From the Institut de Pharmacologie et Toxicologie de l'Université, rue du Bugnon 27, CH 1005-Lausanne, Switzerland
Received for publication, September 26, 2000, and in revised form, January 18, 2001
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ABSTRACT |
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In oligomeric P2-ATPases such as
Na,K- and H,K-ATPases, P-type ATPases represent a family of ubiquitous
transporters which are characterized by the formation of a
phosphorylated intermediate during the catalytic cycle and which are
mainly involved in cation homeostasis. Over 200 members of this family
have been identified (1). Of these, only animal Na,K- and H,K-ATPase isozymes and bacterial K-ATPase isozymes, are oligomeric with 1 and 3 subunits, respectively, in addition to the catalytic subunit. Na,K- and
H,K-ATPase The unique presence of An important issue concerning the structure-function relationship of
Na,K- and H,K-ATPases is the identification of the matching interaction
sites in the Controlled proteolysis assays performed on Na,K-ATPase Interactions in the TM domains of the In this study, we have aimed to identify amino acid residues in the Site-directed Mutagenesis--
Amino acids Ile36 to
Gln56 of Xenopus Na,K-ATPase Expression in Xenopus Oocytes and Immunoprecipitation of Na,K Pump Current Measurements and Determination of Apparent
K+ and Na+ Affinities--
Na,K pump activity
was measured as the K+-induced outward current using the
two-electrode voltage clamp method as described earlier (10). Current
measurements were performed 3 days after injection of oocytes with
Bufo
The K+ activation of the Na,K pump current was determined
in a Na+-containing solution (80 mM sodium
gluconate, 0.82 mM MgCl2, 0.41 mM
CaCl2, 10 mM
N-methyl-D-glucamine (NMDG)-HEPES, 5 mM BaCl2, 10 mM tetraethylammonium
chloride, pH 7.4) or in a nominally Na+-free solution
(sodium gluconate was replaced by 140 mM sucrose). The
current induced by increasing concentrations of K+ (0.3, 1.0, 3.3, and 10 mM K+ in the presence of
Na+ and 0.02, 0.1, 0.5, and 5.0 mM
K+ in the absence of Na+) was measured either
at
Measurements of the half-activation constant for internal
Na+ was performed as previously described (12). Briefly, in
addition to Na,K-ATPase
Intracellular Na+ concentrations were calculated from the
reversal potential of the amiloride-sensitive current obtained from a
pair of I-V curves recorded with and without amiloride in a solution
containing 5 mM Na+ (5 mM sodium
gluconate, 0.5 mM MgCl2, 2.5 mM
BaCl2, 95 mM NMDG-Cl, 10 mM
NMDG-HEPES, pH 7.4). At each intracellular Na+
concentration ([Na]i) the Na,K pump K+-activated
current (IK) was measured in the presence of 20 µM amiloride and in the absence or presence of external Na+ (see above) by addition of 5 or 10 mM
K+. Maximal pump currents (Imax,Na)
and half-activation constants for Na+
(K1/2Na+int) were
determined by fitting the Hill equation [IK = Imax,Na/(1 + (K1/2Na+int/[Na]i)3)]
to the measured IK and [Na]i values.
Six to eight pairs of measurements of [Na]i and of the Na,K
pump K+-activated current were performed successively on
each oocyte at
Measurements of the apparent affinity for external Na+ were
performed using ouabain-sensitive Xenopus
The decrease in the Na,K pump current produced by exposure to external
Na+ was used to determine the half-inhibition constant for
external Na+
(K1/2Na+ext) by fitting
the Hill equation [I = Imax,Na (1-1/(1 + (K1/2Na+ext/[Na])nH)]
to the data of the current (I) observed at each
concentration of external Na+. Means of the Na,K
pump currents produced in oocytes expressing Na,K-ATPase Activity Measurements--
Na,K-ATPase activity was
measured in microsomal fractions prepared as previously described (11)
from oocytes expressing Xenopus Structural Features of the Na,K-ATPase Stabilization of Na,K-ATPase Substitution of Tyr40 and Tyr44 in
the
According to the model presented in Fig. 1B,
Tyr40 and Tyr44 are aligned with each other in
the TM
K1/2K+ values determined in the
presence of external Na+ are only a reflection of the
apparent affinity of K+ for its binding site, since this
parameter is influenced by competing external Na+. To
obtain a more direct estimation of the effect of Tyr40 and
Tyr44 mutations on the real binding affinities of external
K+ to Na,K pumps we also measured
K1/2K+ values in the absence of
external Na+. As expected, in the absence of external
Na+, the Imax value of wild type
pumps was similar but the K1/2K+
value was about 4 times lower than in the presence of external Na+ (Fig. 4, A and B, compare
lanes 1). Significantly, only the replacement of
Tyr40 (Fig. 4B, lane 2) but not of
Tyr44 (lane 3) by tryptophan in the TM domain of
Since the two tyrosine residues are highly conserved in the TM domains
of all
Previous studies have shown that gastric H,K-ATPase
To further characterize the potential functional interaction of the
To gain further insight into the functional effect of the Substitution of Tyr40 and Tyr44 in the
The apparent affinity for intracellular Na+ of wild type
and mutant Na,K pumps was measured as described under "Experimental Procedures." Fig. 5A shows
that Na,K pumps containing
An estimate for the apparent affinity for external Na+
(K1/2Na+ext) of wild
type and mutant Na,K pumps was obtained by measuring the inhibition of
Na,K pump currents as a function of increasing concentrations of
external Na+ (Fig. 5B). When measured at +30 mV,
inhibition of Na,K pump currents by external Na+ was
similar for Na,K pumps associated with wild type or mutant Vanadate Sensitivity of Na,K-ATPases Containing Wild Type By performing a tryptophan-scanning analysis we have identified
two highly conserved tyrosine residues in the TM domain of Na,K-ATPase
Various parameters were tested to assess the functional role of the
Na,K-ATPase All Tryptophan scanning of the TM domain of the Na,K-ATPase The change in the apparent K+ affinitiy of Na,K pumps
containing The tryptophan scanning analysis of the The mechanistic details by which Tyr40 and
Tyr44 in the Interestingly, the amino acids in the subunits play a fundamental role in the
structural and functional maturation of the catalytic
subunit. In
the present study we performed a tryptophan scanning analysis on the
transmembrane
-helix of the Na,K-ATPase
1 subunit to investigate
its role in the stabilization of the
subunit, the endoplasmic
reticulum exit of
-
complexes, and the acquisition of
functional properties of the Na,K-ATPase. Single or multiple tryptophan
substitutions in the
subunits transmembrane domain had no
significant effect on the structural maturation of
subunits
expressed in Xenopus oocytes nor on the level of expression
of functional Na,K pumps at the cell surface. Furthermore, tryptophan
substitutions in regions of the transmembrane
-helix containing two
GXXXG transmembrane helix interaction motifs or a cysteine
residue, which can be cross-linked to transmembrane helix M8 of the
subunit, had no effect on the apparent K+ affinity of
Na,K-ATPase. On the other hand, substitutions by tryptophan, serine,
alanine, or cysteine, but not by phenylalanine of two highly conserved
tyrosine residues, Tyr40 and Tyr44, on another
face of the transmembrane helix, perturb the transport kinetics of Na,K
pumps in an additive way. These results indicate that at least two
faces of the
subunits transmembrane helix contribute to inter- or
intrasubunit interactions and that two tyrosine residues aligned in the
subunits transmembrane
-helix are determinants of intrinsic
transport characteristics of Na,K-ATPase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits have the highest sequence identity among the
P-type ATPases. Based on the membrane topology, the catalytic
subunits of Na,K- and H,K-ATPases belong to the group of P2-ATPases (2)
which according to the crystal structure of the Ca2+-ATPase
contains 10 transmembrane
(TM)1 segments (3). The
subunits associated with Na,K- and H,K-ATPases are type II proteins
with one TM segment, a short cytoplasmic tail, and a large ectodomain
containing several sugar chains and 3 disulfide bridges. So far, three
Na,K-ATPase
isoforms and one H,K-ATPase
subunit have been identified.
