From the Membrane Biology Section, Gene
Therapy and Therapeutics Branch, NIDCR, National Institutes of Health,
Department of Health and Human Services, Bethesda, Maryland 20892, and
§ Laboratoire de Biochimie et de Biologie Cellulaire,
Institut de Pharmacie, Universite Libre de Bruxelles, B 1050 Brussels,
Belgium
Received for publication, December 23, 2002, and in revised form, January 23, 2003
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ABSTRACT |
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The secretory
Na+-K+-2Cl Na+-K+-2Cl NKCC1, the "secretory" isoform of the
Na+-K+-2Cl In a recent series of papers, Forbush and colleagues (6-8) have shown
that it is mainly the central hydrophobic domain of NKCC1 that
determines its apparent affinities for sodium, potassium, and chloride
as well as for the NKCC-specific inhibitor bumetanide (6-8). The
cytosolic N and C termini have little effect on these properties and
are thought to be mainly involved in the regulation of transport
activity. In addition, these authors were able to identify a
number of specific regions within the central hydrophobic domain that
were involved in determining substrate affinities. They did this by
taking advantage of the fact that although the shark and human NKCC1s
are ~75% identical, they have quite different affinities for
transported ions. By examining the properties of various shark-human
chimeras, they were able to establish that sequence differences in MSSs
2, 4, and 7 could account for the differences in ion affinities between
species. From appropriate point mutations, they were then able to
narrow down the list of candidates to 13 residues (7). These residues
are (in the sequence of rat NKCC1) Ser-311 and Val-312 (in MSS 2 associated with sodium affinity differences); Ala-316 and Met-317 (in
MSS 2 associated with potassium affinity differences); Ala-405,
Ile-406, Val-408, Val-410, and Leu-412 (in MSS 4 associated with
potassium and chloride affinity differences); and Val-522, Ile-525,
Ile-527, and Val-529 (in MSS 7 associated with affinity differences of
all of the transported ions). The authors (7) speculate that as few as
four of these residues may be responsible for the affinity differences
between shark and human NKCC1, two residues in MSS 2 and one each in
MSSs 4 and 7. Additional studies to further clarify the roles of these 13 candidate residues have yet to be performed.
The simplest interpretation of the above results is that these 13 residues, or some subset of them, are located at or near the ion
binding sites of NKCC1. However, note that the approach taken in these
experiments necessarily excluded the consideration of residues that are
conserved between the shark and human NKCC1s. It is anticipated that
many of these residues play important functional and structural roles
in NKCC1 (hence their conservation) and that some of them also
influence ion binding and affinities. Mutations of these latter amino
acids would be expected to affect the properties of both shark and
human NKCC1.
In contrast to the relatively simple findings described above for
transported ions, Isenring et al. (7) found that the affinity differences for bumetanide between shark and human NKCC1 were
associated with sequence differences in MSSs 2-7 as well as in MSS 11 and MSS 12. Moreover, a number of their shark-human chimeras had
inhibition constants for bumetanide that were outside the range of both
shark and human NKCC1, making these results difficult to interpret.
In this study, we continue the effort to identify and characterize
important functional residues in NKCC1. To do this, we have made use of
the substituted cysteine accessibility method (9), which involves
replacing potentially interesting residues with cysteine and then
assaying their accessibility and properties using cysteine-specific
reagents. This method has been widely used to study the functional and
structural properties of a number of membrane transporters and
channels. Having only limited information that would allow us to select
residues for mutation, in our initial studies, we chose amino acids
that were predicted to be within the MSSs of NKCC1 but close to the
extracellular surface and that could be conservatively mutated to
cysteine. In a number of cases, we chose residues at or close to those
identified above by Isenring et al. (7). Somewhat
surprisingly, these latter cysteine mutants failed to be affected by
the sulfhydryl reagents tested, possibly indicating that these residues
were located in relatively compact pockets accessible to substrate ions
but otherwise protected from the extracellular solution. However, one
of our mutants (A483C) reacted strongly with sulfhydryl reagents
applied from the extracellular surface and appeared to be exposed only
during certain steps of the transport cycle.
Materials--
The methanethiosulfonate (MTS)
derivatives, MTSEA (2-aminoethyl methanethiosulfonate) and MTSET
(2-(trimethylammonium)ethyl methanethiosulfonate), were from Toronto
Research Chemicals. MTSEA and MTSET were prepared as 100-fold stock
solutions in water on the day of the experiment and were kept on ice
and diluted into appropriate media just before use. HEK-293
cells were from Microbix and were grown in EMEM (Biofluids)
supplemented with 10% heat-inactivated fetal calf serum (Invitrogen),
100 µM penicillin, 100 µM streptomycin, and
2 mM glutamine (all from Biofluids). 86Rb
(0.5-10 mCi/mg) was from Amersham Biosciences.
