npi electronic GmbH) and PClamp 7 soft-
ware (MDS Analytical Technologies) at 21–23°C. Experimental
solutions were (contents in mM): Na+ buffer (see above), NMDG+
Figure 1. Reaction scheme and structural detail of the Na+/
K+-ATPase. (A) Modified Post-Albers reaction cycle of the Na+/
K+-ATPase. Upon intracellular binding of Na+ ions to the E1
conformation, a phosphointermediate with occluded Na+ ions,
E1P(3Na+), is formed, and after a conformational change to
E2P(3Na+), the Na+ ions dissociate to the extracellular space. Sub-
sequently, two K+ ions bind from the extracellular side and become
occluded, a process that stimulates dephosphorylation, and after
a conformational change from E2 to E1, the K+ ions are intracellu-
larly released. The gray box indicates the reaction sequence that
can be studied by voltage pulses at high [Na+]ext and [K+]ext = 0 in
TEVC experiments. (B) Structure of the Na+/K+-ATPase accord-
ing to PDB structure entry 3B8E (Morth et al., 2007). Amino acids
referred to in this work are indicated in ball-and-stick represen-
tation with numbering according to the human Na+/K+-ATPase
2 subunit. Helix M5 is depicted in yellow, the backbone of the
carboxy terminus (V1014-EKETY-Y1020) is in red, and residues of a
carboxy-terminal arginine cluster are in olive. Two Rb+ ions at the
binding sites are shown as magenta spheres. Note that Arg1005 in
the 3B8E structure (pig renal 1 subunit) corresponds to Tyr1009
in the human Na+/K+-ATPase 2 subunit.

Page 4

118C-terminal truncations of the Na+ pump’s  subunit
prepared with PyMOL 1.0r1 (http://www.pymol.org). Data analy-
sis for figure presentation was performed with Origin 7.5 (Origin-
Lab Corporation).
Online supplemental material
Fig. S1 shows an alternative way to determine the ouabain-sensi-
tive K+-induced difference currents and several I-V curves of the
YY deletion construct at pHext 8.1. In Fig. S2, the effects of ex-
tracellular Li+ on ouabain-sensitive currents of the YY construct
and ATP1A2 WT are shown. Fig. S3 depicts the voltage depen-
dence of the ouabain-sensitive stationary and transient currents
mediated by the YY construct over an expanded potential range.
Fig. S4 contains information about the voltage dependence of K+-
induced pump currents and transient currents of the KETYY
deletion construct. Figs. S1–S4 are available at http://www.jgp
.org/cgi/content/full/jgp.200910301/DC1.
R E S U LT S
Dependence of pump-related currents on [K+]ext, [Na+]ext,
and membrane potential
Upon expression in Xenopus laevis oocytes, TEVC exper-
iments were performed in Na+-containing buffers
([Na+]ext = 100 mM) to measure the K+ext-induced oua-
bain-sensitive pump currents of wild-type (WT) human
2/1 Na+/K+-ATPase and of the YY construct, in which
the two tyrosines at the carboxy terminus of the  sub-
unit were deleted. Fig. 2 A shows I-V curves of the
NaIxK(ouab) difference currents for the YY-truncated
Na+/K+-ATPase 2 subunit. In contrast to ATP1A2 WT,
the YY construct mediated inwardly rectifying differ-
ence currents in the absence of extracellular K+. The
amplitudes of these inward currents became smaller
with increasing [K+]ext but were still observed for [K+]ext
up to 2 mM at extremely negative potentials (140 mV).
At 10 mM K+, the ouabain-sensitive pump currents of
the YY construct were positive and increased steadily
with voltage (Fig. 2 A). We then determined the full K+-
induced NaIxK difference currents of the YY construct
at [Na+]ext = 100 mM by calculating the difference be-
tween currents recorded in the presence of a certain
[K+]ext and those measured at [K+]ext = 0 (Fig. 2 B). The
same information can be obtained by subtracting the
NaI0K(ouab) currents at 0 [K+]ext from the NaIxK(ouab) cur-
rents (Fig. S1 A). Because in the case of the YY con-
struct extracellular K+ not only induces normal Na+/K+
outward pumping (as evident from the similarity to WT
pump currents at positive potentials), but also dimin-
ishes the hyperpolarization-activated inward currents,
the full K+-induced NaIxK currents (Fig. 2 B) were mark-
edly different from those of the WT Na+/K+-ATPase
(Fig. 2 D). For the WT enzyme, currents at [K+]ext = 10 mM
increased steadily with voltage, and at [K+]ext below
5 mM, bell-shaped I-V curves were obtained with a maxi-
mum at +40 mV (Fig. 2 D). In contrast, the NaIxK cur-
rents of the YY construct at 10 and 5 mM [K+]ext had a
local minimum around 40 mV, and at lower [K+]ext,
the I-V curves were maximal around 100 mV and
first in Na+ buffer, and then in Na+ buffer containing 10 mM oua-
bain. The resulting difference currents were fitted by a single-expo-
nential function (plus a constant), whereby the first 3–5 ms were
disregarded to exclude artifacts arising from the charging of the
membrane capacitance. This yielded the amplitude A for the start
time of the fit and a decay time constant . The charge Q transported
during a transient current was determined as the integral of fitted
currents, A*·, in which A* was obtained by extrapolating the amp-
litude of the exponential fit curve to the onset of the voltage pulse.
The resulting Q-V curves were fitted to a Boltzmann function:

Q V( )Q
QQ
(zF VV
RT
q
exp
)
,
min
max


min
.
=+
−
⋅
+
−


1
0 5

where Qmax and Qmin are the saturation values of Q(V) at extremely
positive or negative voltages, V0.5 is the midpoint potential, zq is
the slope factor (equivalent charge), F is the Faraday constant, R
is the molar gas constant, T is the absolute temperature, and V is
the transmembrane potential.
Intracellular Na+ concentration achieved by the Na+
loading procedure
To control the efficiency of the applied Na+ loading procedure,
oocytes were injected with RNA mixtures containing cRNAs of the
2 subunit (25 ng/oocyte) and the 1 subunit (2.5 ng/oocyte) of
the Na+/K+-ATPase, and cRNAs of the , , and  subunits (0.3
ng/subunit/oocyte) of the amiloride-sensitive rat renal epithelial
Na+ channel (ENaC), as described previously (Hasler et al., 1998;
Crambert et al., 2000). After injection, oocytes were kept for 3 d
in ORI buffer supplemented with 10 µM amiloride, and Na+ load-
ing was performed as described above with 10 µM amiloride in
the loading/post-loading buffers. The reversal potential of the
amiloride-sensitive ENaC current was measured in the presence
of extracellular buffers containing 10 mM Na+, and [Na+]int was
calculated using the Nernst equation:

