From the Department of Biochemistry, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel
Received for publication, August 28, 2000, and in revised form, November 21, 2000
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ABSTRACT |
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Membrane permeable
N-ethylmaleimide (NEM) and
(2-aminoethyl)methanethiosulfonatehydrobromide (MTSEA) inhibited
the rat brain Na+-Ca2+ exchanger RBE-2
(NCX1.5) expressed in HEK 293 cells in a dose dependent manner.
50% inhibition was obtained at 1 mM MTSEA and 1.65 mM NEM. External application of membrane impermeable
[2-(trimethylammonium) ethyl]methanethiosulfonatebromide (MTSET)
and sodium(2-sulfonatoethyl)methanethiosulfonate (MTSES) did not
inhibit the transport activity in whole cells. Following
reconstitution, however, of RBE-2 transfected cell proteins into
proteoliposomes, external application of MTSET and MTSES led to a
decrease in transport activity to 42.7 (S.D. = 9.1) and 51% (S.D. = 10.14), respectively. Similar results were obtained also when the rat
heart isoform RHE-1 (NCX1.1) or the rat brain isoform RBE-1 (NCX1.4)
was expressed. NEM and MTSEA inhibited Na+
gradient-dependent Ca2+ uptake also in HEK 293 cells expressing RBE-2/C14A/C20S/ C122S/C780S (numbering corresponds
to RBE-2), a mutant in which all putative extracellular
cysteines were exchanged. To study the accessibility of different
cysteines to covalent modification, surface biotinylation of cells
expressing the wild type exchanger and its mutants was carried out
using 3-(N-maleimidylpropionyl)biocytin. Surface
biotinylation revealed immunoreactive protein derived from the wild
type Na+-Ca2+ exchanger only if the transfected
cells were exposed to the reducing agent Tris(2-carboxyethyl)phosphine.
No reduction was needed when the single cysteine mutants of RBE-2,
C14A, C20S, and C780S, were expressed. Treatment of the cells
expressing these mutants with MTSET before biotinylation, led to a
decrease in the amount of exchanger protein that was revealed. No
immunoreactive protein was detected when the quadruple mutant RBE-2,
C14A/C20S/C122S/C780S, was biotinylated, suggesting that no
additional cysteines are accessible directly from the
extracellular face of the membrane. Permeabilizing the cells expressing
RBE-2/C14A/C20S/ C122S/C780S with streptolysin O resulted in
biotinylation of the exchanger protein. Its amount decreased if
exposure to NEM preceded streptolysin O treatment. Our results suggest
that Na+-Ca2+ exchange activity is inhibited by
covalent modification with sulfhydryl reagents of cysteine residues
that are accessible from the cytoplasmic face of the membrane.
Sulfhydryl reagents such as
NEM,1 dithiothreitol,
methylmethane thiosulfonate, p-chloromercuribenoate, and
mersalyl were shown to have considerable effects on
Na+-Ca2+ exchange activity in membrane
preparations derived from sarcolemma and synaptosomes (1-3).
Na+-Ca2+ exchange activity in the squid giant
axon could also be modulated by internal application of
p-chloromercuryphenylsulfonic acid (4). Moreover, the
effects of the thiol reagents on Na+-Ca2+
exchange depended on the ionic composition of the medium (3), which
suggested that ion binding or translocation sites were involved.
Yet examination of the effects of different methanethiosulfonate (MTS)
reagents in conjunction with cysteine-scanning mutagenesis, which was
carried out to study the membrane topology of the dog heart
Na+-Ca2+ exchanger NCX1.1 (5, 6) expressed in
Xenopus oocytes, suggested that none of the membrane
permeable or impermeable sulfhydryl reagents tested had any significant
effect on transport activity. MTSET sometimes stimulated
Na+-Ca2+ exchange (5). In CCL39 cells
expressing the cloned dog cardiac Na+-Ca2+
exchanger only MTSET was tested, but it also did not modulate transport activity. (7).
Recently, based on differences in electrophoretic mobility of the dog
cardiac Na+-Ca2+ exchanger in the presence and
absence of reducing reagents and on site-directed mutagenesis, it was
suggested (8) that disulfide bonds form between cysteine 792 and either
cysteine 14 or 20 in the extracellular (9) N terminus of the protein.
The existence of these disulfide bonds could potentially restrict
access of different reagents to some residues at the extracellular face of the membrane. The possibility that the oxidation-reduction state of these bonds might differ in the different experimental preparations might explain some of the apparent differences between the
various studies using sulfhydryl reagents.