subunits in Na,K- and H,K-ATPases remains
intriguing both from a functional and evolutionary point of view. At
present, we know that the
subunit has two main functions. Of
primary importance is its role as a specific chaperone which favors the
correct membrane insertion and hence the resistance against proteolysis
and cellular degradation of the newly synthesized
subunits of Na,K-
and H,K-ATPases (4-7). Since KdpC subunits of the bacterial KdpFABC
transporter may have a similar function (8), it has been speculated
that the
subunits of Na,K- and H,K-ATPases may be remnants of the
bacterial KdpC subunit that have been eliminated in other P2-ATPases
(1). Based on topology studies on Na,K- and H,K-ATPase
subunits, we
have also suggested that the K+ transport function common
to all oligomeric P-type ATPases, is associated with a particular amino
acid composition that is not compatible with efficient membrane
insertion of the
subunits. This has required that during evolution,
K+ transporting
subunits had to assemble with a helper
protein in order to assist their correct membrane integration (9). In
addition to their chaperone function,
subunits also influence the
cation sensitivity of oligomeric P-type ATPases expressed at the cell
surface. The association of the
subunit with different
isoforms
(10) or N-terminal truncated
subunits (11, 12) produces
Na,K-ATPases with different apparent affinities for Na+ and
K+.
and the
subunits that are responsible for the
chaperone function and/or the transport-modulating effect of the
subunit. Experimental evidence suggests that
and
subunits
interact in the extracytoplasmic, the TM, and the cytoplasmic domains.
In
subunits of both Na,K-ATPase (13) and H,K-ATPase (14), the most
clearly defined interaction site is located in the extracytoplasmic
loop between the TM segment M7 and M8. Interaction of the
subunit
with this region was shown to be important for the correct membrane
insertion and the structural maturation of the Na,K- and H,K-ATPase
subunit (5-7). According to results obtained using the yeast
two-hybrid system, the M7 and M8
-domain interacts with an
extracytoplasmic region of the
subunit contained within the 64 amino acids adjacent to the TM domain (13). However, mutational and
immunological studies suggest that other regions in the
extracytoplasmic domain of the
subunit such as the 10 most
C-terminal amino acids (15) or a YYPYYG sequence conserved in all known
subunits (16, 17) might as well participate in
-
interactions
and contribute to the stabilization of the
subunit. As suggested by
results obtained using chimeras between different
isoforms (12,
18), interactions in the ectodomains of the
and the
subunit
might also be responsible for the
subunits effects on the cation
sensitivity of Na,K-ATPase.
subunits,
indicate that
and
subunits also interact in the cytoplasmic domain (11). A mutational analysis indicates that these interactions are not necessary for the structural maturation of the
subunit (11,
12), nor do they directly influence apparent Na+ or
K+ affinities of Na,K-ATPase (12, 19). On the other hand,
it cannot be excluded that cytoplasmic
-
interactions contribute to some discrete steps in the catalytic cycle of Na,K-ATPase as suggested by Na+ occlusion and electrogenic binding assays
(20).
and
subunits are the
least well understood both in molecular and functional terms. So far,
cross-linking experiments have provided evidence that the TM domain of
the
subunit may be in contact with M8 of the
subunit (21, 22),
but nothing is known on the functional implications of this possible
intersubunit interaction.
TM domain that interact with the
subunit and to determine the
putative functional role of this interaction by using a tryptophan scanning analysis. Tryptophan scanning has previously been used to
determine structural features of integral membrane proteins (23-25).
Tryptophan was chosen because of its moderately hydrophobic properties
and its large size. The results of previous studies are consistent with
the expectations that if tryptophan is introduced at several positions
in a membrane segment, its large side chain is tolerated when facing
the lipid bilayer but, when positioned inside the protein, it may
disrupt function by breaking helix-helix interactions. Based on these
predictions, we aimed to get structural and functional information on
the TM domain of the Na,K-ATPase
subunit by replacing amino acids
individually or in combination by tryptophan.