NaNa
VF
RT
ext
rev amil ,
++
 
= 

⋅




int
exp ,
where Vrev,amil is the reversal potential of amiloride-sensitive differ-
ence currents.
Variation of intracellular [Na+] using Na+ uptake
through the ENaC channel
These experiments used oocytes that simultaneously expressed
Na+/K+-ATPase and ENaC, as described above. After cRNA injec-
tion, cells were kept in a Na+-reduced ORI buffer (contents in
mM: 100 NMDG × Cl, 10 mM NaCl, 5 KCl, 1 MgCl2, 2 CaCl2,
2.5 MOPS, and 2.5 Tris, pH 7.4) for 2–3 d. 18–24 h before experi-
ments, oocytes were incubated in a Na+-free solution (contents in
mM: 110 NMDG × Cl, 5 KCl, 1 MgCl2, 2 CaCl2, 2.5 MOPS, and 2.5
Tris, pH 7.4) to minimize the initial [Na+]int. To successively
increase [Na+]int, oocytes were exposed to buffers containing
10 mM or (for the final increases of [Na+]int) 100 mM [Na+]ext at 30
or 100 mV. Between these Na+ loading steps, sets of measure-
ments were performed in which (1) the [Na+]int, (2) the Na+/K+
pump current in response to 10 mM K+, and (3) the ouabain-sen-
sitive inward currents at 100 mM [Na+]ext and 0 [K+]ext were deter-
mined. [Na+]int was determined from the reversal potentials of
amiloride-sensitive difference currents measured in buffers con-
taining 10 or 100 mM [Na+]ext.
Structural presentations and data analysis
Structural inspections of the Na+/K+-ATPase (PDB structure entry
3B8E) were performed with Swiss PDB viewer 3.7. Figures were

Page 5

Meier et al.119
tion of the “intrinsic” apparent K+ext affinity (Li et al.,
2005) at the two common sites, where K+ ions bind from
the extracellular space during transport. As shown in
Fig. 3 A, the K+-induced currents (NMDGIxK) of the YY
construct were positive for all [K+]ext tested, showing
that in the absence of Na+ext, the YY construct only me-
diates Na+/K+ outward pumping, and that K+ ions ap-
parently do not contribute to hyperpolarization-induced
inward currents of the YY construct. The current am-
plitudes for [K+]ext ≥ 2 mM hardly changed with voltage,
and at lower [K+]ext, the I-V curves showed a shallow de-
crease upon increases in membrane potential, which
can be attributed to the weakly electrogenic binding of
extracellular K+ (Rakowski et al., 1991). In K+-free
NMDG+ buffer, the ouabain-sensitive currents of the
YY construct were very small (Fig. 3 A), and even
smaller than the corresponding currents of ATP1A2
WT (Fig. 3 B), showing that Na+ext is required for the
hyperpolarization-activated inward currents. The large
NMDGI0K(ouab) currents of WT ATP1A2 (in the absence of
extracellular Na+ and K+) can be attributed to the hy-
perpolarization-activated H+ inward currents, as de-
scribed previously (Rakowski et al., 1991; Efthymiadis et
al., 1993; Wang and Horisberger, 1995; Rettinger, 1996;
Vasilyev et al., 2004). Therefore, the YY deletion re-
duces this H+-driven inward current at neutral pH, in
decreased steadily between 100 and +60 mV (Fig. 2 B).
Fitting of the [K+]ext-induced NaIxK currents at each
membrane potential with a Hill equation resulted in
NaK0.5(K+ext) values for the YY construct (Fig. 2 C) and
ATP1A2 WT (Fig. 2 E), which represent the voltage-de-
pendent apparent affinities for extracellular K+ in the
presence of 100 mM Na+ext. Characteristically U-shaped
curves were obtained for both enzymes with similar
minima, but the curve of the YY construct was shifted
by about 80 mV.
Fig. 2 F shows normalized NaI10K(ouab) and NaI0K(ouab) dif-
ference currents of ATP1A2 WT and the YY construct
at pHext 7.4. As indicated above, the YY construct gen-
erated large inwardly rectifying currents in the absence
of K+ext, which were not observed with ATP1A2 WT. The
direct comparison shows that the full K+-induced oua-
bain-sensitive currents of the YY construct at hyperpo-
larizing potentials are larger than the corresponding
currents of the WT enzyme, resulting in a significantly
smaller slope of the respective I-V curve.
To investigate which type of the cation binding site is
affected by the YY deletion, we measured the [K+]ext-
induced currents in Na+-free solutions. Replacement of
extracellular Na+ for a nontransported cation eliminates
the voltage-dependent competition of extracellular Na+
with K+ for the binding sites and allows the determina-
Figure 2. Voltage and [K+]ext dependence of stationary currents at [Na+]ext = 100 mM. (A) I-V curves of normalized ouabain-sensitive
K+-dependent currents NaIxK(ouab) of the Na+/K+-ATPase YY deletion construct measured at 100 mM [Na+]ext and [K+]ext as indicated.
(B) I-V curves of normalized K+-induced difference currents NaIxK (at 100 mM [Na+]ext) of the YY construct. (C) Voltage-dependent
NaK0.5(K+ext) values of the YY construct from fits of a Hill function to the NaIxK currents in B at each membrane potential. The minimal
NaK0.5(K+ext) was 1.02 ± 0.06 mM at 80 mV. The dashed line delineates the corresponding WT data from E for comparison. (D) I-V
curves of normalized NaIxK difference currents for ATP1A2 WT with [K+]ext as indicated. (E) Voltage-dependent NaK0.5(K+ext) values for
ATP1A2 WT from fits of a Hill function to the NaIxK currents in D at each membrane potential. The minimal NaK0.5(K+ext) was 1.10 ± 0.05 mM
at 0 mV. Data for both WT and YY are means ± SE from 14 cells of three oocyte batches. (F) I-V curves of normalized NaI10K(ouab) and
NaI0K(ouab) currents of ATP1A2 WT and the YY construct at pHext 7.4 ([Na+]ext = 100 mM). Data are means ± SE from six (WT) and seven
(YY) cells from two batches.