Molecular Biology--
The wild-type construct used in
these studies was the rat NKCC1 inserted into the mammalian expression
vector pBK-CMVlac Flux Media--
The following media were used in the flux
experiments. "Preincubation medium" contained 135 mM
NaCl, 2.5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 15 mM Na-HEPES (pH 7.4 with NaOH), and 5 mM glucose. "Chloride-free medium"
had the same cationic composition as the preincubation medium but had
all chloride replaced by gluconate. "Uptake medium" contained 135 mM NaCl, 1 mM RbCl, 1 mM
CaCl2, 1 mM MgCl2, 15 mM Na-HEPES (pH 7.4), 5 mM glucose, 0.1 mM ouabain, and ~0.3 µCi/ml 86Rb.
"Termination medium" contained 135 mM potassium
gluconate, 7.5 mM NaCl, 15 mM Na-HEPES (pH
7.4), 0.1 mM ouabain, and 250 µM bumetanide.
Transient Transfections and 86Rb Flux Assays--
On
"day zero," HEK-293 cells growing in 10-cm culture dishes were
transfected with the plasmids indicated using Polyfect (Qiagen) according to the manufacturer's instructions. On "day one," the solution containing the plasmid was removed and the cells were replated
into a 24-well culture dish in fresh culture medium. On "day
three," the 86Rb flux assay was carried out as described
below (variations in the flux protocol, if any, are noted in the figure
legends). The procedure for measuring fluxes was modeled after but not
identical to the methods used by Isenring et al. (6-8): A
row of four wells was used for each experimental point; three wells
were used to measure 86Rb flux (i.e. fluxes were
carried out in triplicate); and the fourth well was used to determine
the protein/well using the BCA protein assay kit (Pierce). All of the
wells were treated identically with the exception that 86Rb
was omitted from the uptake medium in the fourth well. For each row of
wells, the culture medium was removed, the wells were washed twice in
preincubation medium (in every case, additions to the wells were 0.5 ml), and then incubated in preincubation medium for 10 min at room
temperature (20 °C, all steps hereafter were carried out at room
temperature). The wells were washed next with chloride-free medium and
incubated in chloride-free medium for 1 h. The chloride-free
medium was then removed and replaced by chloride-free medium plus 0.1 mM ouabain, and incubation was continued for an additional
10 min. 86Rb uptake was initiated by replacing this medium
with uptake medium. After 7 min of incubation, the uptake medium was
removed and the well was washed four times with termination medium.
Finally, a 0.5-ml aliquot of 1% Triton X-100 was added to each well,
and samples were taken for liquid scintillation counting and protein determination. In control experiments (data not shown), we have verified that 86Rb uptake was linear with time for at least
7 min under the experimental conditions described here.
Data Presentation--
All of the results shown are means ± S.E. for three or more independent
experiments. Theoretical fits to the data were carried out by
non-linear least squares regression using the program SigmaPlot 2000. p values Sensitivity of Cysteine Mutants to MTS Reagents--
Our general
approach in this project was to choose a number of potentially
interesting amino acids in NKCC1 that could be conservatively mutated
to cysteine and then to test the sensitivity of these mutants to
cysteine-specific reagents. Mutants were initially screened with the
highly specific sulfhydryl reagents MTSEA
(C3H10NO2S2) and MTSET
(C6H16NO2S2). Both of
these reagents are positively charged with MTSEA being typically more
widely reactive in all likelihood because of its somewhat smaller size
(9). MTSEA has also been shown to be slightly membrane-permeant,
whereas MTSET is membrane-impermeant (11). As illustrated in Fig.
1, neither 86Rb flux into
mock-transfected HEK-293 cells (V) nor into those transiently transfected with wild-type rat NKCC1 (WT) is
significantly affected by either of these reagents under our test
conditions (3 min of incubation with 3 mM MTS reagent) (see
Fig. 1 legend for details). As illustrated later in the paper (Fig. 5),
these ouabain-insensitive 86Rb fluxes are completely
inhibited by the NKCC-specific inhibitor bumetanide with
K0.5 ~1 µM, indicating that they
are essentially entirely attributed to
Na+-K+-2Cl
As already indicated, the amino acids we have chosen for mutation to
cysteine (Fig. 1) are thought to be located within the MSSs of NKCC1
close to the extracellular surface (5). In most cases, we chose
residues at or close to those identified by Isenring et al.
(7) as being potentially associated with substrate binding sites.
Ser-311, Val-312, and Ala-316 are in the second MSS of NKCC1 and are
thought to be at or near the sites of interaction of the cotransporter
with sodium and potassium, Ala-405 is located in the fourth MSS of
NKCC1 at or near a region associated with the interactions of potassium
and chloride, and Ala-528 and Ser-530 are located in the seventh MSS
close to a region associated with the interactions of all three
substrate ions (see Introduction). Finally, Ala-483 is located in the
sixth MSS of NKCC1, a region of the protein that is highly conserved,
especially between the NKCCs and NCCs (Fig.