In this work, we report that the membrane permeable reagents NEM and
MTSEA inhibited Na+ gradient-dependent
Ca2+ influx in HEK 293 cells expressing the cloned rat
brain exchangers RBE-1 (NCX1.4) and RBE-2 (NCX1.5) and the rat heart
isoform RHE-1 (NCX1.1). As reported previously in oocytes (5, 6),
the membrane impermeant reagents MTSET and MTSES also did not inhibit transport activity in HEK 293 cells when applied from the outside. These reagents did, however, inhibit transport activity when proteins derived from cells expressing RBE-1, RBE-2, or RHE-1 were reconstituted into proteoliposomes. We show that in the wild type exchanger the
putative external cysteine residues were not directly accessible to covalent modification. Reduction, however, of the protein by Tris(2-carboxyethyl)phosphine (TCEP), or exchange of one of the three
cysteine residues Cys-14, Cys-20, and Cys-780 with either alanine or serine, resulted in surface biotinylation with
3-(N-maleimidylpropionyl)biocytin (MB). Pretreatment of the
cells expressing C14A, C20S, or
C780S2 with MTSET
considerably reduced surface biotinylation by MB but did not result in
inhibition of the transporter. Permeabilizing the cells expressing a
mutant in which all putative extracellular cysteines were exchanged
revealed that cysteines facing the cytoplasmic side of the membrane and
derived from the Na+-Ca2+ exchanger are
biotinylated by the reagent. The amount of the biotinylated
Na+-Ca2+ exchanger was considerably reduced
when NEM treatment of cells expressing this mutant preceded
biotinylation. Based on these and other results, we suggest that
cysteines accessible from the cytoplasmic face of the membrane can be
modified covalently by sulfhydryl reagents, which in turn might be
responsible for the impaired transport activity. Some of the
preliminary results were reported in a previously published abstract
(10).
Expression of Rat Isoforms RBE-1 (NCX1.4), RBE-2 (NCX1.5), and
RHE-1 (NCX1.1) or Their Mutants in HEK 293 Cells--
Transfection of
HEK 293 cells and determination of
Na+-dependent Ca2+ uptake was done
exactly as described (11). Transfected cells were preloaded with a
solution containing 0.16 M NaCl, 0.02 M Tris
HCl, pH 7.4, 0.02 M MgCl2, 25 µM
nystatin, and 1 mM ouabain for 10 min. The Na+
preloading solution was replaced by a solution of identical composition except that it did not contain nystatin. When added, NEM (dissolved in
Me2SO) and MTS reagents (Toronto Research Chemicals
Inc.) (dissolved in H2O) at 10-50× concentrations were
added directly to this solution and kept at 25 °C for 30 min.
The concentration of Me2SO was kept below 1%, which by
itself had no effect on the transport assays. The cells were washed
with the same solution (without the sulfhydryl reagents) twice, and
[Na+]-dependent
45Ca2+ uptake was measured as described (11).
In some control experiments, the MTS reagents were added to the cells
expressing the transporter in buffered choline chloride prior to the
Na+ preloading procedure. The change in the ionic
composition of the solution in which the reagents were presented to the
cells expressing the transporter had no effect on the results. All
measurements were done in triplicates, and each experiment was repeated
with three to six different preparations of transfected cells.
Site-directed Mutagenesis--
The method of Kunkel (12) was
used to prepare the different cysteine mutants. Antisense
oligonucleotides that contained the desired mutation flanked by about
15 upstream and downstream bases corresponding to the nucleotide
sequence of the cloned exchanger were used. All of the primers except
that which was used to generate C14A also contained a silent mutation
to introduce restriction sites for selection of mutants. Insertion of
the Flag epitope instead of Asn-9 has been described (9, 11). Usually a
segment of about 600-1000 base pairs containing the desired mutation
was excised from the mutant plasmid and subcloned into a vector derived from the parent clone. The entire insert containing the mutation and
parts of the vector containing the ligation sites where it was
subcloned were sequenced (Dr. Mira Korner, Sequencing Service, Hebrew University).
Reconstitution of Transfected Cell Proteins--
Brain
phospholipids in CHCl3 were dried under N2,
suspended in 0.2 M NaPi, pH 7.4, and dispersed
by sonication to form liposomes. Transfected cells were solubilized in
1% cholate in phosphate-buffered saline and added to the liposomes for
30 min at 37 °C. For confluent transfected cells grown in a 60-mm
culture dish (about 0.75 mg of protein), 2.7 mg of brain phospholipds
were used. Reconstitution and determination of transport activity was
carried out as described (11, 13).