Page 6

120 C-terminal truncations of the Na+ pump’s  subunit
corresponding currents of the YY construct (not de-
picted). The K0.5(K+ext) values determined from the
NMDGIxK(ouab) currents, which represent intrinsic appar-
ent K+ affinities, were much smaller than those mea-
sured at [Na+]ext = 100 mM and did not increase at
hyperpolarizing potentials (Fig. 3 C). The K0.5(K+) val-
ues for the WT enzyme were slightly smaller than those
of the YY construct, but this difference was not signifi-
cant (at P > 0.05 level). Thus, the intrinsic K+ext affinity
at the common sites is apparently not affected by the
YY deletion, in line with findings obtained for the
KESYY construct (Yaragatupalli et al., 2009).
Voltage dependence and kinetics of ouabain-sensitive
presteady-state currents in the absence of extracellular K+
To specifically investigate electrogenic Na+ transport,
we performed voltage pulse experiments under extra-
cellularly high Na+ ([Na+]ext = 100 mM) and K+-free con-
ditions to measure ouabain-sensitive transient currents.
Fits of a single-exponential function to the transient
currents in response to “ON” or “OFF” voltage pulses
were used to determine time constants and the amount
of charge (QON or QOFF from ON or OFF transient cur-
rents) moved during a transient current. For ATP1A2
WT, the ouabain-sensitive ON and OFF transient cur-
rents follow a single-exponential function and decay al-
most completely to 0 (Fig. 4 A). The ON currents of
ATP1A2 WT were substantially faster at negative than at
positive potentials. Consequently, the reciprocal time
constants increased with hyperpolarization (Fig. 4 C).
Several properties of the ouabain-sensitive transient
currents of the YY construct (Fig. 4 B) were markedly
different from the WT enzyme. First, the transient cur-
rents upon voltage pulses to negative potentials showed
a large negative stationary current component (Fig. 4 B),
in line with the hyperpolarization-activated inward
currents described above. Second, the acceleration of
transient current kinetics at negative potentials was not
observed. The reciprocal time constants rather in-
creased at positive potentials (Fig. 4 D). Third, the volt-
age dependence of charge moved during the transient
currents was different, as apparent from the reduced
amplitudes of the ON transient currents at positive po-
tentials (or increased amplitudes at negative poten-
tials) compared with ATP1A2 WT (Fig. 4, A and B).
Accordingly, the V0.5 values from Q-V curves of the YY
construct (Fig. 4 F) were shifted by about 83 mV com-
pared with the WT enzyme (Fig. 4 E) without a signifi-
cant change in zq (Table I).
Whereas for the WT enzyme the QON and QOFF charges
were equivalent (not depicted), in the case of the YY
construct, the QON values (Fig. 4 F, triangles) were sig-
nificantly larger than QOFF (Fig. 4 F, squares) at hyper-
polarizing potentials. The QON and QOFF charges of the
YY construct saturated at positive potentials, but at
negative potentials, due to the large V0.5 shift, no onset
line with observations reported for the KESYY-trun-
cated pump (Yaragatupalli et al., 2009). In contrast to
the YY construct, which exhibited flat I-V curves, the
K+-induced difference currents of ATP1A2 WT increased
at negative potentials for all [K+]ext tested (Fig. 3 B).
This distortion of the I-V curves is most likely due to the
K+-induced inhibition of the H+ inward current, which
occurs in conjunction with K+ext-induced outward pump-
ing. However, when the ouabain-sensitive K+-induced
currents were calculated, the resulting I-V curves were
only weakly voltage dependent and very similar to the
Figure 3. Voltage and [K+]ext dependence of stationary currents
in the absence of Na+ext. (A and B) I-V curves of normalized K+-
dependent NMDGIxK difference currents of the YY construct (A) and
ATP1A2 WT (B) in the absence of extracellular Na+ ([NMDG+]ext
= 100 mM, pH 7.4) with [K+]ext as indicated. Also shown in A and
B are the ouabain-sensitive currents (NMDGIouab) in the absence of
K+ for the YY construct and ATP1A2 WT. (C) K0.5(K+ext) values
for the YY construct and for ATP1A2 WT at [Na+]ext = 0. For
each construct, data are means ± SD from eight oocytes out of
two batches.

Page 7

Meier et al. 121
of saturation was apparent (Fig. 4 F). Only a subset of
cells allowed for sufficiently stable current recordings to
show that QON and QOFF indeed saturate at potentials
below 150 mV (Fig. S3 B). We exclude that the dis-
crepancy between QON and QOFF is a trivial consequence
of the steady-state component of the transient currents
because we applied the sum of an exponential function
and a constant for fitting the transient currents, and
used only the amplitude and time constant from the ex-
ponential component for calculation of the transient
charge. We also do not consider the discrepancy as a
voltage clamp artifact because the difference between
corresponding QON and QOFF values markedly increased
at acidic pHext (see Fig. 5 E).
A direct comparison of several ON transient current
signals of the WT enzyme (from Fig. 4 A) and the YY
construct (from Fig. 4 B) on an expanded time scale in
Fig. 4 G also shows differences in the time course for
the initial phase of the transient currents. Immediately
after the voltage step (Fig. 4 G, dashed line), the arti-
facts from capacitive charging of the membrane are al-
most completely cancelled by the subtraction of current
signals recorded in the presence of 10 mM ouabain. Fit-
ting the sum of two exponential functions to the cur-
rent traces yielded time constants for a fast rising phase
(1) and a slower decaying phase (2). The time con-
stant 2 corresponds to the kinetics of the (slow) release
or deocclusion step of the first Na+ ion released to the
extracellular space, as described previously (Holmgren
et al., 2000). 1 is of 1 ms and reflects the speed of the
voltage clamp in TEVC experiments on oocytes. The
peak of the YY transient current at 140 mV occurred
with a delay of 2 ms, and a significantly slower 1 of
3.3 ms was observed (Fig. 4 G). We exclude that the
larger 1 is due to a slow voltage clamp because in the
same experiment, the rising phase of the transient
current at +60 mV was resolved to 1 ms.
Dependence of hyperpolarization-activated inward
currents on extracellular and intracellular [Na+]
To determine the Na+ dependence of the hyperpolar-
ization-activated inward currents of the YY construct,
experiments were first performed at different [Na+]ext
in K+-free solutions. The resulting I-V curves in Fig. 4 H
show that the amplitudes of the ouabain-sensitive in-
ward currents increase with increasing [Na+]ext, and that
saturation is achieved around 150 mM [Na+]ext. From
the hyperbolic [Na+]ext dependence, a half-maximal
concentration of 70 mM was determined. For com-
parison, the ouabain-sensitive ATP1A2 WT currents at
100 mM [Na+]ext were very small and did not show in-
ward rectification (Fig. 4 H). In a subset of YY-express-
ing cells that allowed for sufficiently stable recordings
even at extremely negative potentials, it could be shown
that the inward leak current amplitudes of the YY con-
struct at [Na+]ext = 100 mM also saturate at potentials
below 150 mV (Fig. S3 A).
Because the mutation or deletion of one or both car-
boxy-terminal tyrosines of the Na+/K+-ATPase  sub-
unit strongly decreased Na+ affinities (Morth et al.,
2009; Toustrup-Jensen et al., 2009), we tested the ap-
plied Na+ loading procedure to exclude effects result-
ing from insufficient saturation of Na+ binding sites
from the intracellular side. For this purpose, we coex-
pressed the YY construct together with the amiloride-
sensitive Na+ channel ENaC and determined [Na+]int
from amiloride-sensitive reversal potential shifts. Be-
fore Na+ loading, no ouabain-sensitive Na+/K+ outward
pump currents were observed, indicating that the ini-
tial [Na+]int was below the apparent KM of 13 mM, as
reported for the human 2/1 Na+/K+-ATPase (Crambert
et al., 2000). After Na+ loading, an average [Na+]int of
40 ± 4 mM was obtained. This value is 10-fold larger
than the K0.5(Na+) from Na+-dependent phosphoen-
zyme formation of the YY-deleted rat 1 isoform
(Toustrup-Jensen et al., 2009). When the ENaC chan-
nel was used to successively elevate [Na+]int, we found
that the hyperpolarization-activated inward currents at
0 [K+]ext saturated at a [Na+]int of 25 mM (Fig. 4 I),
with a half-maximal concentration between 10 and 15
mM, and the Na+/K+ outward pump currents induced
by 10 mM K+ext also did not increase further at a [Na+]int
above 20 mM, with a half-maximal concentration be-
tween 8 and 12 mM (not depicted). Thus, for the YY
construct, the [Na+]int achieved by the Na+ loading
procedure is not limiting for the phosphorylation
reaction that leads to formation of the Na+-occluded
E1P(3Na+) intermediate.
TA B L E I
Parameters of Boltzmann fits to Q-V curves
V0.5
zq
n
(mV)
WT
YY
YY-FF
YY-AA
KETYY
0.5 ± 1.0
83.4 ± 7.9
27.1 ± 0.8
120 ± 18
135 ± 19
0.76 ± 0.02
0.81 ± 0.06
0.81 ± 0.02
0.44 ± 0.03
0.45 ± 0.02
14
14
13
10
8
From transient currents in response to OFF voltage pulses at [Na+]ext = 100 mM and [K+]ext = 0 mM. Means ± SE; n = number of cells.