2). As illustrated in Fig. 1, all of
these cysteine mutants are functional when transiently transfected into HEK293 cells, but only A483C is inhibited by MTSEA and MTSET under our
experimental conditions.
The dependence of the inhibition of A483C on MTSEA and MTSET
concentrations is illustrated in Fig. 3,
A and B. The analyses of these data yield
K0.5 values for the effects of MTSEA and MTSET of 0.36 ± 0.07 and 1.48 ± 0.37 mM, respectively
(see Fig. 3 legend). From Fig. 3A, it can be seen that the
inhibitory effect of MTSEA is essentially complete after 3 min of
incubation with 3 mM reagent. This was confirmed in time
course studies where we found that the t1/2 for the
effect of 3 mM MTSEA was <15s (data not shown, times up to
10 min were studied). After 3 min of incubation with 3 mM
MTSEA, we found that the average residual 86Rb flux into
A483C-transfected cells is 4.7 ± 0.3(n = 17)
nmol/mg protein/min. By comparison, the average 86Rb flux
attributed to HEK-NKCC in mock-transfected cells (3.4 ± 0.2 nmol/mg protein/min (n = 25)) is significantly lower.
Thus, the reaction with MTSEA (and in all likelihood also with MTSET) (cf. Fig. 3B) does not appear to result in
complete inhibition of A483C. These results also indicate that
transient transfection of NKCC1 into HEK-293 cells does not suppress
endogenous HEK-NKCC activity as has been suggested to occur in HEK-293
cells stably transfected with NKCC1 constructs (6, 13).
The above experiments demonstrate that the mutation of Ala-483 to
cysteine renders NKCC1 sensitive to sulfhydryl reagents applied from
the extracellular solution. The fact that reaction with these reagents
leads to transport inhibition indicates that the residue being modified
is at or near an important functional site of NKCC1.
Characterization of A483C--
To better understand the
significance of the A483C mutation, we next compared the behavior of
A483C to that of wild-type NKCC1 and HEK-NKCC. Fig.
4 illustrates the sodium, rubidium, and
chloride dependence of 86Rb fluxes via these three
transporters. In the upper panels, we have plotted
(normalized) total fluxes versus substrate concentration, whereas in the lower panels, wild-type NKCC1 and A483C
fluxes have been corrected for flux via HEK-NKCC. Overall, the
transport characteristics of A483C are very similar to the wild-type
transporter except possibly for a marginally lower affinity for
chloride (see Fig. 4 legend). In contrast, as illustrated in Fig.
5, A483C as well as HEK-NKCC shows a
6-fold higher sensitivity to bumetanide than wild-type NKCC1.
The fact that the mutation of Ala-483 to cysteine has little or no
effect on the affinities of all three substrate ions suggests that this
modification results in only minor perturbations in NKCC1 conformation
and function. The larger effect on bumetanide affinity appears to be
specific to bumetanide, possibly because of a direct modification of
its binding site on NKCC1.
Effects of Inorganic Mercury--
Inorganic mercury is also known
to react strongly with free sulfhydryl groups. Fig.
6 shows the effects of mercury on
wild-type NKCC1, HEK-NKCC, and A483C. It is clear that wild-type NKCC1
and HEK-NKCC have a similar sensitivity to mercury with
K0.5 ~25 µM (see Fig. 6 legend).
However, A483C shows a far greater sensitivity. Approximately, 70% of
the 86Rb flux via A483C is inhibited by mercury with
K0.5 < 1 µM, whereas the
remainder appears to have a similar sensitivity to that of wild-type
NKCC1 (we have not attempted to fit these data to a two-component model
because of the scatter on the points). This behavior is reminiscent of
the effects of MTSEA and MTSET documented in Fig. 3 in that the
inhibition by mercury at its high affinity site does not result in
complete blockade of A483C.
Effects of Substrates and Inhibitors on the Inactivation of A483C
by MTSEA--
Because Ala-483 is located in a highly conserved (Fig.
2) and apparently functionally important region of the NKCCs, we
wondered whether its accessibility to sulfhydryl reagents might be
influenced by substrates or inhibitors of NKCC1. In Fig.
7, we illustrate that when
A483C-transfected HEK-293 cells are exposed to MTSEA in sodium-free or
potassium-free medium or in the presence of 100 µM
furosemide, its effect is not significantly different from that
observed under control (sodium-, potassium-, and chloride-replete) conditions. However, when exposure to MTSEA is carried out in the
absence of chloride, its inhibitory effect is markedly blunted. This
result suggests that the reaction of MTSEA with A483C is dependent on
transporter conformation and, more specifically, that the conformations
of the transporter present in the absence of extracellular chloride are
insensitive to MTSEA. A more detailed discussion of the significance of
this result is given later in the paper.
Cysteine-scanning Mutagenesis of MSS 6--
In Fig.
8, we show the effects of 3 mM MTSEA and MTSET on NKCC1 mutants in which nine residues
surrounding Ala-483 have been individually changed to cysteine.