Surface Biotinylation--
Biotinylation of surface membrane
proteins of transfected HEK 293 cells in situ was done with
3-(N-maleimidylpropionyl)biocytin (Molecular Probes)
essentially as described for surface biotinylation with NHS-SS-biotin
(11, 14). In most of the experiments FN-tagged RBE-2 and its cysteine
mutants were used for transfection. Unless otherwise stated, cells from
a single well of a 12-well plate were used for surface biotinylation.
When pretreatment with MTS reagents preceded biotinylation, a single
well of a 6-well plate was used. At the end of the biotinylation
reaction, an excess of MB was quenched with PBSCM (phosphate-buffered
saline also containing 0.1 mM CaCl2,
1 mM MgCl2) to which 2%
Permeabilization of HEK 293 cells with streptolysin O (Sigma) was done
as described previously (15).
Western Analysis--
SDS- polyacrylamide gel electrophoresis of
biotinylated proteins released from streptavidin beads and total
cell extracts was carried out by standard procedures (16). For analysis
of total cell extracts, 40 µg of cell protein derived from the entire contents of a single well of a 12-well plate was used. The transfection of cells used for biotinylation and for detection of total
immunoreactive protein was done in parallel. To detect protein derived
from the Na+-Ca2+ exchanger AbO-8, a polyclonal
antibody directed against a pentadecapeptide, corresponding to the
amino acids 649-663 of the rat heart exchanger, was used. (9).
Preparation, testing, and purification of the antibodies was done as
described (9, 17). Goat anti-rabbit horseradish peroxidase-conjugated
secondary antibody (Jackson ImmunoResearch Laboratories, Inc.)
was used to detect antigen-antibody complexes using the ECL kit from
Amersham Pharmacia Biotech.
The Effects of Sulfhydryl Reagents on Na+
Gradient-dependent Ca2+ Uptake--
Fig.
1 shows the effects of membrane permeable
MTSEA (panel A) and NEM (panel B) on
Na+ gradient-dependent Ca2+ uptake
in HEK 293 cells transfected with RBE-2. It can be seen that the
inhibition with both reagents is concentration-dependent. Exposure of cells expressing RBE-2 to 2.5 mM MTSEA reduces
Na+ gradient-dependent Ca2+ uptake
to about 25% relative to that measured in untreated cells, and 50%
inhibition is obtained at about 1 mM MTSEA. A higher
concentration of NEM is required to inhibit the transporter. Although
the inhibition of Na+ gradient-dependent
Ca2+ uptake is detectable already at 0.5 mM NEM
and 5 mM NEM reduces Na+ gradient dependent
Ca2+ uptake to below 20% relative to that of the untreated
cells, 50% inhibition is reached when cells are exposed to 1.65 mM NEM.
The inhibition of Na+ gradient-dependent
Ca2+ uptake with the membrane permeable reagents is
obtained both when the WT exchanger is expressed (black
squares in Fig. 1) and when the glycosylation mutant FN-RBE-2, in
which the Flag epitope replaces Asn-9, the single glycosylation site of
the protein (11, 18) (black triangle in Fig. 1A
and black circle in Fig. 1B), is expressed.
Transport activity of the FN-tagged RBE-2 was similar to the WT
exchanger bearing no tag.
The membrane impermeant reagents MTSET and MTSES (Fig.
2) did not inhibit Na+
gradient-dependent Ca2+ uptake when added to
transfected cells in situ (Fig. 2A). In some
experiments, as reported by Nicoll et al. (5), addition of
the reagents led to stimulation of
Na+-dependent Ca2+ uptake.
Because membrane permeable reagents inhibited
Na+-Ca2+ exchange activity in whole cells and
membrane impermeant reagents applied from the outside did not, we
solubilized transfected cell proteins and reconstituted them into
proteoliposomes. Fig. 2B shows the inhibition by 2 mM MTSET and 10 mM MTSES of
Na+-dependent Ca2+ uptake in
proteoliposomes derived from RBE-2-transfected HEK293 cells. The
addition of 0.1, 0.5, and 1 mM MTSET and 3 and 5 mM MTSES to the reconstituted preparation inhibited
Na+-dependent Ca2+ uptake in a
concentration-dependent manner. However, increasing the
concentration of MTSET to 2 mM and above (2-10
mM) and MTSES to 5 mM and above (5-15
mM) did not further increase the extent of inhibition that
was reached in each preparation, which was between 40 and 60% relative
to the transport activities measured in the absence of the sulfhydryl reagents.
