1 Yale University School of Medicine, New Haven, Connecticut 06510; and 2 Nephrology Group, Department of Medicine, Faculty of Medicine, Research Center L'Hôtel-Dieu de Québec, Laval University, Québec, Canada G1R 2J6
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ABSTRACT |
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Mercury alters the
function of proteins by reacting with cysteinyl sulfhydryl
(SH) groups. The
inorganic form (Hg2+) is toxic
to epithelial tissues and interacts with various transport proteins
including the Na+ pump and
Cl
channels. In this study,
we determined whether the
Na+-K+-Cl
cotransporter type 1 (NKCC1), a major ion pathway in secretory tissues,
is also affected by mercurial substrates. To characterize the
interaction, we measured the effect of
Hg2+ on ion transport by the
secretory shark and human cotransporters expressed in HEK-293 cells.
Our studies show that Hg2+
inhibits
Na+-K+-Cl
cotransport, with inhibitor constant
(Ki) values of
25 µM for the shark carrier (sNKCC1) and 43 µM for the
human carrier. In further studies, we took advantage of species
differences in Hg2+ affinity to
identify residues involved in the interaction. An analysis of
human-shark chimeras and of an sNKCC1 mutant
(Cys-697
Leu) reveals that transmembrane domain 11 plays an essential role in Hg2+
binding. We also show that modification of additional
SH
groups by thiol-reacting
compounds brings about inhibition and that the binding sites are not
exposed on the extracellular face of the membrane.
cation-chloride carriers; mutagenesis; binding sites
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INTRODUCTION |
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THE
NA+-k+-cl
cotransporter (NKCC) is an integral membrane protein that couples
the movement of Na+,
K+, and
Cl
across the plasmalemma
of most animal cells (17, 18). Two isoforms of the
Na+-K+-Cl
carrier protein have been described: the widely distributed NKCC1 (32,
42) and the kidney-specific NKCC2 (19, 31). Both isoforms promote the
vectorial movement of salt and fluid across epithelia. NKCC1 is also
present in a variety of nonpolarized cells, where it plays a role in
cell volume regulation (9). The NKCCs belong to the
cation-Cl
cotransporters
(CCCs) (21), a family of proteins that also includes the
K+-Cl
cotransporters (KCCs) and the
Na+-Cl
cotransporter (NCC).
The NKCCs exhibit common structural features, illustrated for the shark NKCC1 by the model shown in Fig. 1. Based on hydropathy analysis, this model predicts 12 putative transmembrane segments (tms) and large intracellular termini. The NKCCs evolved from a presumed primitive cyanobacterial carrier and have maintained high levels of conservation in amino acid sequence. For example, the shark carrier (sNKCC1) is 74% identical to the human carrier (hNKCC1). Similarly, the NKCCs of several teleostean species are highly homologous to those of terrestrial vertebrates (P. Isenring and B. Forbush, unpublished results).
All of the NKCCs described to date are inhibited by loop diuretics
through binding of the drug at a site that is presumably extracellular
(8, 20, 23). Modifying agents such as
N-mustard and
Hg2+ may also block NKCC-mediated
salt transport by direct interaction with the substrates (11, 16, 22,
38). For example, the diuretic effect of
Hg2+ in higher vertebrates could
result from decreased fluid reabsorption in the thick ascending loop of
Henle (4), a
Cl-transporting epithelium
expressing NKCC activity (19, 25, 31).
In the epithelial tissues of many terrestrial species, the NKCC represents one of the major pathways for ion movement across the cell membrane (10, 17, 18, 32). The NKCC1 is also expressed abundantly in the epithelial tissues of aquatic vertebrates; it comprises 2% of the total protein content of the Squalus acanthias rectal gland (36, 42). In this tissue and in the gills of other marine species, the carrier promotes salt and fluid movement from the extracellular milieu to freshwater or seawater (2, 35, 36), thereby contributing to the fish hydromineral balance.
Mercury in both organic and inorganic forms exerts a wide range of toxic effects (13). In humans, acute exposure to inorganic mercurial salts (Hg2+) results in preponderant epithelial toxicity producing corrosive ulceration of the gastrointestinal mucosa and acute renal tubular necrosis (5, 28). Similarly, the respiratory epithelial cells of fish species can be damaged by acute exposure to 0.1- to 0.5-ppm mercuric chloride solutions (1, 30, 33, 34).