Subunit mutants were
expressed in Xenopus oocytes together with
subunits and
the stability and the transport properties of the resulting Na,K-ATPase
-
complexes were analyzed. Our results indicate that interactions
in the TM domain of
and
subunits do not play a role in the
-stabilizing effect of the
subunit. On the other hand, mutations
of two tyrosine residues, highly conserved in the TM domain of all
known
subunits, significantly modulate the transport kinetics of
the Na,K-ATPase. The results of this study thus provide evidence that
the TM domain of the
subunit contributes to intrinsic functional
properties of oligomeric P-type ATPases as opposed to the
transport-modulating interactions in the ectodomain observed for
isoforms.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (
NK) were
individually replaced by tryptophan residues using the polymerase chain
reaction method described by Nelson and Long (26). Briefly, fragments
of
1NK contained in a pSD5 vector (pSD5
1NK) were first amplified
by polymerase chain reaction using sense oligonucleotides containing
mutated sequences coding for tryptophan and an antisense oligonucleotide consisting of nucleotides 628-648 tailed by primer D
of Nelson and Long (26). The amplified fragments were then used as
primers to elongate the inverse DNA strand which in turn was amplified
using a sense oligonucleotide encoding the SP6 sequence of pSD5
NK
and primer D of Nelson and Long (26). The mutated DNA fragments were
introduced into wild type pSD5
1NK using NheI and
BamHI restriction sites. The Y40W mutant was used as a
template for the preparation of Y40W/Y44W. G45W was used as a template for the preparation of G45W/G49W which in turn was used for the preparation of G45W/G49W/G53W. Y40F/Y44F, Y40S/Y44S, Y40C/Y44C, and
Y40A/Y44A were prepared using sense oligonucleotides containing both
mutated sequences. The chimera NK/HK in which the cytoplasmic and TM
domains (Met1-Asp71) are derived from
Xenopus Na,K-ATPase
1 and the ectodomain
(Gln76-Lys291) from rabbit, gastric H,K-ATPase
subunit was prepared as previously described (27). NK/HK Y40W/Y44W
was prepared by amplifying a fragment of
1NK as described above
using a sense oligonucleotide containing both mutated sequences, an
antisense oligonucleotide encoding nucleotides 420-440 tailed by
primer D of Nelson and Long (26) and using pSD5 NK/HK as a template.
NheI and PvuII restriction sites were used to
introduce the amplified fragment into the pSD5 vector containing NK/HK.
3 Y43W/Y47W was prepared by amplifying a fragment of
3NK
contained in a pSD5 vector (pSD5
3NK) using a sense oligonucleotide
containing both mutated sequences, an antisense oligonucleotide
encoding nucleotides 301-320 tailed by primer D of Nelson and Long
(26) and using pSD5
3NK as a template. The mutated DNA fragment was
introduced into wild type pSD5
3NK using NheI and
StuI restriction sites. The nucleotide sequences of all
constructs were confirmed by dideoxy sequencing. cRNAs coding for
Bufo Na,K-ATPase
1 (28), Xenopus Na,K-ATPase
1 (29), Xenopus
1 (29),
3 (30) subunits, and
subunit mutants were obtained by in vitro transcription
(31).
and
Subunits--
Oocytes were obtained from Xenopus
females as described (4). Routinely, 7 ng of Bufo marinus or
and 0.8 ng of Xenopus
subunit cRNAs were injected
into oocytes. Oocytes were incubated in modified Barth's medium
containing [35S]methionine (0.5 mCi/ml) for 6 h and
then subjected to 24 and 72 h chase periods in the presence of 10 mM cold methionine. Digitonin extracts were prepared as
described (11) and the
subunit was immunoprecipitated using a
Bufo
1 subunit antibody (32) under nondenaturing
conditions as described (11) allowing co-immunoprecipitation of the
associated
subunit. The dissociated immune complexes were separated
by SDS-polyacrylamide gel electrophoresis and labeled proteins were
detected by fluorography. Quantification of immunoprecipitated bands
was performed with a laser densitometer (LKB Ultrascan 2202).
1 and either wild type or mutant
cRNAs. One day
before performing measurements, oocytes were loaded with
Na+ in a K+-free solution containing 200 nM ouabain, a concentration that inhibits the endogenous
Na,K pumps but not the ouabain-resistant exogenous Bufo Na,K
pumps (33).
50 mV or during a series of nine 200-ms voltage steps ranging from
130 to +30 mV. To determine the kinetic parameters such as maximal
currents (ImaxK) and half-activation constants (K1/2K+) the Hill equation
was fitted to the data of the current (I) induced by various
K+ concentrations ([K]) using a least square
method: I = ImaxK/(1 + (K1/2K+/[K])nH).
According to previous studies (10), the Hill coefficient (nH) was set
to a value of 1.6 for experiments performed in the presence of external
Na+ and 1.0 for experiments performed in the absence of
external Na+.
and
cRNAs, oocytes were injected with
cRNAs coding for
,
, and
subunits (0.3 ng of each
subunit/oocyte) of the rat epithelial Na+ channel, rENaC
(34), and were incubated for 3 days in a modified Barth's solution
containing 10 mM Na+. One day before
measurements, oocytes were incubated in a Na+-free solution
(50 mM NMDG-Cl, 40 mM KCl, 1 mM
CaCl2, 1 mM MgCl2, 10 mM NMDG-HEPES, pH 7.4) in order to maximally reduce the
internal Na+ concentration.
50 mV. Between each pair of measurements, the oocytes
were allowed to increase their intracellular Na+
concentration by exposure to a 100 mM Na+
solution (100 mM sodium gluconate, 1 mM
MgCl2, 0.5 mM CaCl2, 10 mM Na-HEPES, pH 7.4) in the absence of amiloride and at a
holding potential of
50 to
100 mV.
1 subunits by
measuring the inhibition of the K+-induced current by
external Na+. 3 days after oocyte injection and 1 day
before measurements, oocytes were loaded with Na+ in a
K+-free solution. The Na,K pump current induced by 1 mM K+ was measured in a nominally
Na+-free solution (120 mM NMDG-gluconate, 0.82 mM MgCl2, 0.41 mM CaCl2, 10 mM NMDG-HEPES, 5 mM BaCl2, 10 mM tetraethylammonium chloride, pH 7.4) and in the presence of 10, 30, 60, and 120 mM Na+ (NMDG-gluconate was replaced by sodium
gluconate) during a series of nine 200-ms voltage steps ranging from
130 to +30 mV. The Na,K pump was then blocked by the addition of 100 µM ouabain and the same series of measurements was
repeated. Currents specific for the Na,K pump could be deduced by
subtracting the currents observed in the presence of ouabain from those
observed in its absence. A nonsaturating concentration of
K+ (1 mM) was chosen in order to reveal the
competition of external Na+ with K+ ions for
extracellular cation-binding sites. At each external Na+
concentration, the averaged endogenous, ouabain-sensitive Na,K pump
current was subtracted from total ouabain-sensitive currents measured
in oocytes expressing exogenous Na,K pumps.
subunits and wild type
or mutant
subunits were compared by unpaired Student's
t test.
subunits and wild type
1 subunits or
1 Y40W/Y44W mutants. Before activity measurements,
samples were freeze/thawed twice in liquid nitrogen. Na,K-ATPase
activity was measured in triplicate by an enzyme-linked assay,
according to Schoner et al. (35), in which the resynthesis
of ATP consumed by the ATPase is coupled by the pyruvate and lactate
dehydrogenase reactions to NADH oxidation. The oxidation rate of NADH
was recorded at 340 nm wavelength in the automated enzyme kinetic
accessory of a DU-64 spectrophotometer (Beckman Instruments). The
substrate concentrations of the reaction mixture were 5 mM
KCl, 100 mM NaCl, 4 mM ATP, and 4 mM MgCl2. To reduce nonspecific, mitochondrial
ATPase activities, 5 mM NaN3 were added to the
reaction mixture. Activity measurements were done in the presence or
absence of 10
7-10
4 M
orthovanadate. The specific enzyme activity was calculated as the
difference between samples incubated in the presence or absence of 1 mM ouabain. In the absence of vanadate, ouabain-sensitive activities represented 20-30% of the total enzyme activity.