86Rb uptake into HEK-293 cells transiently transfected with
two of these mutants, P487C and A488C, is not significantly different from that seen with mock-transfected cells (cf. Fig. 1),
indicating that these mutations do not result in a functional
transporter at the plasma membrane. Fluxes via the remaining seven
cysteine mutants are 50-100% of wild-type levels, but only I484C in
addition to A483C is inhibited by MTSEA and MTSET. In Fig.
9, we examine the sensitivity of these
mutants to inorganic mercury. Most of the mutants behave quite
similarly to wild-type NKCC1 (Fig. 9, solid line in all
panels); however, I484C (middle panel,
squares) shows a dramatic stimulation of 86Rb
uptake over the mercury concentration range of 1-10 µM
followed by inhibition at higher mercury concentrations. A similar but blunted effect appears to also occur for S480C (upper panel,
triangles) and F486C (lower panel,
triangles).
We have used substituted cysteine mutagenesis in combination with
sulfhydryl specific reagents to examine the properties of selected
residues in NKCC1. Bumetanide-sensitive 86Rb fluxes via
these mutants were measured in transiently transfected HEK-293 cells.
HEK-293 cells have been extensively employed by previous investigators
to study a number of facets of NKCC1 function (6-8, 12-15). These
past studies were carried out on stably transfected clonal cell lines.
The present experiments demonstrate that comparable 86Rb
fluxes can be obtained from transient transfectants, allowing for a
significant reduction in time and labor in high throughput-type experiments such as the screening of mutants.
In their experiments, Isenring et al. (6-8, 12) have
extensively characterized human NKCC1 in this expression system. They found Km values of 15, 1.95, and 31 mM
for the substrates sodium, rubidium, and chloride, respectively, and
K0.5 values of 56 µM for mercury
and 0.28 µM for the NKCC-specific inhibitor bumetanide.
In the present experiments with wild-type rat NKKC1, we found
Km values of 61, 1.85, and 48 mM for
sodium, rubidium, and chloride, respectively, and
K0.5 of 29 µM for mercury and 2.4 µM for bumetanide (Figs. 4-6). In their 86Rb
flux measurements, Isenring et al. (6-8) used 5 mM unlabeled rubidium, whereas we have used 1 mM. Because higher rubidium concentrations are expected to
increase the affinity of NKCC1 for sodium, chloride, and bumetanide,
this may account for at least some of the differences between our
results and theirs. In their studies, Isenring et al. (12)
also found that HEK-NKCC, the endogenous
Na+-K+-2Cl A number of the residues we have mutated to cysteine in this study
(S311, Val-312, Ala-316, Ala-405, Ala-528, and Ser-530) have been
previously suggested to be at or near the binding sites for sodium,
potassium, or chloride on NKCC1 (7). Interestingly, none of these
mutations rendered NKCC1 sensitive to MTSEA or MTSET under our
experimental conditions (Fig. 1). In additional experiments (data not
shown), we have confirmed that the sensitivity of these mutants to
inorganic mercury is likewise not significantly altered from that of
wild-type NKCC1. These results indicate either that the cysteines
introduced at these sites are inaccessible to the sulfhydryl reagents
we have tested or that the modifications resulting from these reagents
have no effect on transport. Although additional experiments are
required to distinguish between these possibilities, given the rather
convincing evidence that modifications in at least some of these
residues can dramatically affect substrate affinities (7), we would
suggest that the former explanation is more likely to be the correct
one. If this is the case, it would appear that the NKCC1 ion binding
sites may be located in regions that are relatively inaccessible except
to substrates, for example, in pockets buried below the protein surface.
In contrast to the above mutations, we found that the NKCC1
mutant A483C was inhibited by MTSEA and MTSET (Figs. 1 and 3) and
showed a dramatically higher sensitivity to inorganic mercury than
wild-type NKCC1 (Fig. 6). Because MTSET is membrane-impermeant, its
reaction with A483C must be from the extracellular solution. After 3 min of incubation with 300 µM MTSEA, we found that
it has achieved ~50% of its maximal effect, indicating a
t1/2 of ~3 min. However, reaction rates of MTSEA
and MTSET with accessible protein cysteines can exceed 104
M The relatively conservative mutation of Ala-483 to cysteine results in
little or no change in substrate affinities (Fig. 4), but it does
produce a 6-fold increase in the affinity of NKCC1 for bumetanide (Fig.
5). The fact that this mutation produces a bumetanide-specific effect
suggests that it may result in a relatively small change that only
affects the bumetanide binding site. In their studies, Isenring
et al. (7) found that the residues associated with the
differences in bumetanide affinity between human and shark
NKCC1 were in MSS 2-7 as well as MSS 11 and MSS 12. The present
results indicate that MSS 6 is also associated with bumetanide binding.