The mechanism of Hg2+ toxicity
remains unclear. Hg2+ alters
protein function by interacting with cysteinyl sulfhydryls
(SH). Although the NKCCs
contain conserved cysteine residues (19, 22, 31, 32, 42), it is unknown
whether mercurial salts target these carriers. Silva et al. (38) showed
that fluid secretion in the S. acanthias rectal gland, which depends on NKCC1 activity (7), was repressed by Hg2+ with an
IC50 of ~25 µM. However,
bumetanide-sensitive secretion in this tissue is also influenced by
other transport systems including the
Na+ pump (39),
K+ channels (37), and
Cl
channels (15). The
Na+ pump, in particular, is a
well-documented Hg2+ target
(41).
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In this study, we used a heterologous expression system to determine the effect of Hg2+ on NKCC1s. We provide strong evidence that Hg2+ inhibits the activities of sNKCC1 and hNKCC1. Molecular targets within the transporter sequence were further defined by analyzing the Hg2+ sensitivity of mutant and chimeric NKCC1s. We find that a cysteine residue in transmembrane domain 11 (tm11) plays an important role in Hg2+ binding and that other residues are also involved. These binding sites are localized in the transmembrane segments of the carrier or in the COOH terminus.
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MATERIALS AND METHODS |
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Nomenclature Used to Designate Mutant NKCCs
As previously described (20-23), we use single letters in lowercase to designate domains from an NKCC species (h = human; s = shark) that are included in a chimera. A number between 0.x and 13.x indicates the position of the chimeric junction in the NH2 terminus (0.x), the 12 putative transmembrane domains (1.x to 12.x), or the COOH terminus (13.x). Each domain is further divided into 10 subdomains from x.0 to x.9. In the chimera hsh10.5/13.1, for example, residues between the start codon and the middle region of tm10 are from hNKCC1, residues between this region (junction 10.5) and the beginning of the COOH terminus (junction 13.1) are from sNKCC1, and residues in the remainder of the sequence are from hNKCC1.All the point mutations described here are in sNKCC1 and consist of
cysteineglycine or cysteine
leucine substitutions. A number in parentheses indicates the site of the mutations following the
nomenclature above. In Cys
Leu (11.8), for example, a leucine residue replaces a cysteine residue in the distal region of tm11.
Models of the chimeras used in these studies and sites of the residue substitutions are shown in Fig. 1.
Vectors and cDNAs
The wild-type constructs sNKCC1/ptz18U, sNKCC1/pJB20M, and hNKCC1/pJB20M were as described in Refs. 20-23. Mutations in the shark cDNA were created with the sNKCC1/ptz18U construct as the template. For the expression of wild-type and mutant NKCC1s in HEK-293 cells, we used the vector pJB20M (20), which contains the strong cytomegalovirus promoter and a G418 resistance cassette.The chimera hsh10.5/13.1 was as described in Ref. 22. Briefly, human and shark fragments were exchanged at an Nde I restriction site (bp 2410 in sNKCC1NDE and 2218 in hNKCC1) and at a Pml I site (bp 2703 in sNKCC1 and 2511 in hNKCC1). The Nde I junction point (10.5) corresponds to the junction between Tyr-686 and Ala-687 (Tyr-686/Ala-687) in hNKCC1 and to Tyr-659/Ala-660 in sNKCC1, and the Pml I junction point (13.1) corresponds to His-756/Val-757 in sNKCC1 and His-783/Val-784 in hNKCC1. The chimera hsh0.9/13.1 was as in Ref. 20. Here, human and shark fragments were exchanged at a Bst1 107I restriction site (bp 1175 in sNKCC1BST and 986 in hNKCC1) and at a Pml I site (as described above). The Bst1 107I junction point (0.9) corresponds to His-247/Val-248 in sNKCC1 and to Tyr-275/Thr-276 in hNKCC1.
Mutations were generated by the Kunkel method as previously
described (20). Replacement of cysteine residues in the
N-glycosylated loop (residues 515, 536, 541, 550, and 555) and in tm11 (residue 696) of sNKCC1 resulted
in cotransporters with
86Rb+
activity slightly below background. The substitution CysLeu (11.8) at position 697 resulted in a functional transporter. The oligonucleotide used to generate this mutant was CT ATC TTG TGC TTG GGA
GTG ATG TTT GTC ATT AAT TGG TGG GCA G. The transfer of the
Cys
Leu (11.8) mutation to
sNKCC1/pJB20M was done by
exchanging BspE
I-Pml I fragments (bp 1579 to 2703).