Statistical analysis was done by unpaired Student's t test.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Subunits TM
Domain--
Fig. 1A shows the
membrane-spanning domain of the
1 subunit of Xenopus
Na,K-ATPase, as previously defined (19). The TM domain of the
Xenopus
1 subunit comprises 23 amino acids and shows a
sequence identity of 43% with Xenopus
2, 65% with
3, and 43% with the
subunit of gastric H,K-ATPase. Secondary
structure prediction on the
1 TM domain revealed an
-helical
structure with at least two distinct faces (Fig. 1B). One
face is characterized by three aligned residues with large lateral side
chains (Tyr40, Tyr44, and Phe51)
(numbering of amino acids correspond to Xenopus
1
subunits) whereas another face contains three aligned glycine residues
(Gly45, Gly49, and Gly53) forming a
cleft in the
-helix. The two tyrosine residues are conserved in all
known
subunits whereas the three glycine residues are only found in
Na,K-ATPase
1 isoforms and certain
3 isoforms. Other Na,K-ATPase
isoforms and H,K-ATPase
subunits contain 1 or 2 glycine
residues corresponding to either Gly45 and/or
Gly49. Three phenylalanine residues (Phe39,
Phe43, and Phe51) are present in the TM domain
of all Na,K-ATPase
isoforms whereas the H,K-ATPase
subunit
contains a conservative substitution of tyrosine for Phe39.
Na,K-ATPase
2 and
3 isoforms contain a fourth phenylalanine residue which is replaced by a cysteine residue (Cys46) in
1 isoforms (Fig. 1, A and B). In order to
identify the amino acid residues in the
TM domain that interact
with the
subunit and to determine the putative functional role of
this interaction, amino acids encompassing Ile36 to
Gln56 were substituted individually or in combination by
tryptophan.
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Fig. 1.
Modeling of the TM domain of the
Xenopus Na,K-ATPase 1
subunit. A, linear model of the
1 subunit of
Na,K-ATPase with indications of the cytoplasmic (Cyt), TM,
and extracellular (Ext) domains. Sugar residues are indicated as
"Y." The C-terminal end of the TM domain of the
1 subunit has
been determined previously (19). B, helical wheel
representation and space-filling model, obtained using the Rasmol
program of the TM domain of the
1 subunit. Tyr40 and
Tyr44 are indicated in gray, Gly45,
Gly49, and Gly53 are spotted, and
Cys46 is checked. The space-filling model starts
with Lys35 delimiting the N-terminal end of the TM domain
which is represented at the bottom of the model.
Subunits by
Subunit Tryptophan
Mutants--
We first tested whether tryptophan substitutions in the
TM domain of the
subunit might interfere with the structural
maturation of the
subunit. Bufo
subunits were
expressed in Xenopus oocytes alone or together with
Xenopus wild type or mutant
1 subunits and the cellular
expression of the
-
complexes was followed by immunoprecipitation
of metabolically labeled proteins after a pulse and various chase
periods. As expected,
subunits expressed without
subunits were
degraded during a 48-h chase period (Fig. 2, lanes 10 and
11). Similar to wild type
1 subunits (Fig. 2, lanes
1-3), all
-mutants assembled with and stabilized
-subunits as illustrated for the triple glycine mutant G45W/G49W/G53W
(lanes 4-6) and the double tyrosine mutant Y40W/Y44W
(lanes 7-9). In addition, all
subunit mutants assembled
with
subunits became fully glycosylated during the chase periods
(lanes 5, 6, 8, and 9) indicating that the
-
complexes were able to leave the ER. Thus, these results show
that introduction of tryptophan residues into the
1 TM domain does
not significantly affect the structural maturation of the
subunit.
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Fig. 2.
Assembly, stabilization, and intracellular
transport of Na,K-ATPase -
complexes containing tryptophan mutations in the
subunit's TM domain. Xenopus
oocytes were injected with 7 ng of Bufo
NK cRNA alone
(lanes 10 and 11) or together with 0.8 ng of
either Xenopus wild type (
1wt) (lanes 1-3) or
mutant
1 (lanes 4-9) cRNAs. After a 6-h pulse period
with [35S]methionine (lanes 1, 4, 7, and
10) and after 24 h (lanes 2, 5, 8, and
11) and 72 h (lanes 3, 6, and 9)
chase periods, digitonin extracts were prepared and
immunoprecipitations were performed using an anti-
subunit antibody
under nondenaturing conditions which allowed co-immunoprecipitation of
associated
subunits.
and
subunits were revealed by
SDS-polyacrylamide gel electrophoresis and fluorography. One out of two
to four similar experiments is shown. cg, core glycosylated
subunit; fg, fully glycosylated
subunit.
Subunits TM Domain by Tryptophan Modifies the K+
Activation Kinetics of Na,K Pumps--
To determine the functional
expression and the transport properties of Na,K pumps associated with
wild type or mutant
subunits, K+-induced outward
currents were measured on intact oocytes by the two electrode voltage
clamp technique. Maximal pump currents (Imax) varied but were not consistently different in oocytes expressing wild
type or mutant
-
complexes (Fig.
3A), indicating that none of
the
tryptophan mutants affected the overall transport activity. However, measurements of the apparent affinities for external K+ revealed a significant increase in the half-activation
constants for K+
(K1/2K+) for pumps associated with
1 mutants I36W, Y40W, and Y44W (Fig. 3B), compared with
wild type pumps. K1/2K+ values of
pumps associated with other
tryptophan mutants, including the
double G45W/G49W or the triple G45W/G49W/G53W mutant, were similar to
those of wild type pumps.
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Fig. 3.
Functional expression and K+
activation of wild type and mutant
-
complexes. Oocytes
were injected with 7 ng of Bufo
NK and 0.8 ng of either
Xenopus wild type (
1wt) or mutant
cRNAs.