When residues surrounding Ala-483 were mutated to cysteine, only I484C
showed a dramatic change in sensitivity to MTS reagents and inorganic
mercury (Figs. 8 and 9). Surprisingly, I484C showed increased transport
activity in the presence of low concentrations (1-10 µM)
of mercury (Fig. 9), whereas A483C showed inhibition (Fig. 6). The
reason for this dramatic difference in transport behavior
remains to be determined, but it is again consistent with the
hypothesis that MSS 6 plays an important role in the function of NKCC1.
When the residues surrounding Ala-483 are plotted on a helical wheel
(Fig. 10), it is clear that Ala-483 and
Ile-484 lie on a relatively polar face of a (putative) helix whose
opposite side is strongly hydrophobic. Interestingly, Ser-480 whose
cysteine mutant also shows some stimulation at low concentrations of
mercury (Fig. 9) and Pro-487 whose cysteine mutant shows no function
(Fig. 8) also lie on the same face (Ser-480, Ala-483, Ile-484, and
Pro-487 are identified by asterisks in Fig. 10). Because
little is really known regarding the structure of NKCC1, it is
difficult to speculate about the involvement of MSS 6 in helix packing;
however, the highly hydrophobic face of MSS 6 on the left side in Fig.
10 almost certainly faces the membrane lipid, indicating that MSS 6 lies on the periphery of the intact NKCC1 molecule.
cotransporter
(NKCC1) is a member of a small gene family of electroneutral salt
transporters that play essential roles in salt and water homeostasis in
many mammalian tissues. We have identified a highly conserved residue
(Ala-483) in the sixth membrane-spanning segment of rat NKCC1
that when mutated to cysteine renders the transporter sensitive to
inhibition by the sulfhydryl reagents 2-aminoethyl methanethiosulfonate
(MTSEA) and 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET).
The mutation of Ala-483 to cysteine (A483C) results in little or no change in the affinities of NKCC1 for substrate ions but produces a
6-fold increase in sensitivity to the inhibitor bumetanide, suggesting
a specific modification of the bumetanide binding site. When residues
surrounding Ala-483 were mutated to cysteine, only I484C was sensitive
to inhibition by MTSEA and MTSET. Surprisingly I484C showed increased
transport activity in the presence of low concentrations of mercury
(1-10 µM), whereas A483C showed inhibition. The
inhibition of A483C by MTSEA was unaffected by the presence or absence
of sodium and potassium but required the presence of extracellular
chloride. Taken together, our results indicate that Ala-483 lies at or
near an important functional site of NKCC1 and that the exposure of
this site to the extracellular medium is dependent on the conformation
of the transporter. Specifically, our results indicate that the
cysteine introduced at residue 483 is only available for interaction
with MTSEA when chloride is bound to NKCC1 at the extracellular surface.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cotransporters (NKCCs)1
mediate the electroneutral transport of Na+,
K+, and Cl
across cell membranes with a
stoichiometry of 1Na+:1K+:2Cl
(1,
2). By providing a concentrative chloride entry step in chloride
secreting and absorbing epithelia, these transporters play a central
role in trans-epithelial salt movements across these tissues (1, 2).
The NKCCs belong to a small gene family with homologues in vertebrates,
crustaceans, insects, worms, plants, and some microorganisms. Nine
members of this family have been identified in vertebrates, and of
these, seven have been shown to be electroneutral cation-chloride
cotransporters (3). These include two
Na+-K+-2Cl
cotransporter isoforms
(NKCC1 and NKCC2), a Na+-Cl
cotransporter
(NCC), and four K+-Cl
cotransporter isoforms
(KCC1, KCC2, KCC3, and KCC4). The function of the remaining two
vertebrate sequences as well as all of the homologues identified in
lower organisms remains to be definitively established.
cotransporters, is the
most extensively studied member of the cation-chloride cotransport
family at the molecular level. This transporter is relatively widely
expressed in both epithelial and non-epithelial tissues and has been of
considerable interest because of its roles in cell volume regulation as
well as trans-epithelial chloride secretion (1, 2). The activity of
NKCC1 typically is highly regulated by physiological stimuli that can
result in its phosphorylation as well as other as yet uncharacterized
modifications (1, 4). Hydropathy analyses indicate that all of the
vertebrate cation-coupled cotransporters share a common membrane
topology consisting of large hydrophilic N and C termini (15-35 and
~50 kDa, respectively) on either side of a central hydrophobic
transmembrane domain (~50 kDa). Topology studies of NKCC1 (5) have
established that this hydrophobic domain consists of 12 (presumably
-helical) membrane-spanning segments (MSSs).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Stratagene) prepared as described
previously (10). The NKCC1 sequence was modified to include an
N-terminal histidine tag2 and
cloned between the EcoRI and XhoI sites of
pBK-CMVlac
. Mutations in the NKCC1 sequence were made
using the Stratagene QuikChange mutagenesis kit used according to the
manufacturer's instructions with 5% Me2SO added to
all PCR reactions.