Because none of the sulfhydryl reagents had a significant effect on the
transport activity of the dog sarcolemmal
Na+-Ca2+ exchanger NCX1.1 expressed in oocytes
(5-7) and because the dog heart isoform has an extra cysteine in
position 879 instead of a serine in the corresponding rat isoform, we
prepared a mutant of RHE-1, RHE-1/S879C. RHE-1/S879C was expressed in
HEK 293 cells, and Na+ gradient-dependent
Ca2+ uptake activity of this mutant was determined in the
absence and in the presence of the sulfhydryl reagents. Inhibition of transport activity was obtained by the membrane permeable reagents NEM
and MTSEA added to whole cells from the outside and by MTSET and MTSES
only following reconstitution, in a manner similar to the results
presented in Figs. 1 and 2 for the rat isoforms.
Accessibility of Cysteines to Sufhydryl Reagents--
To further
examine the functional and structural implications that the effects of
membrane permeable and impermeable sufhydryl reagents had on the
Na+-Ca2 exchanger, we determined the
accessibility of the different cysteines to covalent modification. This
was done by surface biotinylation of adherent cells expressing the
Na+-Ca2+ exchanger and some of its cysteine
mutants with MB directly or following the permeabilization of these
cells in situ with SLO. Biotinylated proteins were isolated
from lysed cells on immobilized streptavidin; Western analysis of the
proteins released from the beads was carried out to detect the
Na+-Ca2+ exchanger or its mutants. To examine
whether glycosylation of the exchanger played a role in determination
of accessibility of the protein to covalent modification, transfection
was carried out both with glycosylated and nonglycosylated
(FN-tagged) RBE-2 and its cysteine mutants. As also seen with transport
activity (see Figs. 1 and 2), no differences in surface expression were detected between the glycosylated and nonglycosylated exchangers.
Fig. 3A shows the location of
the different cysteines. Because the topology of the protein has
not been fully established, we based the model in Fig. 3A on
a combination of the hydropathy analysis model and possible changes
suggested by the neural network algorithm-based analysis of secondary
structure of the protein and experimental data based on
immunocytochemistry (9) and cysteine-scanning mutagenesis (5, 7). We
exchanged the 14 cysteines of the rat isofoms of NCX1 with either
alanine or serine alone or in different combinations. Each one of the
single cysteine mutants was functional. Based on the results shown in
Figs. 1 and 2, we have studied in this work the accessibility of the
four putative extracellular cysteines to surface biotinylation by MB. The location of these cysteines is marked by an arrow in Fig. 3A.
Fig. 3B shows the relative transport activities of the
different putative extracellular single and multiple cysteine mutants that were generated. It can be seen that although relative transport activities of these mutants varied, all were functional.
Fig. 4 shows an immunoblot of surface
biotinylated FN-RBE-2 (panel A) and some of its FN-tagged
cysteine mutants (panels B and C). Biotinylation
was carried out with MB, which botinylates cysteine residues. The
amount of "total" immunoreactive protein derived from transfected
cell extracts is also shown. It can be seen (Fig. 4A), that
in the surface membrane, only traces of immunoreactive protein were
detected when cells expressing the FN-RBE-2 were biotinylated. To
examine whether disulfide bonds block the access of the biotinylating
reagent MB to extracellular cysteines and thereby prevent the isolation
of exchanger protein from FN-RBE-2-transfected cells, we used the
reducing agent TCEP. TCEP, unlike dithiothreitol or
Fig. 4C shows the immunoblot derived from the
Na+-Ca2+ exchanger proteins that were detected
when cells expressing the double mutants FN-RBE-2/C14A/C20S and
FN-RBE-2/C20S/C122S, the triple mutant FN-RBE-2/C14A/C20S/C122S, and
the quadruple mutant FN-RBE-2/C14A/C20S/C122S/C780S were biotinylated.
Immunoreactive protein was revealed when each of these mutants was
biotinylated except for the quadruple mutant FN-RBE-2/C14A/C20S/C122S/C780S.