A, Imax values of Na,K pumps
containing wild type
1 subunits or
1 tryptophan mutants. Three
days after injection, maximal K+-induced Na,K pump currents
(Imax) were measured at
50 mV in the presence
of external Na+ as described under "Experimental
Procedures." Measurements were performed in the presence of 200 nM ouabain which blocks endogenous, oocyte Na,K pumps but
not the exogenous, moderately ouabain-resistant Bufo Na,K
pumps. The Imax values for Na,K pumps containing
wild type
1 subunits (691.6 ± 62 nA) or
1 tryptophan
mutants were extrapolated from K+ activation curves
permitting estimations of K1/2K+
values shown in B. Shown are mean ± S.E. of data
obtained from 14 to 28 oocytes from two to four different
Xenopus females. B, apparent affinities for
external K+ of Na,K pumps containing wild type
1
subunits or
1 tryptophan mutants. Half-activation constants for
external K+
(K1/2K+) were determined at
50 mV
in the presence of external Na+ as described under
"Experimental Procedures." The
K1/2K+ value for Na,K pumps
containing wild type
1 subunits (651 ± 23 µM)
was arbitrarily set to 1. Shown are mean ± S.E. of data obtained
from 14 to 28 oocytes from two to four different Xenopus
females. *, p < 0.01 compared with wild type
-
complexes.
-helix suggesting that they may cooperate in the same
functional effect. To test this possibility, we measured the apparent
K+ affinity of Na,K pumps containing
1 subunits in which
both tyrosine residues were replaced by tryptophan.
-
Complexes
containing the
1 double mutant Y40W/Y44W produced maximal pump
currents similar to that of wild type
-
complexes (see insert
Fig. 4A) and exhibited a
significantly higher K1/2K+ value
than that of
-
complexes containing either the Y40W or the
Y44W
-mutant (Fig. 4A) indicating that Tyr40
and Tyr44 act in concert in the K+ effect.
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Fig. 4.
Apparent affinities for external
K+ of Na,K pumps associated with
1 or
3 mutants or
with
NK/HK chimera. A and
B, K+ activation of wild type and mutant
-
complexes. Oocyte cRNA injection was as described in the legend to Fig.
3. Half-activation constants for external K+
(K1/2K+) were determined at
50 mV
in the presence (A) or absence (B) of external
Na+ as described under "Experimental Procedures." Shown
are mean ± S.E. of data obtained from 10 to 20 oocytes from two
to three different Xenopus females. *, p < 0.01. Insets, maximal Na,K pump currents
(Imax) extrapolated from K+
activation curves. C, influence on the K+
activation of Na,K pumps containing
subunits in which
Tyr40 and Tyr44 were replaced by various amino
acids. Oocyte cRNA injection as described in the legend to Fig. 3.
K1/2K+ values were determined at
50 mV in the presence of external Na+. Shown are
mean ± S.E. of data obtained from 10 to 15 oocytes from two to
three different Xenopus females. *, p < 0.01 compared with wild type
-
complexes (lane 1);
NS, statistically not significantly different from wild type
-
complexes. Inset, maximal Na,K pump currents
(Imax) extrapolated from the K+
activation curves. D, voltage dependence of
K1/2K+ values.
K+-induced currents were measured during a series of nine
200-ms voltage steps ranging from
130 to +30 mV in oocytes injected
with
and wild type
1 (squares) or
1 Y40W/Y44W
mutant (triangles) cRNAs.
K1/2K+ values were determined after
each voltage step in the presence of external Na+ as
described under "Experimental Procedures." Shown are mean ± S.E. of data obtained from 14 oocytes from two different
Xenopus females. *, p < 0.01 compared with
wild type Na,K pumps. Inset, maximal Na,K pump currents
(Imax).
1 subunits significantly increased the
K1/2K+ value of
-
complexes in
the absence of external Na+ and the K+ effect
of the Tyr40 tryptophan substitution was less pronounced
than in the presence of external Na+ (compare Fig. 4,
A and B, lanes 1 and 2). These results
indicate that tyrosine mutations produce highly specific effects on
discrete steps in the transport cycle.
subunits identified so far, we also investigated K1/2K+ values of Na,K pumps
associated with tyrosine mutants of other
isoforms, namely of the
Na,K-ATPases
3 isoform. The corresponding tyrosine residues of the
Xenopus
3 isoform, Tyr43 and
Tyr47, were replaced by tryptophan residues (
3
Y43W/Y47W) and K1/2K+ values of
mutant
-
3 complexes were compared with those of wild type
-
3 complexes, both in the presence and absence of external Na+. As previously reported (10),
K1/2K+ values measured in the
presence, but not in the absence of external Na+, were
about 2-fold higher for wild type
1-
3 complexes than for wild
type
1-
1 complexes (Fig. 4, A and B,
compare lanes 1 and 5). Similar to the results
obtained with the
1 Y40W/Y44W mutant, the
3 Y43W/Y47W mutant
produced an increase in K1/2K+
values of Na,K pumps, which was greater in the presence than in the
absence of external Na+ (Fig. 4, A and B,
lanes 5 and 6).
subunits can
associate with Na,K-ATPase
subunits and produce
-
HK complexes
which exhibit higher K1/2K+ values
than
-
1NK complexes (10). As suggested by analysis of chimeras
between
NK and
HK, the reduced apparent K+ affinity
of
-
HK complexes compared with
-
NK complexes is mainly
mediated by the ectodomain of
HK (12). To test whether the
K+ effect of the TM domain and the ectodomain of
subunits is additive, the two tyrosine residues in the TM domain of a
NK/HK chimera (containing the cytoplasmic and the TM domain of
1NK and the ectodomain of
HK) were substituted by tryptophan. The
NK/HK chimera, rather than the wild type
HK, was chosen for
these studies since
HK only produces a small population of pumps
which are functionally active (12). As previously reported (12), Na,K pumps containing
NK/HK chimeras produced high
Imax values and the
K1/2K+ value was similar to
-
HK complexes and about 2.5-fold higher than that of Na,K pumps
containing wild type
1 subunits both in the presence and absence of
external Na+ (Fig. 4, A and B,
compare lanes 1 and 7). Compared with
-
NK·HK complexes, Na,K pumps which were associated with the
NK/HK
chimera containing the Y40W/Y44W mutations exhibited
K1/2K+ values which were slightly
but significantly increased in the absence of external Na+
(Fig. 4B, compare lanes 7 and 8).