0.05 (Student's t test) were
taken to represent statistically significant differences.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cotransport. The
presence of an endogenous NKCC transporter in the HEK-293 cells
(HEK-NKCC) has been well documented in previous studies (12). As also
seen in Fig. 1, 86Rb fluxes in HEK-293 cells transiently
transfected with wild-type NKCC1 are 3-4 times greater than those
observed in mock transfectants, indicating a good level of
functional NKCC1 expression under our assay conditions.
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Fig. 1.
Effects of MTSEA and MTSET on
86Rb fluxes via NKCC1 cysteine mutants.
86Rb fluxes were measured in HEK-293 cells transiently
transfected with wild-type NKCC1 (WT), empty
pBK-CMVlac vector (V), or the NKCC1 mutants
indicated following preincubation in the absence (open bars)
or the presence of 3 mM MTSEA (gray bars) or 3 mM MTSET (black bars). Fluxes were carried out
as described under "Experimental Procedures" with the exception
that the 10-min incubation in preincubation medium was immediately
followed by a 3-min incubation in the presence or absence of 3 mM MTSEA or MTSET in preincubation medium.
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Fig. 2.
Comparison of the sequence of the sixth
membrane-spanning segment of the NKCCs, NCCs, and KCCs. The amino
acids shown are those that are completely conserved in all of the
presently known members of the cation-coupled chloride transporter
families indicated. Note that MSS 6 is completely conserved in all of
the NKCCs sequenced to date (human, rat, mouse, bovine, and shark
NKCC1; and human, rat, mouse, rabbit, and shark NKCC2). Two unconserved
residues in MSS6 of the KCCs are indicated by dots. Ala-483
of rat NKCC1 is indicated by an asterisk.
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Fig. 3.
Dose responses of the effects of MTSEA and
MTSET on A483C. 86Rb fluxes were measured in HEK-293
cells transiently transfected with A483C following preincubation with
the concentrations of MTSEA (A) or MTSET (B)
indicated. Fluxes were carried out as described in Fig. 1 legend. The
data were fit to a model that assumes that one component of the flux is
blocked by the sulfhydryl agent at a single site, whereas a second
component is unaffected. These fits yielded K0.5
values of 0.36 ± 0.07 mM for MTSEA and 1.48 ± 0.37 mM for MTSET.
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Fig. 4.
Sodium, rubidium, and chloride dependence of
fluxes via wild-type NKCC1, HEK-NKCC, and A483C. 86Rb
fluxes were measured in HEK-293 cells transiently transfected with
wild-type NKCC1 (circles), empty pBK-CMVlac
vector (squares), and A483C (triangles) as a
function of the sodium, rubidium, and chloride concentrations
indicated. Ionic strength was held constant by replacing sodium and
rubidium with N-methyl-D-glucamine and chloride
with gluconate. In determinations of sodium dependence, Na-HEPES was
also replaced with Tris-HEPES (pH 7.4). Other details are as described
under "Experimental Procedures." Total fluxes are shown in the
upper panels, and wild-type and A483C fluxes corrected for
endogenous HEK-NKCC fluxes are shown in the bottom panels.
In each data set, fluxes were normalized to those observed at the
maximum concentration of the test ion. Sodium and rubidium fluxes were
fit to the Michaelis-Menten equation, and chloride fluxes were fit to
the Hill equation. The lines shown in the upper
panels are fits to the HEK-NKCC data. They yield
Km values of 41.2 ± 5.5 mM for
sodium, 1.47 ± 0.18 mM for rubidium, and 63.6 ± 3.4 mM with a Hill coefficient of n = 2.20 ± 0.15 for chloride. The solid lines in the
lower panels are fits to the wild-type NKCC1 results. They
yield Km values of 60.8 ± 4.0 mM
for sodium, 1.85 mM ± 0.26 mM for rubidium,
and 48.1 ± 4.7 mM with n = 2.47 ± 0.45 for chloride. The dashed lines in the lower
panels are fits to the A483C results, yielding
Km values of 60.2 ± 17.6 mM for
sodium, 2.42 ± 0.64 mM for rubidium, and 62.4 ± 8.2 mM with n = 2.60 ± 0.60 for
chloride.
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Fig. 5.
Dose response of the effect of bumetanide on
wild-type NKCC1, HEK-NKCC, and A483C. 86Rb fluxes were
measured in HEK-293 cells transiently transfected with wild-type NKCC1
(circles), empty pBK-CMVlac vector
(squares), and A483C (triangles) in the presence
of the concentrations of bumetanide indicated. In these experiments,
bumetanide was present with 86Rb in the uptake medium as
well as during the 10-min incubation with chloride-free medium plus 0.1 mM ouabain (see "Experimental Procedures" for other
experimental details). Wild-type NKCC1 and A483C fluxes have been
corrected for 86Rb uptake via HEK-NKCC, and all of the
fluxes have been normalized to that observed in the absence of
bumetanide. The data were fit to a model that assumes a single
inhibitory site for bumetanide. The solid line is a fit to
the wild-type NKCC1 results (K0.5 = 2.4 ± 0.7 µM), and the dashed line is a fit to the
A483C data (K0.5 = 0.37 ± 0.02 µM). A fit to the HEK-NKCC data (not shown) yielded
K0.5 = 0.43 ± 0.09 µM.