Cell Lines
Lines expressing hNKCC1, sNKCC1, hsh0.9/13.1, hsh10.5/13.1, and pJB20M were the same as those previously described (20, 22). The point mutations were transfected into HEK-293 cells by calcium phosphate precipitation, and stable lines were isolated by G418 resistance (20). Western blotting showed that sNKCC1-, sNKCC1-, hsh0.9/13.1-, hsh10.5/13.1-, and CysFlux Studies
As in previous experiments (20), the HEK-293 cells were subcultured onto 96-well plates precoated with polylysine and grown to confluence. Ion transport rates were determined by 86Rb+ influx measurements. All experiments were done at room temperature, and the solutions were at pH 7.4. The ion concentrations of different incubating media were adjusted by replacing cations with N-methylglucamine (NMG) and anions with gluconate.Before each flux assay, the cells were incubated in a hypotonic
low-Cl (163 mosM, 2 mM)
medium for 45-60 min to activate the cotransporter (42). To
examine the effects of mercuric chloride
(HgCl2), 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA), or 2-trimethylammonium ethyl
methanethiosulfonate bromide (MTSET) on transport activity and the
effects of dithiothreitol (DTT) or reduced glutathione (GSH) on
Hg2+-mediated changes, the cells were subjected to
additional preincubation periods of 5 min. Agents were diluted in an
isosmotic NMG-gluconate solution, and NaCl replaced
HgCl2 in control experiments. To
determine the time course of the
Hg2+ effect on NKCC-mediated
86Rb+
uptake, different preincubation periods (0.5-12 min each) were used.
Each influx lasted 1 min in (in mM) 135 NaCl, 5 RbCl (2 µCi/ml 86RbCl), 1 CaCl2, 1 MgCl2, 1 Na2HPO4, 1 Na2SO4, 15 Na+-HEPES, and 0.1 ouabain. To determine NKCC-specific influx, 250 µM bumetanide was added directly to the medium. 86Rb+ uptake was terminated by three rinses in ice-cold stop solution (135 mM potassium gluconate, 5 mM sodium gluconate, 250 µM bumetanide, and 0.1 mM ouabain). Cells were solubilized in 2% SDS and assayed for 86Rb+ by counting Cerenkov radiation.
As previously described (20-23), each time- or concentration-vs.-influx curve was generated from measurements carried out in single rows of 96-well plates. In a typical experiment, there were four to eight wells per row and two to six replicate rows. Counts from each row were normalized to the value of uninhibited flux and were expressed as means ± SE among all rows. Inhibitor constant (Ki) values and Hill numbers were determined by nonlinear least-squares analysis for each data row and were also expressed as means ± SE. When appropriate, differences between groups of variables were analyzed by Student's two-tail t-tests.
Because the 86Rb+ activity in the transfected cell lines is severalfold above that of the nontransfected HEK cells, we have not attempted to subtract the component of the bumetanide-sensitive background flux that may be contributed by endogenous cotransporters. As described previously (20), we noted that in cells expressing inactive NKCC mutants, the native HEK-293 cell cotransport rate was often greatly decreased. Similarly, we have not subtracted the Hg2+-sensitive bumetanide-insensitive background because it represents a minor fraction (<5%) of the absolute 86Rb+ flux rates after activation of the cell lines. In this system, nonspecific background was not found to affect NKCC-specific kinetic parameters substantially (results not shown).
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RESULTS |
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Effect of Hg2+ on Cotransport Activity
The effect of Hg2+ on cotransporter activity was first determined in sNKCC1-transfected HEK-293 cells. 86Rb+ influx was measured after incubating the cells for various time periods in an isosmotic NMG-gluconate solution containing HgCl2. This experiment is shown in Fig. 2A. A 5-min preincubation in 100 µM Hg2+ led to 65% inhibition of the 86Rb+ uptake by the sNKCC1-transfected cells. The half-life of the 86Rb+ activity vs. the time of preincubation in 100 µM Hg2+ was ~2 min, and the on rate constant for Hg2+ [Kon(Hg2+)] was ~100 M
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We also compared the inhibitory effect of Hg2+ (250 µM; 5-min incubation) on the sNKCC1 and the hNKCC1 expressed in HEK-293 cells with its effect on the mock-transfected HEK-293 cells. Under such conditions, over 80% of the sNKCC1-dependent 86Rb+ activity (Fig. 2B) and of the hNKCC1 activity (data not shown) is suppressed. The endogenous cotransporter of the HEK-293 cells is also sensitive to Hg2+. The results suggest that conserved residues or domains in the NKCC1 family mediate the inhibition by Hg2+.