These values were more than two times higher in the presence of
external Na+ (Fig. 4A, compare lanes
7 and 8). These results suggest that the effect of the
subunits TM domain and ectodomain on the apparent K+
affinity are complementary. Since the relative increase in
K1/2K+ values of Na,K pumps
containing double tryptophan mutants of
1 isoforms,
3 isoforms,
and NK/HK chimeras was similar, our results also indicate that the
K+ effect of the TM domain is of general functional relevance.
subunit with the
subunit in the TM domain, Tyr40 and
Tyr44 were replaced by several amino acids other than
tryptophan.
-
Complexes containing double phenylalanine, serine,
cysteine, or alanine
-mutants produced similar Na,K pump currents as
wild type Na,K pumps (see inset in Fig. 4C).
K1/2K+ values of Na,K pumps
associated with double serine, cysteine, or alanine
1 mutants were
significantly higher than that of wild type Na,K pumps (Fig.
4C, compare lane 1 to lanes 4-6), but
were lower than that of Na,K pumps associated with the double
tryptophan
1 mutant (compare lane 2 to lanes
4-6). On the other hand, a double phenylalanine
1 mutant
produced Na,K pumps with a K1/2K+
value which was similar to that of wild type pumps (compare lanes 1 and 3). These results indicate that correctly
positioned aromatic side chains, present in tyrosine and phenylalanine
but not in serine, cysteine, and alanine residues, are essential for
correct interaction of the
subunits TM domain with the
subunit
and the inherent Na,K pump function. The most pronounced, functional perturbations produced by the double tryptophan
mutant could be due
to the large side chain of tryptophan which as predicted (23) may favor
disruption of helix-helix interactions by inducing local structural changes.
subunits
TM domain on the transport properties of the Na,K-ATPase
subunit,
we next compared the voltage dependence of Na,K pump currents in the
presence of external Na+ and in particular of
K+ binding to Na,K pumps associated with wild type
1
subunits or
1 Y40W/Y44W mutants. As previously observed (10),
maximal Na,K pump currents of wild type Na,K pumps composed of
1 and
1 subunits are slightly voltage-dependent (Fig.
4D, inset). Na,K pumps associated with
1 Y40W/Y44W
mutants exhibited a stronger voltage dependence and produced lower
Imax values than wild type Na,K pumps, in
particular at highly negative membrane potentials (Fig. 4D,
inset). K1/2K+ values of mutant
Na,K pumps were significantly higher than those of wild type Na,K pumps
over the whole potential range and the difference was particularly
pronounced at very negative membrane potentials (Fig.
4D).
Subunits TM Domain by Tryptophan Modifies Na+ Translocation
Kinetics of Na,K Pumps--
Since differences in
K1/2K+ values between Na,K pumps
associated with wild type
1 subunits or
1 Y40W/Y44W mutants were
more pronounced when measured in the presence rather than in the
absence of external Na+, it is likely that mutations in the
subunits TM domain not only directly influence the binding site for
external K+ but also modify Na+ translocation
kinetics. To verify this hypothesis we investigated the effect of
Tyr40/Tyr44
mutations on the apparent
affinities of Na,K pumps for intra- and extracellular
Na+.
1 Y40W/Y44W mutants had a slightly higher
apparent affinity for intracellular Na+ than wild type Na,K
pumps.
View larger version (21K):
[in a new window]
Fig. 5.
Apparent affinities for intracellular and
extracellular Na+ of Na,K-ATPase
-
complexes containing
wild type
1 subunits or
1 Y40W/Y44W mutants. A, activation
of wild type and mutant Na,K pumps by intracellular Na+.
Oocytes were injected with Na,K-ATPase
and
cRNAs (7 and 0.8 ng,
respectively) together with cRNAs coding for
,
, and
subunits
of the rat epithelial sodium channel (0.3 ng of each). Half-activation
constants for intracellular Na+
(K1/2Na+int) were
determined as described under "Experimental Procedures." Shown are
mean ± S.E. of data obtained from 11 oocytes from 3 different
Xenopus females. *, p < 0.01 compared with
wild type
-
complexes (lane 1). Inset,
maximal Na,K pump currents (Imax). B,
inhibition of wild type and mutant Na,K pumps by extracellular
Na+. Oocytes were injected with Xenopus
cRNA
and wild type
1 (squares) or
1 Y40W/Y44W mutant
(triangles) cRNAs. Na,K pump currents (I) were
determined at
130,
50, and +30 mV in the presence of various
concentrations of extracellular Na+, as described under
"Experimental Procedures." Shown are mean ± S.E. of data
obtained from 9 and 11 oocytes from two different Xenopus
females for wild type and mutant pumps, respectively. *,
p < 0.01 compared with wild type Na,K pumps.
I values measured in the absence of external Na+
were arbitrarily set to 1. Half-inhibition constants for extracellular
Na+
(K1/2Na+ext) estimated
from the Na,K pump inhibition curves at
130,
50, and +30 mV, were
37.9 ± 7.3, 78 ± 6.2, and 55 ± 10 mM,
respectively, for wild type Na,K pumps, and 9.8 ± 5.7, 35.3 ± 5.7, and 43.6 ± 6.8 mM, respectively, for mutant
Na,K pumps.
subunits. However, when Na,K pump currents were measured at a membrane
potential of
130 or
50 mV, external Na+ had a
significantly stronger inhibitory effect on Na,K pumps containing than
on those containing the wild type
1 subunit (Fig. 5B).
These results indicate that mutant
-
complexes have a higher, voltage-dependent, apparent affinity for external
Na+ than wild type
-
complexes.
Subunits or
1Y40W/Y44W Mutants--
The parallel decrease and
increase in the apparent affinity for external K+ and
internal Na+, respectively, of Na,K pumps containing
1
Y40W/Y44W mutants compared with those containing wild type
1
subunits, indicates that tyrosine mutations in the
1 subunit may
shift the E1-E2 conformational equilibrium to the E1 state. To test
this hypothesis, we investigated the sensitivity to vanadate of the
Na,K-ATPase activity measured in microsomes of oocytes expressing wild
type
1 subunits or
1 mutants. Vanadate competes with
Pi for binding to the E2 conformation (36) and can be used
as a conformational probe (37, 38). The results shown in Fig.
6 indicate that the
1 Y40W/Y44W mutant
significantly decreases the sensitivity of Na,K pumps to vanadate, with
estimated Ki values of 7 and 100 µM
for Na,K-ATPases containing wild type
1 subunits and
1 mutants,
respectively. This result is consistent with a change in the
steady-state E1/E2 distribution toward E1 of the Na,K pump containing
the
1 Y40W/Y44W mutant.
View larger version (17K):
[in a new window]
Fig. 6.