Page 8

122C-terminal truncations of the Na+ pump’s  subunit
Figure 4. Properties of transient currents and hyperpolarization-induced inward currents of the YY construct. (A and B) Ouabain-
sensitive presteady-state currents NaI(ouab) at [Na+]ext = 100 mM and [K+]ext = 0, pHext 7.4, upon voltage steps from 30 mV to potentials
between +60 and 140 mV (in 20-mV decrements) for WT (A) and the YY construct (B). (C and D) Voltage dependence of reciprocal
time constants from ON transient currents for ATP1A2 WT (C) and YY (D). Data for WT and YY are means ± SE from 14 cells out of
three batches. Results from a fit of a polynomial function to the WT or YY data are superimposed in C and D as a dashed or dotted line,
respectively. (E) Q-V curves obtained from QOFF charges of transient currents from 14 cells expressing ATP1A2 WT. The correspond-
ing QON charges were equivalent (not depicted). The fit of a Boltzmann function to Q-V distribution is superimposed as a dashed line
(fit parameters: Qmin = 6.65 ± 0.12 nC; Qmax = 2.34 ± 0.04 nC; V0.5 = 0.5 ± 1.0 mV; zq = 0.76 ± 0.02). (F) Q-V curves obtained from

Page 9

Meier et al.123
small difference current at pH 7.4, but a substantial
ouabain-sensitive inward current occurred upon acidi-
fication to pH 5.5 (Fig. 5 B). This inward current,
which can only be carried by protons in the absence of
Na+ext, reaches 30% of the amplitude measured upon
the same pH change in the presence of Na+. The I-V
curves of various pump-related currents of the YY
construct and ATP1A2 WT at different pHext are com-
pared in Fig. 5 (C and D). At pH 7.4, the exclusively
H+-carried NMDGI0K(ouab) currents were small, both for
the YY mutant (Fig. 5 C) and ATP1A2 WT (Fig. 5 D).
Extracellular acidification to pH 5.5 increased the
NMDGI0K(ouab) currents for ATP1A2 WT, but even more
so for the YY mutant. Acidification to pHext 5.5 at
100 mM [Na+]ext substantially increased the inward-rec-
tifying NaI0K(ouab) currents of the YY mutant (Fig. 5 C),
whereas the same pH change hardly affected the cor-
responding currents of ATP1A2 WT (Fig. 5 D). Com-
parison of the NaI0K(ouab) current amplitudes of the YY
construct at pH 5.5 in the absence and presence of
Na+ext shows that at least 25–30% of the inward cur-
rents is carried by H+. The large increase of the inward
current amplitudes observed upon acidification in the
presence of high [Na+]ext indicates that extracellular
protons stimulate the Na+-dependent inward leak cur-
rent. This agrees with the observation that the leak
currents of the YY construct decreased at alkaline pH
(Fig. S1 B).
Increased difference between QON and QOFF charges
at low pHext
To investigate the pH dependence of presteady-state
charge translocation by the YY construct, we measured
ouabain-sensitive transient currents at pH 7.4 and 5.5
with 0 and 100 mM [Na+]ext. Fig. 5 E depicts ON and
OFF transient currents of the YY construct upon volt-
age pulses from 30 to 140 mV and back. At [Na+]ext =
100 mM and pH 7.4, the ON current was composed of
Temperature dependence of pump-related currents
of the YY construct
To investigate whether the Na+-dependent inward leak
currents were due to a transporter-like rather than an
ion channel–like function of the mutant enzyme, we
measured the temperature dependence of the hyper-
polarization-induced inward currents and of the Na+/
K+ pump currents. The resulting Arrhenius plots for
temperatures between 18 and 30°C (Fig. 4 J) showed
that the inward leak currents and the Na+/K+ pump
currents share a very similar temperature dependence
with a high activation energy of 145 kJ/mol, similar
to values determined previously (Tavraz et al., 2008).
The high activation energy strongly suggests that the
inward currents of the YY construct are not con-
trolled by diffusion, as would be expected for a pas-
sively conducting ion channel.
Dependence of pump-related currents of the YY
construct on extracellular pH
To explore correlations between the hyperpolariza-
tion-activated inward currents of the YY construct
and the previously described H+ currents of the Na+/
K+-ATPase in the absence of extracellular Na+ and K+,
we tested whether the inward currents of the YY con-
struct were modulated by pHext. Fig. 5 shows current
recordings at 30 mV from an oocyte expressing the
YY construct, first at high [Na+]ext (100 mM; Fig. 5 A)
and then in the absence of Na+ext (Fig. 5 B). In the
presence or absence of extracellular Na+, the addition
of 10 mM K+ induced similar Na+/K+ pump current
amplitudes (Fig. 5, A and B). Whereas in the absence
of K+ hardly any current change could be observed
upon the addition of 10 mM ouabain at pH 7.4, a large
ouabain-sensitive inward current was induced when
the pH of the Na+-rich buffer was lowered to 5.5
(Fig. 5 A). Upon replacement of Na+ by NMDG+, the
addition of 10 mM ouabain again induced only a
QON and QOFF charges of 14 cells expressing the YY construct. The fit of a Boltzmann function to the QOFF values is superimposed as
a dashed line (fit parameters: Qmin = 1.14 ± 0.17 nC; Qmax = 4.72 ± 0.63 nC; V0.5 = 83.4 ± 7.9 mV; zq = 0.81 ± 0.06). The short dashed
line represents the inverted QOFF fit curve for comparison with QON values. QON and QOFF values of the YY construct were significantly
different at hyperpolarizing potentials (*, P > 0.05; **, P > 0.005; Student’s t test). The WT Q-V curve from E is included as a dotted line
in F after appropriate scaling. (G) Transient currents in response to ON voltage steps to +60 and 140 mV from WT data in A and YY
data in B on an expanded time scale. The voltage step occurred at 58 ms (dashed line). Fits of the sum of two exponential functions to
the data (from t0 = 59 ms) yielded time constants for the fast rise, 1, and the slower decay, 2. The peak of the transient current of YY
at 140 mV appeared with a delay of 2 ms (gray bars). (H) [Na+]ext dependence of normalized ouabain-sensitive inward currents of
the YY construct at [K+]ext = 0. The shallow dashed line shows the I-V curve of NaI(ouab) currents of ATP1A2 WT at [Na+]ext = 100 mM and
[K+]ext = 0. For each construct, data are means ± SE from 14 cells of four batches. (I) [Na+]int dependence of ouabain-sensitive NaI(ouab)
currents (at [Na+]ext = 100 mM and [K+]ext = 0), measured on an oocyte coexpressing the YY construct and the amiloride-sensitive Na+
channel ENaC. Na+ loading was achieved by exposing oocytes to [Na+]ext-containing solutions in the absence of amiloride, and [Na+]int
was determined after each loading step from the amiloride-sensitive reversal potential shift (see Materials and methods). Data from one
out of three experiments with a similar [Na+]int range are shown. (J) Temperature dependence (Arrhenius plots) of Na+/K+ pump cur-
rents (NaI10K) at 0 mV (filled squares) and hyperpolarization-induced inward currents (NaI(ouab)) at 140 mV (open squares) of the YY
construct. Data are from one out of four experiments across a similar temperature range (18–30°C). For both data sets, the amplitudes
were normalized to the respective current at 21°C. Activation energies were derived from linear fits to the data.
 