To determine the kinetics of Hg2+
inhibition, NKCC activity was measured after 5-min exposures to an
increasing Hg2+ concentration
([Hg2+]). In these
experiments, the ligand was also diluted in an NMG-gluconate solution.
The relationships between
[Hg2+] and
86Rb+
transport by sNKCC1- and hNKCC1-transfected cells are illustrated in
Fig. 3A,
and kinetic data summarizing these relationships are presented in Fig.
3B. It is seen that
Hg2+ inhibits NKCC1 activity in a
concentration-dependent fashion. The normalized counts are best fitted
with a model of Hg2+ binding at a
single site for sNKCC1-transfected HEK-293 cells (Hill number ± SD = 1.3 ± 0.4) and binding at two sites for hNKCC1-transfected cells
(Hill number ± SD = 2.0 ± 0.7). Interestingly, the dependence of
86Rb+
influx on Hg2+ also translates
into inhibition constants that are significantly different among
species (Fig. 3B). The
Ki values for
Hg2+
[Ki(Hg2+)]
are 24.7 ± 1.9 µM for sNKCC1 and 43.1 ± 1.4 µM for
hNKCC1. Because the cell lines are identical to one
another except for the (over)expressed foreign NKCC1, these differences
in Hg2+ affinities are consistent
with a model of ligand binding directly at a cotransporter site.
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Molecular Targets
The human and the shark cotransporters contain 12 conserved cysteine residues, 3 of which are in the COOH terminus and 9 of which are in the central domain (Fig. 1A). Because most forms of mercury permeate cellular membranes and accumulate in cells (14, 25), several cysteinyl SHRole of tm11 in
Hg2+ binding.
As noted above, sNKCC1 has an apparent affinity for
Hg2+ that is twofold higher than
that of hNKCC1. Taking advantage of this difference, we measured
Hg2+ inhibition of
86Rb+
influx in a cell line expressing hsh0.9/13.1 (Fig.
1C) to determine whether the central
domain and/or the intracellular termini mediate the variant kinetic
behaviors. As illustrated in Fig.
4A, the response of the chimera hsh0.9/13.1 to 5-min preincubations with Hg2+ is very similar to that of
the shark transporter
[Ki(Hg2+) = 20.6 and 24.7 µM, respectively]. The data demonstrate
that residues within the central domain specify the kinetic
characteristics of the Hg2+-NKCC1
interaction and that the NH2 and
COOH termini have little if any role in determining the species'
variant behavior.
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Role of the extracellular domains compared with that of the
transmembranous and intracellular domains.
The CysLeu (11.8) substitution in sNKCC1 alters the sensitivity
to Hg2+ but does not prevent
inhibition of
86Rb+
influx, indicating that other
SH
binding sites are
involved. The extracellular cysteine residues, which in the NKCC1s are
confined to the N-glycosylated loop
(between tm7 and tm8), are the putative binding sites that are
theoretically among the most accessible. Single substitutions
(Cys
Gly) in this domain at all five locations (Fig. 1) resulted
in complete loss of cotransport function. Here, a mutational approach
was not useful in determining the role of these amino acids in
Hg2+ binding, because there may
have been carrier misfolding due to failure to form proper disulfide bonding.
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DISCUSSION |
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We have shown that Hg2+ inhibits ion transport by the NKCC1. The activities of different forms of the carriers (sNKCC1, hNKCC1, and endogenous NKCC) expressed in HEK-293 cells are partially suppressed at 6.25 µM Hg2+ and are completely inhibited at 250 µM. For sNKCC1, the Hg2+-vs.-cotransport activity curve translates into a Ki of ~25 µM. This value is similar to that reported by Silva et al. (38), who investigated the effect of Hg2+ on fluid secretion by the S. acanthias rectal gland. In this tissue, however, transepithelial water movement is not solely dependent on sNKCC1 (7, 15, 37, 39).