Vanadate sensitivity of Na,K-ATPase
-
complexes containing
wild type
1 subunits or
1 Y40W/Y44W mutants. Oocytes were injected
with Xenopus
cRNA and wild type
1 cRNA (black
bars) or
1 Y40W/Y44W mutant cRNA (white bars). Four
days after injection, microsomes were prepared and the Na,K-ATPase
activity was measured as described under "Experimental Procedures"
in the absence (control) or presence of
10
7-10
4 M vanadate. Data are
represented as percent of control Na,K-ATPase, which amounted to
32 ± 4 and 20 ± 2 µmol/mg of protein/h
1 for
microsomes from oocytes expressing wild type
subunits and
mutants, respectively. Na,K-ATPase activities were 10-30 times higher
in microsomes from cRNA-injected oocytes than in microsomes from
non-injected oocytes. The results shown are mean ± S.E. of two
independent measurements performed in triplicate on microsomes from 2 different batches of oocytes. A statistically significant difference
(p < 0.01) between the vanadate sensitivity of
Na,K-ATPase in microsomes from oocytes expressing wild type
subunits and
mutants is indicated by an asterisk.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits which are implicated in Na,K pump function. This result
provides compelling evidence for interactions of
and
subunits
in the TM domain and highlights the role of the
subunit as a
determinant of intrinsic Na,K-ATPase transport properties.
subunits TM domain including its role in stabilizing
the Na,K-ATPase
subunit, in favoring a folding state that permits
ER exit of
-
complexes, and in conveying particular transport
properties to Na,K-ATPase.
mutants containing individual or combined tryptophan
substitutions in the TM domain were able to stabilize
subunits and
to permit ER exit of
-
complexes, indicating that no single amino
acid, nor Tyr40/Tyr44 and
Gly45/Gly49/Gly53 when mutated in
combination are necessary for the structural maturation of Na,K-ATPase.
These results apparently contrast with previous results obtained with
chimeras between Na,K- and H,K-ATPase
subunits, which showed that
the presence of the TM domain of the Na,K-ATPase
subunit is needed
for a correct interaction with the Na,K-ATPase
subunit permitting
ER exit of
-
complexes (12). Although it cannot entirely be
excluded that tryptophan substitutions may allow for proper
assembly, it is more likely that the results obtained in this and
previous studies indicate that a certain overall integrity rather than
individual amino acids may be necessary for
-
interactions that
mediate some specific steps in the maturation process. This conclusion
is also supported by results obtained by Renaud et al. (39)
who showed that deletions of 3 amino acids in the TM domain permits
assembly of
-
complexes and their cell surface expression whereas
deletions of 5-11 amino acids in the TM domain of
subunits only
allows for assembly but not for ER exit of the
-
complexes.
1 subunit
revealed that substitution of 3 amino acids, Ile37,
Tyr40, and Tyr44, perturbs the inherent,
apparent affinities for K+ of associated Na,K-ATPase
subunits. The functional effects observed with Tyr40 and
Tyr44 substitutions are likely to be a reflection of the
disruption of a functionally active site. This is supported by the
observations that the two tyrosine residues are aligned in the TM
-helix, they produce additive effects, and the functional effects
can be reproduced with different amino acids. Moreover, the
K+ effect produced by Tyr replacements in the
subunits
TM domain varies depending on experimental conditions, e.g.
in the presence or absence of external Na+. The cause
underlying the K+ effect produced by substitution of
Ile37 with tryptophan is less clear. In membrane proteins,
tryptophan residues are enriched at both ends of TM domains (40) and
preferentially interact near the lipid carbonyl moiety (41, 42).
Introduction of tryptophan, in positions flanking model TM helices,
tend to "push" the TM helix into the membrane (43). In the context
of our result, these observations may indicate that tryptophan
substitution of Ile37, located adjacent to
Lys36 which delimits the TM domain of
1 subunits, could
produce a local conformational perturbation in the
helix that may
be transmitted to the aligned Tyr residues and thereby produce an
indirect functional effect. Consistent with this idea is that the
K+ effect of Ile37 substitutions is observed
both in the presence and absence of external Na+ (data not shown).
subunits with Tyr40 and Tyr44
substitutions are more prominent when measurements are performed in the
presence rather than in the absence of external Na+
indicating that substitution of the tyrosine residues in the
subunits TM domain may not only alter the K+-binding step
itself, but also other discrete steps in the ion transport cycle. In
both wild type and mutant Na,K pumps, a parallel exists between the
decrease in the apparent affinity for external K+ and a
gain in the apparent affinity for external Na+ during
hyperpolarization. This result reflects the interplay between
Na+ binding and release and K+ binding to
external sites and the voltage dependence of these steps. Furthermore,
the difference in the apparent affinities for external Na+
between Na,K pumps associated with
subunit tyrosine mutants and
those associated with wild type
subunits are largest at very
negative membrane potentials. This result is consistent with the idea
that Tyr mutations in the
subunits TM domain may favor the
transition from an E2 to a Na+-carrying E1 conformation
which prevails during hyperpolarization. A shift of the E1-E2
equilibrium to the E1 conformation may also explain the higher apparent
affinity for intracellular Na+ and the lower sensitivity
for vanadate observed for the mutant Na,K pumps. The two tyrosine
residues are highly conserved in the TM domain of all known
subunits of both Na,K- and H,K-ATPases. Experimental confirmation is
needed to show whether, as expected, these residues influence a basic
step in the catalytic cycle common to Na,K and H,K pumps.
1 subunits TM domain was
complemented by substitution of the two functionally relevant tyrosine
residues by other amino acids. An increase in
K1/2K+ values was also observed in
mutant Na,K pumps containing
subunits in which Tyr40
and Tyr44 in the TM domain were replaced by serine,
cysteine, or alanine residues. On the other hand, no K+
effect was observed by replacing the tyrosine residues with
phenylalanine. These results indicate that a correctly positioned
aromatic moiety present in the native tyrosine and substituting
phenylalanine residues, but absent in serine, cysteine, and alanine
residues, may be important in producing the wild type phenotype. Since
tryptophan substitutions produce the largest functional perturbations,
it is also possible that the size of the side chain may be critical for
a correct K+ effect.
subunits TM domain affect intrinsic
transport properties of Na,K-ATPase are not known. According to our
results, it is likely that the two tyrosine residues in the
subunits TM domain interact with a TM segment of the
subunit and
produce a conformational effect. The primary interactions that specify
TM helix packing which mediates subunit interactions and the correct
folding of membrane proteins are still largely unknown (44). Based on
energetic considerations, the possible driving forces for TM helix
interactions are van der Waals interactions between closely packed
helices and interhelical polar interactions. Since TM helices of
membrane proteins contain only few polar amino acids and TM helix
interactions can be very stable in the absence of any hydrogen bonds or
salt bridges (45), van der Waals interactions may play a major role in
governing interhelical interactions. However, recent work has
implicated polar residues in membrane helix interactions. For instance,
charged residues in the membrane domain are necessary for subunit
assembly in the T-cell receptor (46). In the product of the
neu oncogene, Val to Glu mutations in the TM domain result
in dimer formation and constitutive activation (47). Furthermore,
formation of hydrogen bonds, e.g. between Asn residues,
mediates strong interactions of TM helices (48, 49). Little is known on
the role of tyrosine residues in mediating TM helix interactions.