Page 10

124 C-terminal truncations of the Na+ pump’s  subunit
Influence of extracellular Li+ on pump-related currents
To investigate whether Li+ ions contribute to the inward
leak currents of the YY mutant, we measured ouabain-
sensitive currents at [Li+]ext = 100 mM. It is known that
electrogenic outward pumping of the Na+/K+-ATPase is
stimulated by extracellular as well as intracellular Li+
(Hermans et al., 1997). In accordance, we observed
positive ouabain-sensitive currents for ATP1A2 WT in
solutions containing [Li+]ext = 100 mM already in the
absence of K+ext (Fig. S2 A). In the absence of K+ext and
at positive potentials, the YY construct also mediated
positive pump currents (Fig. S2 B). At hyperpolarizing
potentials, however, negative LiI(ouab) current amplitudes
were observed, which were larger than the NMDGI(ouab)
currents (Fig. S2 B). Therefore, Li+ ions apparently con-
tribute to inward leak currents of the YY construct, al-
beit not as efficiently as Na+. Because currents at negative
potentials are most likely a superposition of Li+-depen-
dent hyperpolarization-activated inward transport and
Li+-dependent outward pumping, the absolute contri-
bution of Li+ ions to the inward currents cannot be
quantified. The addition of 10 mM K+ to the Li+-con-
taining solution resulted in a further increase of pump
a transient and a stationary inward current. The small
discrepancy between QON and QOFF charge is difficult to
recognize at this pH. At pH 5.5 and [Na+]ext = 100 mM,
the same voltage pulse induced a transient current with
a substantially increased stationary component, and the
charge integral within the peak of the ON transient cur-
rent was by far larger than that of the corresponding
OFF current. The OFF transient currents at pH 5.5 and
7.4 are nearly identical, indicating that at both pH val-
ues a fixed amount of QOFF charge is released. Again,
the ON current peak at acidic pH occurred with a delay
of 2 ms with respect to the peak at pH 7.4. The subse-
quent relaxation to the stationary amplitude occurred
with a time constant of 20 ms, which was about four-
fold slower than the relaxation at pH 7.4, showing that
the pHext profoundly influences transient current kinet-
ics. In contrast to the transient currents measured in
the presence of Na+ext, the ouabain-sensitive H+-carried
transient current signal (in the absence of Na+ext) at
pH 5.5 did not show a peak, but increased steadily ac-
cording to a single-exponential function (time constant
3 ms), and the subsequent OFF transient current
relaxed to 0 with a similar time course.
Figure 5. Voltage dependence of stationary and ouabain-sensitive transient currents of the YY construct under different ionic condi-
tions and pHext. (A and B) Current recordings at 30 mV from a YY-expressing oocyte in the presence (A) or absence (B) of extracellu-
lar Na+ at pH 7.4 and 5.5. If present, [Na+]ext and [NMDG+]ext were 100 mM, and [K+]ext and [ouabain] were 10 mM. The composition of
the extracellular solution is indicated above the current traces. Dashed lines indicate the “zero pump current” level (at 10 mM ouabain)
as a reference for pump-related currents. (C and D) I-V curves of stationary currents in different extracellular solutions from oocytes
expressing YY (C) or ATP1A2 WT (D), each normalized to the NaI10K(ouab) current at 0 mV. Similar data were obtained on at least three
different cells. (E) Ouabain-sensitive transient currents of YY at pH 7.4 (NMDGI(ouab) and NaI(ouab)) and pH 5.5 (NMDGI(ouab)5.5 and NaI(ouab)5.5)
in the absence of K+ext upon 250-ms voltage steps from 30 to 140 mV. The peak of the NaI(ouab) current is indicated by a dotted line.
Note the time axis break (hatched bar).

Page 11

Meier et al.125
more, the YY-AA mutant showed ouabain-sensitive hyper-
polarization-activated inward currents (albeit smaller
than in the case of YY), whereas the YY-FF mutant did
not (Fig. 6 E). These characteristic similarities contin-
ued regarding the properties of ouabain-sensitive tran-
sient currents (Fig. 7). The reciprocal time constants
for the YY-FF mutant were only slightly elevated com-
pared with WT values (Fig. 7 A), and the V0.5 of the
Q-V distribution was only moderately shifted by about
25 mV, without a change in zq (Fig. 7 C and Table I).
The reciprocal time constants for the YY-AA mutant
(Fig. 7 B) were similar to those of the YY construct
(Fig. 4 D), with a strong increase at positive potentials.
Compared with the WT enzyme, the Q-V curve of mu-
tant YY-AA showed a larger V0.5 shift (by 112 mV) and
a smaller slope (reduced zq; Fig. 7 D and Table I) than
the YY construct.
D I S C U S S I O N
Recent biochemical studies have supported the hypoth-
esis that the highly ordered carboxy terminus of the
Na+/K+-ATPase  subunit contributes to the formation
of Na+ binding site III (Toustrup-Jensen et al., 2009), as
proposed previously (Morth et al., 2007). Deletions or
currents (Fig. S2, A and B), but for ATP1A2 WT as well as
the YY construct, the pump currents induced by 10 mM
K+ in the presence of 100 mM Li+ were smaller than in
Na+-containing solutions, in line with previous results
(Hermans et al., 1997).
Replacement of the carboxy-terminal tyrosines by alanines
or phenylalanines
To explore which properties of the two carboxy-termi-
nal tyrosines are essential for function, we mutated the
tyrosines to phenylalanines (to preserve the aromatic
rings but remove the OH groups) and to alanines (to
eliminate the aromatic moieties), which resulted in
mutants YY-FF and YY-AA. The K+-dependent NaIxK cur-
rents (at [Na+]ext = 100 mM) of the YY-FF mutant (Fig. 6 A)
were similar to those of the WT enzyme (Fig. 2 D),
whereas currents of the YY-AA mutant (Fig. 6 B) re-
sembled those of the YY construct (Fig. 2 B). The
voltage-dependent NaK0.5(K+ext) values for the YY-FF
mutant were slightly elevated, and the minimum of the
curve was shifted by about 20 mV (Fig. 6 C) com-
pared with the corresponding WT data. In contrast,
the minimal NaK0.5(K+ext) value for the YY-AA mutant
was smaller (Fig. 6 D), and the NaK0.5(K+ext) curve was
shifted by about 80 mV compared with WT. Further-
Figure 6. Properties of stationary
currents of ATP1A2 mutants YY-
FF and YY-AA. (A and B) Normal-
ized I-V curves for NaIxK currents
of ATP1A2 mutants YY-FF (A) and
YY-AA (B), with [K+]ext as indicated
([Na+]ext = 100 mM, pH 7.4). (C and
D) NaK0.5(K+ext) values from NaIxK cur-
rents for mutant YY-FF (C) and YY-
AA (D) from fits of a Hill function
to the data in A and B at each mem-
brane potential. The correspond-
ing WT data from Fig. 2 E (dashed
lines in C and D) and YY data from
Fig. 2 C (dotted lines in C and D)
are superimposed. (E) I-V curves
of normalized ouabain-sensitive
NaI(ouab) currents ([Na+]ext = 100 mM
and [K+]ext = 0, pH 7.4) for ATP1A2
WT and mutants YY-FF, YY-AA, and
YY. Data are means ± SE of 10–14
cells from three batches.