In our study, we have found that the effect of
Hg2+ on
86Rb+
influx by transfected HEK-293 cells is due to inhibition of cotransport activity and not to inhibition of an accessory pathway. For example, 86Rb+
uptake by these cells was measured in the presence of ouabain, indicating that the Hg2+ effect
was not mediated by a change in the activity of the
Na+ pump. Also, it is unlikely
that inhibition of Cl
channel function during Hg2+
incubation decreased cotransport activity, because intracellular Cl
was already reduced at
the time of the Hg2+ exposure due
to prior incubation in a
low-Cl
medium. Another
argument supporting a direct
NKCC1-Hg2+ interaction is based on
the finding of a single-residue mutation in the sNKCC1 tm11 that leads
to a twofold decrease in Hg2+
affinity without altering the kinetics of ion transport.
Finally, the relatively rapid onset of
Hg2+ inhibition
[Kon(Hg2+) = 100 M
1s
1],
which is well described by first-order binding kinetics, is also
consistent with the hypothesis that
Hg2+ acts on a cotransporter site
(41) and not on an accessory protein involved in turnover regulation;
in the latter case, activation or deactivation of the transport process
would presumably occur over a wider time range (6, 20).
We have previously noted that the shark transporter differs from the
human transporter in having Na+,
K+, and
Cl affinities that are
four- to sixfold lower (20-23). Here, in examining the effect of
Hg2+ on cotransporter function, we
find that sNKCC1 also differs from hNKCC1 in having higher
Hg2+ affinity. Thus
Km(ions) and
Ki(Hg2+) for each of the transporters are inversely related, a kinetic pattern
suggesting that the ions may be competitive or noncompetitive inhibitors of Hg2+ binding.
However, our results also show that
Hg2+ affinity and ion affinities
can change independently of one another. For example, the
Cys-697
Leu substitution in sNKCC1 and the human
shark
substitution of tms 10-12 in hNKCC1 do not affect the
Km(Na+), Km(Rb+),
and
Km(Cl
) of the parent transporters (21), but the substitutions do change the
Ki(Hg2+)
values twofold. Taken together, the data strongly suggest that the
differences in Hg2+ affinity among
species are not due to differences in ion affinities.
A significant finding of this report is the identification of a
putative Hg2+ binding site in the
predicted tm11 of the NKCC. Indeed, the CysLeu mutation at
position 697 of sNKCC1 results in a phenotype of
Hg2+ inhibition that is
indistinguishable from that of the hNKCC1. The importance of tm11 in
Hg2+ binding is also supported by
the similar behaviors of hsh10.5/13.1 and sNKCC1, which have the tms
10-12 of the shark sequence in common, whereas the remaining
proteins are from different cotransporter species.
Because the cysteine residues in tm11 are conserved (Cys-696 and
Cys-697 in sNKCC1 and Cys-723 and Cys-724 in hNKCC1), the difference
between sNKCC1 and hNKCC1 in Hg2+
affinity suggests that Cys-724 is less accessible in the hNKCC1 sequence. An alternative explanation is that the CysLeu (11.8) mutant has a disrupted conformation that alters the accessibility of a
remote cysteinyl SH
in the
sNKCC1 sequence. We consider this possibility less likely because the
Cys
Leu (11.8) mutant has a conservative single-residue substitution, exhibits the hallmark of a functional cotransporter, and
shares the ion transport characteristics of sNKCC1. Similarly, we have
previously shown that mutations in the tm11 of sNKCC1 had no effect on
ion binding parameters (21).
The CysLeu (11.8) substitution could have resulted in a subtle
intrahelix conformational change, affecting the accessibility of the
contiguous Cys-696 but not that of the other cysteine residues in tm1
and tm8 or in the COOH terminus. Because a Cys-696
Gly mutation
(as described above) and a Cys-696
Ser mutation (B. Forbush and
F. Dewaersegger, personal communication) resulted in
nonfunctional carriers, it was not possible to determine the importance
of Cys-696 in Hg2+ binding.
Nevertheless, the data presented here suggest that the tm11 region of
NKCC1 plays an essential role in
Hg2+ binding.
Various pertinent observations in this study suggest
Hg2+ binding at more than one site
on the NKCC. For example, the CysLeu substitution decreases
Hg2+ sensitivity but does not
prevent inhibition of ion transport. It is also apparent that the
effect of Hg2+ on hNKCC1- and
Cys
Leu (11.8)-mediated
86Rb+
influx is better characterized by fitting the Hill equation to represent a model of ligand binding at more than one site. Similarly, the sigmoidal dependence of
86Rb+
transport rates on another
SH
-reacting compound
(MTSEA) indicates a model in which a minimum of two binding sites are occupied.