Aromatic amino acids such as Tyr, Phe, or Trp may undergo "amino
aromatic" interactions in which neutral NH-containing groups tend to
be positioned near the aromatic rings (50) and even more favorably
"cation aromatic" interactions (cation-À interactions) with
positively charged residues (51). Sequence analysis of TM segments of
Na,K-ATPase
subunits does not provide conclusive evidence for the
existence of a potential interaction site matching the two tyrosine
residues in the
subunit. Highly conserved, neutral NH-containing
amino acids are indeed present in M5, M7, and M8 of Na,K- and
H,K-ATPases but they are unique or not aligned in a putative
-helix
to permit accommodation of the two tyrosine residues of the
subunits TM domain. Furthermore, interactions of Tyr40 and
Tyr44 with positively charged residues are not very likely
since arginine residues are absent in the TM domain of all Na,K- and
H,K-ATPase
subunits and conserved lysine residues are only present
at the N-terminal border of M5 whereas a single lysine residue within M5 is present in H,K-ATPases but not in Na,K-ATPase
subunits.
subunits TM domain, which
according to our analysis have an effect on the Na,K-ATPase function
and therefore are likely to interact with the
subunit, are located
on the face of the TM
helix which is opposite to the face which
contains the cysteine residue that was shown to cross-link with a
cysteine residue in M8 of the
subunit (21, 22). This result may
suggest that two faces of the TM helix interact with the
subunit
and that the
subunits TM domain is not or minimally exposed to
lipids. Based on cross-linking studies and other biochemical evidence
(21, 22, 52), possible models for the spatial organization of the
transmembrane segments of Na,K-ATPases include contacts of the
subunits TM domain with M9 and/or M7. Provided that the organization of
the transmembrane segments of the Na,K-ATPase
subunit is similar to
that deduced from the crystal structure of the Ca2+-ATPase
(3), and that the cysteine residue of the
subunits TM is positioned
toward M8 of the Ca2+-ATPase, then the two tyrosine
residues would be directed toward M10 or alternatively M7 of the
subunit as outlined in Fig. 7. Mutational
analysis on the
subunit should shed more light on the potential
intersubunit interaction site mediated by the tyrosine residues in the
TM domain of the
subunit.
View larger version (28K):
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Fig. 7.
Possible positioning and orientation of the
Na,K-ATPase 1 subunits TM helix with respect
to the TM domain of P2-ATPases. Shown are views from the
cytoplasmic side of the transmembrane helix of the Na,K-ATPase
1
subunit, and of the 10 transmembrane helices (M1 to M10) of the
sarcoplasmic Ca pump
subunit as deduced from the crystal structure
by Toyoshima et al. (3). The Na,K-ATPase
1 subunits TM
helix was oriented with Cys46 directing toward M8 of the
subunit as suggested by cross-linking studies (21, 22). In this
model, Tyr40 and Tyr44 in the
subunits TM
domain, which contribute to intrinsic transport properties of Na,K
pumps, are directed toward M10 or M7 of the
subunit and the
putative TM helix dimerization motif, GXXXGXXXG
is freely exposed.
A view from the cytoplasmic side (Fig. 7) reveals that the 3 aligned
glycine residues which form a grove in the TM helix (Fig. 1) may
represent a third helix face. In the models presented in Fig. 7, these
glycine residues are freely exposed, away from the
subunit.
Interestingly, the 3 glycine residues are part of a TM helix
interaction motif which was initially identified in glycophorin A (53)
but which occurs frequently in other helical membrane proteins (54).
This motif consists of a LIXXGXXXGXXXT sequence where GXXXG was found to be necessary and
sufficient for dimerization and corect packing of glycophorin A into a
right-handed pair of
helices (55). A
LIXXGXXXGXXXG motif is present in the
TM domain of all Na,K-ATPase
1 isoforms and the basic
GXXXG motif in that of Xenopus and
Bufo
3 isoforms, strongly suggesting that the TM domains
of these
subunits interact with another TM
-helix containing a
similar motif. Since TM domains of Na,K-ATPase
subunits lack
GXXXG motifs, there is the potential for dimer formation via
this motif, with a yet unknown protein or with the
subunit itself.
The possibility exists that the GXXXG motif mediates the
formation of an 2
2
structure, proposed by several studies to be
the functional unit of Na,K-ATPases (56, 57), by forming a
-
-
-
complex. However, if this would be the case, monomeric
and dimeric
-
complexes must exhibit very discrete differences in
transport properties since in this study, we could not detect any
impairment of the Na,K pump function after disrupting the putative
-
interaction site.
In conclusion, modeling of the TM helix accompanied by tryptophan
scanning reveal that the TM domain of 1 subunits of Na,K-ATPase contain two distinct helix faces which are probably both buried in the
interior of the protein and participate in membrane protein-protein interactions. Whereas no functional role could be attributed to TM
-
interactions in the face of the
subunits TM helix
containing the cysteine residue which is closely located to M8 of the
subunit, putative TM interactions in another face of the
subunits TM helix, which are mediated by two highly conserved tyrosine
residues, contribute to intrinsic functional properties of oligomeric
P-type ATPases. Finally, a putative third face, containing a basic
GXXXG motif, may be of potential functional relevance, in
permitting
1 subunit dimerization.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Francesca Fanelli and Peter Greasley
for help in the modeling of the subunits TM domain and for
critically reading the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Swiss National Fund for Scientific Research Grant 31-53721.98.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Institut de
Pharmacologie et de Toxicologie, rue du Bugnon 27, CH-1005 Lausanne, Switzerland. Tel.: 41-21-692-54-10; Fax: 41-21-692-53-55; E-mail: kaethi.geering@ipharm.unil.ch.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M008778200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TM, transmembrane; NMDG, N-methyl-D-glucamine; ER, endoplasmic reticulum.
![]() |
REFERENCES |
---|
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