Page 12

126C-terminal truncations of the Na+ pump’s  subunit
is equivalent to a twofold reduction in Na+ext affinity
(Holmgren et al., 2000; Holmgren and Rakowski, 2006).
The Q-V curve of the YY construct was shifted by
83 mV compared with the WT enzyme, which together
with a zq of 0.75–0.8 indicates a 12- to 14-fold reduced
affinity for extracellular Na+. This agrees well with the
ninefold reduction in apparent Na+ affinity of phospho-
enzyme formation for the YY deletion of the rat 1 iso-
form (Toustrup-Jensen et al., 2009). The negative V0.5
shift also indicates an increased preference of the YY
enzyme for the E2P state at 0 mV, in agreement with the
substantial E2P stabilization found in biochemical stud-
ies (Toustrup-Jensen et al., 2009). Due to the decreased
affinity for extracellular Na+, more strongly hyperpolar-
izing potentials are required to saturate the Na+ binding
sites of the YY construct from the extracellular side
and to drive the enzyme subsequently from E2P to E1P.
The half-maximal [Na+]int from the stimulation of Na+/
K+ pump currents was between 8 and 12 mM, which is
similar to the value of 10.8 ± 0.6 mM reported for the
Na+ dependence of phosphoenzyme formation for the
KETYY construct (Toustrup-Jensen et al., 2009). How-
ever, this half-maximal Na+int concentration is compara-
ble to the value of 12.8 ± 2.2 mM reported for the WT
mutations within the carboxy-terminal WVEKETYY mo-
tif of the rat 1 isoform largely decreased the Na+ affin-
ity from either side of the membrane (Toustrup-Jensen
et al., 2009). Reduction was ninefold for the YY dele-
tion, 26-fold for the KETYY deletion, and even 32-fold
for the YY-AA mutation. In light of these data, our re-
sults regarding the consequences of deletions or muta-
tions of the two carboxy-terminal tyrosines raise three
major questions. Do the observed functional alterations
reflect changes in cation affinity, and which type of cat-
ion binding site is affected by the YY deletion? Which
mechanisms could give rise to the Na+- and pH-depen-
dent inward leakage currents? Which side chain property
of the carboxy-terminal tyrosines is crucial for correct
function of the Na+/K+-ATPase?
Influence of the carboxy-terminal tyrosines on common
and unique cation binding sites
Shifts of the Q-V curves obtained from the slow compo-
nent of presteady-state currents directly indicate changes
in the apparent affinity for extracellular Na+. Based on
models for extracellular release or reverse binding of
Na+ ions through an access channel with a fractional
depth (or zq) of 0.7, a shift in the Q-V curve by 25 mV
Figure 7. Properties of transient currents for mutants YY-FF and YY-AA. (A and B) Voltage dependence of reciprocal time constants
from ON transient currents for mutant YY-FF (A) and YY-AA (B) ([Na+]ext = 100 mM and [K+]ext = 0, pH 7.4). Data are means ± SE from
13 (YY-FF) or 10 (YY-AA) oocytes from at least three batches. Curves delineating the corresponding WT data from Fig. 4 C (dashed lines
in A and B) and the YY data from Fig. 4 D (dotted line in B) are superimposed. (C and D) Q-V curves for QOFF charge integrals from
oocytes expressing ATP1A2 mutants YY-FF (C) and YY-AA (D). Dashed lines show fits of a Boltzmann function with the following param-
eters: YY-FF (C): Qmin = 2.73 ± 0.05 nC; Qmax = 2.26 ± 0.03 nC; V0.5 = 27.1 ± 0.8 mV; zq = 0.81 ± 0.02; YY-AA (D): Qmin = 1.15 ± 0.13
nC; Qmax = 5.59 ± 0.15 nC; V0.5 = 120 ± 18 mV; zq = 0.44 ± 0.03. The Q-V distribution of ATP1A2 WT from Fig. 4 E is superimposed as a
dotted line in C and D after appropriate scaling.