We have determined that the additional
SH binding site(s) is
localized in the transmembrane segments or in the COOH termini of the
NKCC1s, and not in the extracellular domains. This topological assignment is based on the findings that impermeant thiol-reacting agents have little or no effect on
Hg2+ inhibition (GSH) or on
cotransporter function (MTSET). Furthermore, the initial rate of
Hg2+ inhibition that was deduced
from a time course of incubation in
Hg2+ (Fig.
2A) is in a lower range than would
be expected for interactions involving readily accessible
SH
groups, such as those in
extracellular domains (41). The data presented here do not concur with
a suggestion of Kinne-Safran and Kinne (26) that the action of
Hg2+ is extracellular.
An interesting issue raised in this study is whether the cysteine
residues involved in Hg2+
inhibition are accessible to modification by other
SH-reacting compounds. For
example, N-ethylmaleimide (NEM) is an alkylating reagent that is frequently used to elicit
K+-Cl
cotransport in the red blood cell (24), and it is also known to inhibit
Na+-K+-Cl
cotransport in secretory tissues (12). It has been suggested that these
actions of NEM on ion transport by the CCCs are mediated through
inactivation of a regulatory kinase (24). Based on our findings, an
alternative explanation is that the reagent exerts its effects by
interacting with key regulatory domains in the CCC proteins via
intracellular cysteine residues. These domains could harbor a conserved
Cl
-sensitive site that
modulates the transport activities of the NKCCs (3) and of the KCCs.
The NKCCs are abundantly expressed in various epithelia, where they
play key roles in transcellular salt and water movement (17, 18, 32).
Here, we have shown that Hg2+,
which is especially toxic to epithelial cells (1, 5, 13, 28, 30, 33,
34), affects the activity of the secretory cotransport proteins at
[Hg2+] of
~102 M. In humans, blood
Hg2+ concentration at this level
is usually associated with clinical toxicity (14), and, on the basis of
our studies with the HEK-293 expression system, the level could be
sufficiently high to alter NKCC1 activity in epithelial tissues.
Further studies are necessary to determine if inhibition of
Na+-K+-Cl
cotransport plays a role in
Hg2+-related toxicity.
In conclusion, the work presented here describes the effect of
Hg2+ on ion movement by the NKCC.
We have found that Hg2+ inhibits
the activities of different forms of the carrier by binding to
SH groups and that a
cysteine residue in tm11 is essential for the interaction. We have also
determined that additional
SH
groups, in the tms or in
the COOH termini of the NKCC1s, are involved in the inhibition.
Characterization of the Hg2+-NKCC
interaction may lead to interesting insight towards a better understanding of toxicological mechanisms related to mercurial compounds. It may also provide us with a very useful premise to study
structure-function relationships for the CCC family (29).
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the support of Dr. Bliss Forbush III (Department of Cellular and Molecular Physiology at the Yale University School of Medicine). We also thank Dr. John H. Grose, Rachel D. Behnke, Sue R. Brill, Deborah Lynn, Ignacio Giménez, and Dr. Andreas Flemmer for reading the manuscript.
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FOOTNOTES |
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This study was supported by the Medical Research Council of Canada (MRC) and the National Institutes of Health (Grants DK-47661 and ES-3828). P. Isenring is an MRC Clinician Scientist Scholar.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. Isenring, 10 rue McMahon (Room 3852), Québec, PQ, Canada G1R 2J6 (E-mail: paul.isenring{at}crhdq.ulaval.ca).
Received 30 March 1999; accepted in final form 17 June 1999.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Allen, P.
Distribution of mercury in the soft tissues of the blue tilapia Oreochromis aureus (Steindachner) after acute exposure to mercury chloride.
Bull. Environ. Contam. Toxicol.
53:
675-683,
1994[Medline].
2.
Avella, M.,
and
J. Ehrenfeld.
Fish gill respiratory cells in culture: a new model for Cl-secreting epithelia.
J. Membr. Biol.
156:
87-97,
1997[Medline].
3.
Breitwieser, G. E.,
A. A. Altamirano,
and
J. M. Russell.
Elevated [Cl]i and [Na+]i inhibit Na+, K+, Cl
cotransport by different mechanisms in squid giant axons.
J. Gen. Physiol.
107:
261-270,
1996[Abstract].
4.
Burg, M.,
and
N. Green.
Effect of mersalyl on the thick ascending limb of Henle's loop.