Because no immunoreactive exchanger protein was revealed by surface
biotinylation of cells expressing FN-RBE-2/C122S (Fig. 4B)
and FN-RBE-2/C14A/C20S/C122S/C780S (Fig. 4C) with MB and
because both mutants exhibited Na+
gradient-dependent Ca2+ uptake in whole
transfected cells (Fig. 3B), we carried out surface biotinylation of cells expressing these mutants with NHS-SS-biotin. This membrane impermeable reagent covalently labels free amino groups
such as N-terminal amines and
The addition of MTSET to cells expressing FN-RBE-2 (Fig. 2A)
or its mutants FN-RBE-2/C14A, FN-RBE-2/C20S, FN-RBE-2/C122S, and
FN-RBE-2/C780S from the outside (Fig.
6A) did not inhibit Na+-Ca2+ exchange activity; this could result
either from insensitivity of the FN-RBE-2 exchanger and the different
cysteine mutants to covalent modification by MTSET or the fact that the
cysteines were inaccessible to the reagent. To distinguish between
these possibilities, we determined the cross-reactivity between MTSET and MB. Cells expressing FN-RBE-2 and the different cysteine mutants were biotinylated directly or incubated first with 5 mM
MTSET for 30 min and biotinylated after appropriate washes with PBSCM. Because protein derived from cells expressing FN-RBE-2 was not revealed
by biotinylation without reduction (Fig. 4A), 1 mM TCEP was included in the biotinylating reaction mixture
of cells expressing this protein. Fig. 6B shows the results
of these experiments. It can be seen that pretreatment of the cells
with MTSET considerably reduced the amount of biotinylated protein that
was revealed. suggesting that MTSET and MB bind to the same site. These
experiments also suggest that MTSET binds to the remaining cysteine(s)
after the mutation of the single cysteine in each mutant, but this
binding does not result in inhibition of their transport activity.
To determine whether intracellular cysteines are accessible to covalent
modification, we expressed mutant FN-RBE-2/RBE-2C14A/C20S/C122S/C780S, in which all the cysteines that could potentially be biotinylated by
external addition of the reagent were exchanged. Biotinylation of cells
expressing this mutant with MB (Fig. 4C) did not lead to
detection of immunoreactive Na+-Ca2+ exchanger
protein. Fig. 7A shows an
experiment in which cells expressing
FN-RBE-2/RBE-2C14A/C20S/C122S/C780S were permeabilized with SLO and
then biotinylated with MB. Following cell lysis (see "Experimental
Procedures") and isolation of the biotinylated proteins on
streptavidin beads, Western analysis was carried out. It can be seen,
that immunoreactive protein derived from the
Na+-Ca2+ exchanger was revealed. Treatment of
the cells with either 5 mM MTSEA (not shown) or 5 and 10 mM NEM prior to the biotinylation reduced the amount of
biotinylated immunoreactive exchanger protein that was detected. Fig.
7B shows a parallel experiment in which the effect of NEM on
the transport activity of this mutant was examined. It can be seen that
Na+ gradient-dependent Ca2+ uptake
was reduced when cells expressing the mutant were treated with 5 mM NEM. A similar inhibition was obtained when 2.5 mM MTSEA was added to cells expressing
FN-RBE-2/C14A/C20S/C122SC780S.
In this work we have shown that the transport activity of the rat
isoforms of the NCX1 gene expressed in HEK 293 cells
is inhibited by membrane permeable sulfhydryl reagents such as NEM and
MTSEA applied to adherent cells from the outside and with the membrane
impermeable reagents MTSET and MTSES, following reconstitution. We used
reconstitution of transfected cell proteins to gain access to the
cytoplasmic face of the membrane, because this procedure "scrambles" the topology of membrane proteins and at least some of
the transporters might assume "inside-out" orientation and undergo
covalent modification with membrane impermeable reagents added from the
outside. Applying the membrane impermeable sulfhydryl reagents to the
reconstituted preparation resulted in a
concentration-dependent inhibition of
Na+-dependent Ca2+ uptake up to
about 2 mM MTSET and 5 mM MTSES. At these
concentrations, a 40-60% inhibition of the transport activity was
obtained, which did not increase with a further increase in the
concentration of the reagents. This finding suggests that in our
preparation of the reconstituted proteoliposomes, about half of the
transporters have an "inside-out" orientation and only these
transporters are amenable to covalent modification by membrane
impermeable reagents. When all of the externally accessible cysteines
are covalently modified, a further increase in the concentration of the
membrane impermeable reagents cannot lead to further inhibition.