Page 13

Meier et al.127
struct are not exclusively Na+/K+ outward pump currents
(in contrast to the WT pump), and a substantial contri-
bution arises from the inhibition of the hyperpolariza-
tion-activated inward currents. It is likely that both
processes rely on the same event, namely the binding of
extracellular K+ to the pump in E2P (which at the same
time stimulates outward pumping and prevents the
pump from entering the inward leak current mode),
and therefore occur with the same apparent K+ affinity.
Whereas we propose that the YY deletion changes
Na+ affinity at the unique binding site without a major
effect on the common sites, Yaragatupalli et al. (2009)
arrived at the opposite conclusion based on their find-
ings on a KESYY deletion of the Xenopus Na+/K+-
ATPase 1 isoform. These authors reported a V0.5 shift
of the Q-V curve by about 100 mV and observed simi-
lar ouabain-sensitive inward leak currents. In contrast
to the YY-deleted Na+ pump, the voltage-dependent
NaK0.5(K+ext) values of the KESYY construct were not
only shifted to hyperpolarizing potentials, but also the
minimum of the curve was significantly lower. We tested
whether the decreased minimum of the NaK0.5(K+ext)
curve could be the result of the more extensive five–
amino acid deletion and found this indeed to be the
case (Fig. S4, A and B). Functional analysis of a homolo-
gous KETYY deletion of the human 2 isoform showed
that the NaK0.5(K+ext) values were significantly lower than
those of the YY construct (Fig. S4 B) and similar to the
values reported for the KESYY-truncated Xenopus 1
isoform. Therefore, the KETYY deletion apparently
causes a larger structural disruption with additional
long-range consequences also on the distant common
sites, whereas the YY deletion mainly affects the proxi-
mal unique Na+ binding site. The properties of the
KETYY construct were similar to the more severe phe-
notype of the YY-AA mutation, which further indicates
that the functional consequences of the KETYY dele-
tion are more substantial. The NaK0.5(K+ext) values of the
KETYY construct and mutant YY-AA were similar (com-
pare Fig. 6 D and Fig. S4 B), and the V0.5 shift of the Q-V
curve from transient currents (Fig. S4 F) was larger than
that of the YY construct with a similarly reduced slope
factor zq (Table I). Regarding the properties of ouabain-
sensitive transient currents, the KETYY and YY con-
structs were equivalent. Stationary inward leak currents
reached similar amplitudes (Fig. S4 D), and the recipro-
cal time constants showed the same reversed voltage de-
pendence (Fig. S4 E). Furthermore, the amount of QON
charges at negative potentials also exceeded the QOFF
values (Fig. S4 F).
The assignment of the consequences of the KESYY
deletion to the two common cation binding sites resided
on two experimental aspects: the 100-mV shift of the
Q-V curve and the overlapping I-V curves of ouabain-
sensitive, 10-mM K+-induced currents for the WT enzyme
and the KESYY construct (Yaragatupalli et al., 2009).
human 2/1 pump from pump current measurements
(Crambert et al., 2000), which indicates that pump cur-
rent measurements might not be adequate to reveal
changes in the affinity for intracellular Na+.
The shifted Q-V curve of the YY construct can be at-
tributed to a change in affinity at the unique Na+ bind-
ing site III (see below). Because the slow presteady-state
charge movement reflects the strongly electrogenic
extracellular deocclusion/release of the first Na+ ion
(Holmgren et al., 2000), the major electrogenic event is
most likely Na+ deocclusion/release from site III. Alter-
natively, if the deletion affected the Na+ affinity at the
two common sites, the shifted Q-V curve could result
from the fact that more hyperpolarizing potentials are
required to saturate these sites, a process that has to be
complete before the major electrogenic reuptake of the
third Na+ to site III can take place. However, we disfavor
the interpretation of an affinity change at the two com-
mon sites for the following reasons. The  subunit’s car-
boxy terminus is proximal to the unique Na+ binding
site. Therefore, it is unlikely that the YY deletion would
exclusively affect the more distant sites I and II. Further-
more, we observed that the YY deletion changed nei-
ther the intrinsic apparent affinity for extracellular K+
in the absence of extracellular Na+ (K0.5(K+ext) values)
nor the maximal apparent K+ affinity in the presence of
high [Na+]ext (minimum of the NaK0.5(K+ext) values).
Thus, even under conditions that allow for competition
of extracellular Na+ with K+ ions at the common binding
sites, the YY deletion does not change the maximal K+
affinity and probably does not change the cation affinity
at the common sites in general. Of note, the voltages at
which the NaK0.5(K+ext) curves are minimal, and the shift
between these minima, absolutely coincide with the V0.5
values from the corresponding Q-V curves obtained
from transient currents of the WT enzyme and the YY
construct. It is conceivable that the decreased affinity at
the unique Na+ binding site III, which leads to a shifted
voltage dependence of charge release/reuptake from/
to site III, is simultaneously responsible for the shift of
the NaK0.5(K+ext) curves. This modulatory role of site III
on the voltage dependence of the NaK0.5(K+ext) values
can be explained as follows. Potentials more positive
than V0.5 promote the release of Na+ from site III, which
in turn allows for Na+ release from the two common
sites. The increase of the NaK0.5(K+ext) values at positive
potentials can then be attributed to the subsequent
weakly electrogenic binding of K+ ions to the common
sites. At potentials more negative than V0.5, which pro-
mote Na+ reuptake to site III, increasing concentrations
of K+ would be required to overcome the inhibitory
effect of extracellular Na+ on Na+/K+ pumping, thus
giving rise to the increase of NaK0.5(K+ext) upon hyperpo-
larization. However, the NaK0.5(K+ext) values at negative
potentials should be considered with some caution be-
cause the full K+ext-induced currents of the YY con-

Page 14

128C-terminal truncations of the Na+ pump’s  subunit
tudes of the YY construct shows that the inward leak
currents at 140 mV are only 1.2-fold (pH 7.4) or
12-fold (pH 5.5) larger than the maximal Na+/K+ pump
currents at 0 mV. This, together with a Na+/K+ pump
turnover rate of 13 s1 for the human 2/1 Na+/K+-
ATPase (Tavraz et al., 2008) would yield ion perme-
ation rates of 15 to 160 s1 per pump molecule, which
is by several orders of magnitude smaller than in ion
channels. Third, the high activation energy of the in-
ward leak currents argues against a channel-like, diffu-
sion-controlled mechanism. Because the activation
energy is equivalent to that of Na+/K+ outward pump-
ing, a major conformational transition might be simi-
larly involved for the inward leak currents. It seems
possible that after extracellular Na+ binding to the
pump in E2P, the E2P↔E1P conformational change is
involved. This can be inferred from the equivalent
voltage dependences of the inward leak currents and
the Q-V distributions of the YY construct (Fig. S3)
under the assumption that for the YY-truncated
pump, the Q-V distribution also reflects the poise of
the dynamic equilibrium between E1P and E2P, as com-
monly accepted for the WT enzyme (Holmgren et al.,
2000). In this interpretation, the saturating inward
leak currents at hyperpolarizing potentials coincide
with maximal abundance of the Na+-occluded E1P
state. Under these conditions for the dynamic equilib-
rium, the enzyme would toggle between the Na+-loaded
E2P state and the Na+-occluded E1P state, and the in-
ward currents could then arise from cation release
(“leak”) from the Na+-occluded E1P state. As will be
outlined below, we suggest that the YY deletion desta-
bilizes the Na+-occluded E1P state of the enzyme.
Yet, one cannot exclude that cation leak already oc-
curs from E2P, although such a mechanism would lack
the direct coupling to a major conformational transi-
tion or to a change in accessibility of cation binding
sites. Furthermore, the inward leak currents could be
due to a Na+ext-dependent E2P-E2-E1 cycle, which might
be correlated with the increased Na+-dependent ATP
hydrolysis rate of carboxy-terminally truncated pumps
(Toustrup-Jensen et al., 2009). However, these rates
were found to still be 10-fold smaller than the maximal
hydrolysis rates under Na+/K+ turnover conditions
(Toustrup-Jensen et al., 2009). Because the YY-trun-
cated pump apparently works with a 3Na+/2K+ stoichi-
ometry under Na+/K+ turnover conditions, such an
E2P-E2-E1 cycle would imply that each extrusion of 3Na+
ions must be followed by inward translocation of much
more than three cations to account for the inward cur-
rent amplitudes. The ATP/cation inward transport ra-
tio might even be variable because pHext has a profound
influence on the inward current amplitudes. It remains
to be determined whether the inward cation leak mech-
anism requires ATP hydrolysis at all, and which fractions
of the current are carried by Na+ and H+.
Based on a kinetic scheme for Na+/K+-ATPase function,
these authors argued that a shift of identical magnitude
would occur in both the I-V and the Q-V curves if the
deletion only affected Na+ binding at the unique bind-
ing site. In contrast, an effect only at the common sites
would shift the Q-V curve, but not the I-V curve. How-
ever, the comparison between the NaI10K(ouab) curves of
the WT enzyme and those of the KESYY construct crit-
ically assumes that, even at the most hyperpolarizing
potentials, the NaI10K(ouab) currents of KESYY are exclu-
sively Na+/K+ outward pump currents. Any residual in-
ward current component would result in a downward
deflection of the NaI10K(ouab) curve and conceal a possible
horizontal shift. Our data show that the I-V curve of the
NaI10K(ouab) currents of the YY construct is indeed shifted
compared with WT data (Fig. 2 F), which supports our
conclusion that the YY deletion changes the Na+ affin-
ity at the unique site also by kinetic arguments (Yara-
gatupalli et al., 2009).
Mechanisms involved in the generation of inward
leak currents
The hyperpolarization-activated inward current of the
YY construct occurs in the absence of extracellular K+,
a condition that prevents Na+/K+ outward pumping.
In the absence of K+ext, the WT Na+/K+-ATPase hardly
generates any ouabain-sensitive current, except for the
pH-sensitive ATP-dependent and mainly H+-driven cur-
rent, which only occurs if extracellular Na+ is also absent
(Rakowski et al., 1991; Efthymiadis et al., 1993; Wang
and Horisberger, 1995; Rettinger, 1996; Li et al., 2006).
The inward leak currents of the YY construct de-
pended on intracellular [Na+] in a very similar way as
the normal outward pump currents, indicating that
Na+int-dependent phosphoenzyme formation is equally
involved. Furthermore, the inward leak currents re-
quired extracellular Na+ and showed saturating con-
centration dependence.
Yaragatupalli et al. (2009) also observed such hyper-
polarization-induced inward currents for the KESYY-
truncated pump and attributed these currents to a
“low affinity Na+ passive flow” through the Na+ pump
because saturation of inward current amplitudes could
not be observed at [Na+]ext up to 125 mM. However,
our data vote against a passive, ion channel–like trans-
port mechanism for the following reasons. First, the
inward current amplitudes of our YY construct satu-
rated at hyperpolarizing potentials and at rather mod-
erate [Na+]ext, which would not be expected for a
channel-like conductance. Because the KESYY-trun-
cated pump conceivably has an even lower affinity for
extracellular Na+ than the YY construct (reduction
26-fold vs. ninefold; Toustrup-Jensen et al., 2009),
much higher [Na+]ext than 125 mM would be required
to achieve saturation of the KESYY inward currents.
Second, a comparison of the relative current ampli-