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Fig. 6.
Dose response of the effect of inorganic
mercury on wild-type NKCC1, HEK-NKCC, and A483C. 86Rb
fluxes were measured in HEK-293 cells transiently transfected with
wild-type NKCC1 (circles), empty pBK-CMVlac
vector (squares), and A483C (triangles) in the
presence of the concentrations of mercury (as HgCl2) as
indicated. In these experiments, mercury was present with
86Rb in the uptake medium as well as during the 10-min
incubation with chloride-free medium plus 0.1 mM ouabain
(see "Experimental Procedures" for other experimental details).
Wild-type NKCC1 and A483C fluxes have been corrected for
86Rb uptake via HEK-NKCC, and all of the fluxes have been
normalized to that observed in the absence of mercury. The wild-type
NKCC1 and HEK-NKCC data were fit to a model that assumes a single
inhibitory site for mercury. The solid line is a fit to the
wild-type NKCC1 results (K0.5 = 27.8 ± 8.9 µM). A fit to the HEK-NKCC data (not shown) yielded
K0.5 = 24.4 ± 2.9 µM.
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Fig. 7.
Effects of substrates and inhibitors of NKCC1
on the inactivation of A483C by MTSEA. 86Rb fluxes
were measured in HEK-293 cells transiently transfected with A483C
following preincubation with 0.3 mM (hatched
bars) or 3.0 mM (gray bars) MTSEA. Fluxes
were carried out as described under "Experimental Procedures" with
the exception that the 10-min incubation in preincubation medium was
immediately followed by a 3-min incubation in the presence or absence
of MTSEA in preincubation medium (Control and
Fur), preincubation medium containing 100 µM
furosemide (+Fur), or in preincubation medium without
sodium, potassium, or chloride as indicated. In sodium-free
(Na-free) preincubation medium, sodium was replaced with
N-methyl-D-glucamine and Na-HEPES was replaced
with Tris-HEPES (pH 7.4). In potassium-free (K-free)
preincubation medium, potassium was replaced with
N-methyl-D-glucamine, and in chloride-free
preincubation medium (Cl-free), chloride was replaced with
gluconate. In each case, wells were washed with the appropriate
modified preincubation medium without MTSEA before incubation in the
presence of MTSEA. In each data set, fluxes were normalized to those
observed without MTSEA treatment (open bars).
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Fig. 8.
Effects of MTSEA and MTSET on
86Rb fluxes via NKCC1 cysteine mutants in MSS 6. 86Rb fluxes were measured in HEK-293 cells transiently
transfected with the NKCC1 mutants indicated following preincubation in
the absence (open bars) or the presence of 3 mM
MTSEA (gray bars) or 3 mM MTSET (black
bars). Fluxes were carried out as described in Fig. 1
legend.
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Fig. 9.
Dose response of the effect of inorganic
mercury on NKCC1 cysteine mutants in MSS 6. 86Rb
fluxes were measured in HEK-293 cells transiently transfected with
various NKCC1 mutants in the presence of the concentrations of mercury
(as HgCl2) as indicated. Upper panel, F479C
(circles) and S480C (triangles); middle
panel, V481C (circles), F482C (triangles),
and I484C (squares); and lower panel, F485C
(circles) and F486C (triangles). Fluxes were
carried out as described in Fig. 6 legend. All of the fluxes have been
normalized to that observed in the absence of mercury. The solid
line is a fit to the wild-type NKCC1 results as determined in Fig.
6.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cotransporter of the
HEK-293 cells (a human cell line), had rather different properties from
their human NKCC1 clone. More specifically, they found
Km values of 35, 15, and 42 mM for
sodium, rubidium, and chloride, respectively, and
K0.5 values of 133 µM for mercury
and 0.08 µM for bumetanide. In our hands, the behavior of
HEK-NKCC is more similar to that of the rat NKCC1. We found Km values of 41, 1.47, and 63.6 mM for
sodium, rubidium, and chloride, respectively, and
K0.5 values of 24 µM for mercury and 0.4 µM for bumetanide (Figs. 4-6). This difference
may be related to the source of HEK-293 cells used in our experiments,
but we have not explored this matter further.
1s
1 (11, 16, 17) in which case
the t1/2 for reaction with 300 µM
reagent would be 0.23 s. The much longer t1/2
we observe with A483C suggests that the reactive cysteine is not
directly accessible from the extracellular solution. Consistent with
this finding, hydropathy analyses and topology studies (5) indicate
that Ala-483 is located within MSS 6 of NKCC1 close to but not at the
extracellular surface. This region of the protein is highly conserved
among NKCCs and their homologues (Fig. 2), suggesting an important role in transporter structure or function.
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Fig. 10.