To study further the effects of sulfhydryl reagents on Na+
gradient-dependent Ca2+ uptake, we monitored
the accessibility of different cysteines of the
Na+-Ca2+ exchanger to covalent modification.
This was done by surface biotinylating transfected cells that express
the protein with MB and detection of immunoreactive biotinylated
Na+-Ca2+ exchanger. Our studies show that only
traces of immunorective protein derived from the FN-RBE-2 could be
revealed by biotinylation with MB directly, although the transporter
was functional. This result is puzzling for the following reasons.
First, our previous studies indicated that surface-expressed FN-tagged
Na+-Ca2+ exchanger of similar transport
activity is detected by indirect immunofluorescence using the anti-Flag
antibody and also by surface biotinylation with NHS-SS-biotin, which
reacts with the free N-terminal and The inhibition with membrane permeable reagents persisted also when all
of the putative external cysteines were mutated. Taken together with
the effects of the membrane impermeable reagents in the reconstituted
preparation, our results suggest that covalent modification of
intracellular cysteines was involved. Support for this proposal
comes from the experiment shown in Fig. 7 in which we incubated cells
expressing FN-RBE-2/C14A/C20S/C122S/C780S without and with NEM,
permeabilized them with SLO, and then exposed them to MB.
Permeabilization of cells expressing FN-RBE-2/C14A/C20S/C122S/C780S before biotinylation resulted in detection of biotinylated
immunoreactive protein derived from the
Na+-Ca2+ exchanger. Pretreatment of the cells
with NEM led to a considerable decrease in the amount of immunoreactive
protein that was revealed.
These results suggest that intracellular cysteines are accessible to
covalent modification by membrane permeable sulfhydryl reagents.
Because these sulfhydryl reagents impair transport activity not only of
the WT transporter but also of its RBE-2/C14A/C20S/C122S/C780S mutant,
which has no cysteines accessible from the extracellular face of the
membrane, we suggest that the covalent modification of intracellular
cysteines is responsible for the impaired
Na+-Ca2+ exchange activity. Further studies are
required to identify which and how many of the intracellular cysteines
are sensitive to the sulfhydryl reagents.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mercaptoethanol was added. Surface biotinylation with NHS-SS-biotin (Pierce, catalog no. 21331), was carried out exactly as described (11, 14). The
cells were lysed with a solution containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton
X-100, 1% SDS, and 0.1 mM phenylmethylsulfonyl fluoride
(Sigma), 0.01 mg/ml pepstatin A (Sigma), and 0.02 mM
leupeptin (Sigma). The SDS concentration was lowered by a 10-fold
dilution of the lysate with a solution of identical composition to that
used to lyse the cells except that it did not contain SDS loaded on
streptavidin-agarose beads (Pierce) and was gently shaken for 4 h
at 4 °C. Washing of the beads was done as described (14).
Biotinylated proteins were released from the beads by heating for 10 min at 85 °C with Laemmli sample buffer and separated by
SDS-polyacrylamide gel electrophoresis. Western analysis was carried
out as described below.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The effect of membrane permeable sulfhydryl
reagents on Na+ gradient-dependent
Ca2+ uptake in RBE-2 transfected HEK 293 cells. HEK
293 cells were transfected with wild type RBE-2 cloned in pcDNA3.1
(black squares) or with FN-RBE-2 in which the Flag epitope
replaces Asn-9 (black triangle in A and
black circle in B). 24 h post-transfection
Ca2+ uptake was measured (see details under "Experimental
Procedures") without and with different concentrations of membrane
permeable MTSEA (A) and NEM (B). The data were
compiled from six different transfections; individual measurements were
done in triplicate. The transport activity in each experiment without
the sulfhydryl reagent was taken as 100%, and other data were
normalized. Bars represent standard deviations.
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Fig. 2.
The effect of membrane impermeable reagents
MTSET and MTSES on Na+ gradient-dependent
Ca2+ uptake in transfected HEK 293 cells, measured in whole
cells (A) and following reconstitution of solubilized
cell proteins into proteoliposomes (B). HEK 293 cells were transfected with wild type plasmid DNA or with FN-tagged
RBE-2. 24 h post-transfection, Ca2+ uptake was
measured (see details under "Experimental Procedures") in the
presence of membrane impermeable reagents MTSET or MTSES. The average
transport activity without the reagents in each experiment was defined
as 100%; the transport activity with the reagents is presented in
relative values: A, in whole cells; B, following
reconstitution of transfected cell proteins into proteoliposomes.