Page 15

Meier et al.129
Consequently, the kinetic model (Scheme 2 in Fig. 8
and Appendix) included that at acidic pHext, the Na+-
occluded state of the pump is kinetically accessible from
both the extracellular side (passage along a deep ion
well of fractional depth ) as well as from the intracellu-
lar side (through a shallow access channel of fractional
depth ). As a result, the rate coefficients ktot of the tran-
sient currents (Eq. 2 in Appendix) increase at negative
potentials as well as at positive potentials. The destabili-
zation of the intracellular Na+ occlusion gate was con-
firmed by tracer flux measurements, which showed that
in addition to protons, Na+ ions also contributed to
the inward currents with a H+/Na+ permeation ratio of
3 × 104 (Vasilyev et al., 2004).
Correlation of the Na+-dependent leak currents to acid-
induced inward currents of the Na+/K+-ATPase
From the pHext dependence and the similar behavior of
the reciprocal time constants from transient currents,
we propose that the inward leak currents of the YY-
truncated pump and the acid-activated inward currents
Interference with the intracellular ion occlusion gate
of the Na+ pump
Active transporters avoid energy-dissipating leakage
currents by strictly coordinated operation of two access
gates for the transport sites from either side of the mem-
brane (Gadsby, 2009). Holmgren and Rakowski (2006)
analyzed the underlying mechanism of the Na+ pump
by measuring the effects of intracellular and extracellu-
lar Na+ on ouabain-sensitive presteady-state currents
(Scheme 1 in Fig. 8 and Appendix). Because intracellu-
lar Na+ binding was shown to be weakly electrogenic
(Wuddel and Apell, 1995; Pintschovius et al., 1999), it
was asked whether nonsaturating [Na+]int could change
kinetics, voltage dependence, or amplitudes of the pre-
steady-state currents. However, [Na+]int had no effect
on the V0.5 or zq of the Q-V distributions, but rather
determined the total amount of charge Qtot available
for presteady-state charge translocation. At depolarizing
potentials, neither charge transients that reflected electro-
genic intracellular binding of Na+ nor increasing rate
coefficients of transient currents (ktot) were observed. It
was concluded that the transient charge movement
arises only from extracellular Na+-sensitive current, and
that the voltage dependence of the pseudo first-order
rate coefficient for reverse binding of Na+ through a
deep extracellular ion well largely determined the ob-
served ktot (Eq. 1 in Appendix). To explain why intracel-
lular Na+ binding does not affect the extracellular
Na+-sensitive current, it was proposed that a slow occlu-
sion step follows intracellular Na+ binding. This slow oc-
clusion reaction kinetically isolates intracellular Na+
binding from the rapid extracellular release steps, which
is the kinetic equivalent to closure of an intracellular
occlusion gate (Holmgren and Rakowski, 2006).
At this point, the reversed voltage dependence of the
reciprocal time constants from presteady-state currents
of the YY mutant needs consideration (Fig. 4 D). Ac-
cording to Holmgren and Rakowski (2006), exactly
such a behavior would result if the intracellular Na+ oc-
clusion step were not rate limiting. Vasilyev et al. (2004)
reported such an effect on the reciprocal time constants
of presteady-state currents of the Na+/K+-ATPase as a
consequence of extracellular acidification. Whereas
lowering the extracellular pH did not modify V0.5 and zq
of the Q-V distributions from transient currents, the re-
ciprocal time constants at pHext ≤ 6.6 exhibited a suspi-
cious increase at positive potentials. In addition,
steady-state components were observed in the transient
current signals at acidic pH, which correspond to the
acid-induced inward currents described previously
(Rakowski et al., 1991; Efthymiadis et al., 1993; Rettinger,
1996). Vasilyev et al. (2004) suggested that protonation
at some regulatory site reduces the activation energy of
the 3Na++E1+ATP↔E1P(3Na+) occlusion reaction, which
would in effect lead to a proportional increase of the
rate coefficients for the forward and backward reaction.
Figure 8. Partial reaction schemes for the Na+ transport limb of
the Na+/K+-ATPase cycle. Scheme 1 was introduced by Holmgren
and Rakowski (2006) to describe the dependence of Na+/K+-
ATPase presteady-state currents on intracellular [Na+]. At saturat-
ing intracellular concentrations of Na+ and ATP, the intracellular
Na+ binding (and phosphorylation) is at rapid equilibrium, and
the subsequent slow occlusion step kinetically isolates the intracel-
lular Na+ binding step from the more rapid extracellular Na+ re-
lease steps. Scheme 2 refers to Vasilyev et al. (2004), who provided
a concept for the mainly H+-driven inward currents of the Na+/K+-
ATPase observed in Na+- and K+-free extracellular solutions. At
low pHext, the energy barrier for equilibration of occluded Na+
ions with the intracellular space is reduced so that the occluded
state can be reached from the extracellular as well as the intracel-
lular solution. See Appendix for details.