Helical wheel representation of residues
479-488 of rat NKCC1. These amino acids represent the
extracellular (N-terminal) end of MSS 6 (see text and Fig. 2). Residues
Ser-480, Ala-483, Ile-484, and Pro-487 are identified by
asterisks (see "Discussion").
Because there are other native cysteines on NKCC1, it is possible in principle that mutating Ala-483 could result in a change in conformation that leads to the exposure of a previously hidden cysteine and that the effects we observe here are because of reactions at that site. In fact, there are only two candidates for this putative cysteine. Of the 12 native cysteines on wild-type rat NKCC1 (18), five are thought to be intracellular and therefore inaccessible to MTSET and five are located in a long glycosylated extracellular loop between MSS 7 and MSS 8 and therefore already accessible to extracellular reagents in wild-type NKCC1. The remaining two cysteines are located near the middle of MSS 11 (5). There are several reasons why we feel that it is very unlikely that our results could be the result of the exposure a native cysteine. First, the mutation of Ala-483 to cysteine is a relatively conservative one and is unlikely to result in a major conformational change in the protein. Thus, the reactive cysteine in A483C is much more likely to be Cys-483 than a previously non-reactive native cysteine. Second, the transport properties of A483C are almost identical to those of wild-type NKCC1, again arguing strongly against a major change in conformation. Third, the effects of mercury on A483C and I484C are dramatically different (Figs. 6 and 9). It is very unlikely that these two disparate effects could both be accounted for by the exposure of a previously inaccessible cysteine.
The mutant A483C has one other very interesting property. It is
insensitive to inhibition by 3 mM MTSEA and MTSET in the
absence of extracellular chloride (Fig. 7). This result indicates that the reactivity of the cysteine introduced at position 483 is dependent on transporter conformation. The significance of this observation can
be best appreciated by considering a model for the function of NKCC1.
Fig. 11 shows the NKCC1 transport cycle
proposed by Lytle et al. (19). The binding of substrates is
ordered with sodium binding first to the empty transporter on the
extracellular side followed by a chloride, potassium, and then a second
chloride. NKCC1 is thought to have "mirror symmetry," meaning that
substrate dissociation on the intracellular side occurs in the same
order that the substrates bind on the extracellular side,
i.e. sodium dissociates first from the fully loaded
transporter followed by chloride, potassium, and chloride (see
Fig. 11). Coupling is "tight," i.e. only the completely
empty and fully loaded transporters are capable of the conformational
change that reorients the substrate binding pocket between its
outward-facing and inward-facing forms (the transitions
between conformations I and X and between V and VI, respectively).
During incubation in sodium-, potassium-, and chloride-replete medium,
all of the conformations of NKCC1 will be populated so that the
inhibition of A483C by MTS reagents (Fig. 3) under these conditions
could be attributed to a reaction with any or all of them. In the
absence of extracellular chloride, conformations III, IV, and V can
still be populated from conformation VI; however, conformations III and
V will only be present transiently because chloride will dissociate
from them and cannot be replaced. Accordingly, because the reaction
with 3 mM MTSEA is rapid (t1/2 < 15 s, see above) and chloride loss from HEK-293 cells in isotonic chloride-free medium is much slower (t1/2 ~40 min;
see Ref. 12), we expect that all of the conformations of NKCC1 other
than III and V would be available for reaction with MTSEA when these
cells are switched to chloride-free medium. Because MTSEA is in fact
without effect under these conditions (Fig. 7), we conclude that only
conformations III and V could be sensitive to MTSEA. By similar
arguments, conformations III and V would be expected to be available
for reaction in extracellular sodium-free and potassium-free
conditions, whereas conformations II and IV, respectively, would not.
Consistent with our argument that only conformations III and/or V are
MTSEA-sensitive, MTSEA is in fact inhibitory in sodium-free and
potassium-free media (Fig. 7). Other models of NKCC1 function would
obviously lead to appropriately modified conclusions, but in general,
our results indicate that the cysteine introduced at site 483 is only
available for interaction with MTSEA when NKCC1 is in an externally
oriented conformation with chloride bound.
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ACKNOWLEDGEMENT |
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We thank Dr. Bruce J. Baum for many helpful discussions during the course of this work.
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FOOTNOTES |
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* 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: National Institutes of Health, Bldg. 10, Rm. 1A01, 10 Center Dr., MSC 1190, Bethesda, MD 20892-1190. Tel.: 301-402-1060; Fax: 301-402-1228; E-mail: rjturner@nih.gov.
Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M213148200
2 M. Saitoh and R. J. Turner, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
NKCC, Na+-K+-2Cl cotransporter;
NCC, Na+-Cl
cotransporter;
KCC, K+-Cl
cotransporter;
HEK-NKCC, endogenous
HEK-293 cell Na+-K+-2Cl
cotransporter;
MSS, membrane-spanning segment;
MTS, methanethiosulfonate;
MTSEA, 2-aminoethyl methanethiosulfonate;
MTSET, 2-(trimethylammonium)ethyl methanethiosulfonate.
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