Bars represent standard deviations.
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Fig. 3.
A, topological model of the
Na+-Ca2+ exchanger NCX1 and putative location
of the cysteine residues. This model is based on hydropathy analysis
(25, 26) and experimental data compiled previously (5, 7-9).
The numbering of cysteine residues is shown as in the rat isoform
NCX1.5. B, Na+ gradient-dependent
Ca2+ uptake in transfected HEK 293 cells expressing
different cysteine mutants. HEK 293 cells were transfected with
different single cysteine mutants, derived from FN-RBE-2, C14A, C20S,
C122S, and 780S; double cysteine mutants C14A/C20S and C20S/C122S; the
triple cysteine mutant C14A/C20S/C122S; and the quadruple mutant
C14A/C20S/C122S/C780S. In each individual experiment, parallel
transfection with the WT FN-RBE-2 DNA was also carried out, and its
transport activity was defined as 100%. The transport activity of the
mutants is presented in relative values.
-mercaptoethanol, can be added together with MB without quenching it
(19-21) and used for the reduction of disulfide bonds. The addition of
an equimolar concentration (500 µM) of TCEP to the
biotinylating reaction mixture led to detection of immunoreactive
exchanger protein. The addition of an excess of TCEP (750 µM) over the concentration of MB (500 µM)
that was used led to detection of higher amounts of exchanger protein.
Biotinylation of cells expressing the single cysteine mutants
FN-RBE-2/C14A, FN-RBE-2/C20S, and FN-RBE-2/C780S (Fig. 4B)
resulted in detection of immunoreactive exchanger protein without any
reduction. No immunoreactive protein was detected when mutant
FN-RBE-2/C122S was biotinylated, although the mutant was functional and
protein derived from it was detected in total cell extracts.
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Fig. 4.
Expression of surface biotinylated proteins
and total cell proteins derived from HEK 293 cells expressing FN-RBE-2
and its cysteine mutants. HEK 293 cells grown in 12-well plates
were transfected with FN-RBE-2 plasmid DNA (A) and with some
of its cysteine mutants (B and C). 24 h
post-transfection, cells were surface-biotinylated and isolated by
binding to immobilized streptavidin, and following release from the
beads, Western analysis was carried out. Total immunoreactive protein
derived from parallel transfections (40 µg of cell protein/lane) is
also shown (see "Experimental Procedures"). A,
immunoreactive protein derived from surface-biotinylated cells
expressing FN-RBE-2 without reduction and with 500 µM or
750 µM TCEP. Immunoreactive protein derived from total
transfected cell extracts prepared in parallel is also shown.
B, immunoreactive biotinylated protein derived from cells
expressing single cysteine mutants C14A, C20S, and C780S
(lanes marked A) and from parallel total cell
lysates derived from transfections with the same mutants
(lanes marked B). C, the same as
B but with different double, triple, and quadruple cysteine
mutants.
-amino lysines (11). Fig. 5 shows that biotinylated protein derived
from cells transfected with both RBE-2/C122S and
RBE-2/C14A/C20S/C122S/C780S is detected in the surface membrane
(lanes A) and not only in total cell extracts (lanes
B). The surface expression and total cell expression of exchanger
protein derived from RBE-2 is also shown.
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Fig. 5.
Surface expression of the cysteine mutants
RBE-2/C122S and RBE-2/C14A/C20S/C122S/C780S. HEK293 cells were
transfected with the WT Na+-Ca2+ exchanger
RBE-2 and its cysteine mutants C122S and C14A/C20S/C122S/C780S. 24 h post-transfection, the cells were biotinylated with NHS-SS-biotin,
biotinylated proteins were isolated by streptavidin beads, biotinylated
immunoreactive protein was derived from the
Na+-Ca2+ exchanger, and its mutants was
analyzed by SDS-polyacrylamide gel electrophoresis (lanes
marked A). Total immunoreactive protein derived from
parallel transfection of cells expressing the
Na+-Ca2+ exchanger and its mutants is also
shown (lanes marked B).
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Fig. 6.
Na+
gradient-dependent Ca2+ uptake and immunoblot
of biotinylated surface expressed cysteine mutants expressed in HEK 293 cells. HEK 293 cells were transfected with FN-RBE-2 or its single
cysteine mutants C14A, C20S, and C780S. A, 24 h
post-transfection, transport activity was measured without and with 5 mM MTSET. B, immunoreactive biotinylated protein
of cells expressing the WT exchanger and the same single cysteine
mutants without and with preincubation with 5 mM MTSET
before biotinylation. 1 mM TCEP was also included in the
biotinylation mixture of the WT exchanger.
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Fig. 7.
The effect of NEM on transport activity and
on the detection of immunoreactive biotinylated proteins derived from
cells expressing the quadruple cysteine mutant FN-RBE-2
C14A/C20S/C122S/C780S. A, HEK 293 cells were
transfected with the quadruple cysteine mutant
FN-RBE-2/C14A/C20S/C122S/C780S. 24 h post-transfection, the cells
were permeabilized with SLO (300 units/well of a 6-well plate) and then
biotinylated, or they were treated with 5 or 10 mM NEM,
permeabilized with SLO, and then biotinylated. Isolation and detection
of biotinylated immunoreactive protein was carried out as described
under "Experimental Procedures." B, transport activity
of cells expressing the quadruple cysteine mutant without and with 5 mM NEM.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino lysines (11).
Second, MB was successfully used to monitor extracellular cysteines in
different expression systems and with different transporters (15, 22,
23). Third, based on hydropathy analysis (24, 25), the position of the
glycosylation site at Asn-9 (18) and indirect immunofluorescence
studies with N-Flag-tagged Na+-Ca2+ exchanger
(9), Cys-14 and Cys-20 are in the extracellular N terminus of the
protein (Fig. 3A) and should be accessible to surface
biotinylation. Fourth, Santacruz-Toloza et al. (8) suggested
that, in addition to Cys-14 and Cys-20, Cys-780 also is extracellular
and Cys-780 is connected by a disulfide bond to either Cys-14 or
Cys-20. Yet, if this disulfide bond alternates between Cys-14 and
Cys-20 to Cys-780, one of these three cysteines (or a fraction from
each at a time) that is not involved in sulfhydryl bond formation
should be free for biotinylation. Because exposure of the cells
expressing the transporter to the reducing agent TCEP led to detection
of biotinylated immunoreactive RBE-2, it is possible that an additional
cysteine might be involved in disulfide bond formation, which together
with Cys-14, Cys-20, and Cys-780 prevents free access to the
extracellular cysteines. The existence of an additional cysteine that
participates in disulfide bond formation is also consistent with the
finding that immunoreactive protein was detected without reduction when
Cys-14 alone, Cys-20 alone, or Cys-780 alone (Fig. 4B) was
exchanged with either Ala or Ser. From current topological
models, cysteine 122 could have been an appropriate candidate to
interact with Cys-14 or Cys-20. Yet mutation of this residue alone did
not result in detection of immunoreactive protein (Fig. 4B),
suggesting that it was not involved in disulfide bond formation and it
was not responsible for blocking the access of the biotinylating
reagent to the WT exchanger.
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ACKNOWLEDGEMENTS |
---|
We thank Doron Rozenzweig for his assistance with some of the experiments and for preparation of some of the figures and Dr. Arie Dagan for his suggestion to use TCEP.
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FOOTNOTES |
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* This work was supported in part by a fellowship (X. R.) from the Bernard Katz Minerva Center for Cell Biophysics.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. Tel.: 972-2-6758511;
Fax: 972-2-6784010; E-mail: Hannah@cc.huji.ac.il.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M007823200
2 The numbering corresponds to the amino acid sequence of RBE-2 (NCX1.5).
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ABBREVIATIONS |
---|
The abbreviations used are: NEM, N-ethylmaleimide; RBE-1 (NCX1.4) and RBE-2 (NCX1.5), rat brain exchanger 1 and 2 (GenBankTM accession no. X68812 and x68813, respectively); RHE-1 (NCX 1.1), rat heart exchanger 1 (GenBankTM accession no. X68191); WT, wild type exchanger; MTS, methanethiosulfonate; MTSEA, (2-aminoethyl)methanethiosulfonatehydrobromide; MTSET, [2-(trimethylammonium)ethyl]methanethiosulfonatebromide; MTSES, sodium(2-sulfonatoethyl)methanethiosulfonate; FN, N-terminal Flag (DYKDDDDK)-tagged; MB, 3-(N-maleimidylpropionyl)biocytin; TCEP, Tris(2-carboxyethyl)phosphine; SLO, streptolysin O.
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