Kidney Int.
4:
245-251,
1973[Medline].
5.
Diamond, G. L.,
and
R. K. Zalups.
Understanding renal toxicity of heavy metals.
Toxicol. Pathol.
26:
92-103,
1998[Medline].
6.
Dunham, P. B.,
J. Klimczak,
and
P. J. Logue.
Swelling activation of K-Cl cotransport in LK sheep erythrocytes: a three-state process.
J. Gen. Physiol.
101:
733-765,
1993[Abstract].
7.
Forbush, B.,
M. Haas,
and
C. Y. Lytle.
Na+-K+-Cl cotransport in the shark rectal gland. I. Regulation in the intact perfused gland.
Am. J. Physiol.
262 (Cell Physiol. 31):
C1000-C1008,
1992
8.
Forbush, B.,
and
H. C. Palfrey.
[3H]bumetanide binding to membranes isolated from dog kidney outer medulla. Relationship to the Na+-K+-Cl co-transport system.
J. Biol. Chem.
258:
11787-11792,
1983
9.
Fujise, H.,
K. Abe,
M. Kamimura,
and
H. Ochiai.
K+-Cl cotransport and volume regulation in the light and the dense fraction of high-K+ dog red blood cells.
Am. J. Physiol.
273 (Regulatory Integrative Comp. Physiol. 42):
R991-R998,
1997
10.
Gagnon, F.,
S. N. Orlov,
J. Tremblay,
and
P. Hamet.
Complete inhibition of Na+-K+-Cl cotransport in Madin-Darby canine kidney cells by PMA-sensitive protein kinase.
Biochim. Biophys. Acta
1369:
233-239,
1998[Medline].
11.
Garay, R. P.,
J. P. Labaune,
D. Mesangeau,
C. Nazaret,
T. Imbert,
and
G. Moinet.
CRE 10904 [2-(p-fluorophenoxy),1-(o-hydroxyphenyl)-ethane]: a new diuretic and anti-hypertensive drug acting by in vivo sulfation.
J. Pharmacol. Exp. Ther.
255:
415-422,
1990[Abstract].
12.
George, J. N.,
and
R. J. Turner.
Inactivation of the rabbit parotid Na/K/Cl cotransporter by N-ethylmaleimide.
J. Membr. Biol.
112:
51-58,
1989[Medline].
13.
Gerstner, H. B.,
and
J. E. Huff.
Clinical toxicology of mercury.
J. Toxicol. Environ. Health
2:
491-526,
1977[Medline].
14.
Graef, J.
Mercury, parts I and II.
Clin. Toxicol. Rev.
2:
7-8,
1980.
15.
Greger, R.,
E. Schlatter,
and
H. Gogelein.
Chloride channels in the luminal membrane of the rectal gland of the dogfish (Squalus acanthias). Properties of the "larger" conductance channel.
Pflügers Arch.
409:
114-121,
1987[Medline].
16.
Grunicke, H.,
W. Doppler,
J. Hofmann,
H. Lindner,
K. Maly,
H. Oberhuber,
H. Ringsdorf,
and
J. J. Roberts.
Plasma membrane as target of alkylating agents.
Adv. Enzyme Regul.
24:
247-261,
1985[Medline].
17.
Haas, M.
Properties and diversity of Na+-K+-Cl cotransporters.
Annu. Rev. Physiol.
51:
443-457,
1989[Medline].
18.
Haas, M.
The Na+-K+-Cl cotransporters.
Am. J. Physiol.
267 (Cell Physiol. 36):
C869-C885,
1994
19.
Igarashi, P.,
G. B. Vanden Heuvel,
J. A. Payne,
and
B. Forbush.
Cloning, embryonic expression, and alternative splicing of a murine kidney-specific Na+-K+-Cl cotransporter.
Am. J. Physiol.
269 (Renal Fluid Electrolyte Physiol. 38):
F405-F418,
1995
20.
Isenring, P.,
and
B. Forbush.
Ion and bumetanide binding by the Na+-K+-Cl cotransporter: importance of transmembrane domains.
J. Biol. Chem.
272:
24556-24562,
1997
21.
Isenring, P.,
S. C. Jacoby,
J. Chang,
and
B. Forbush.
Mutagenic mapping of the Na+-K+-Cl cotransporter for domains involved in ion transport and bumetanide binding.
J. Gen. Physiol.
112:
549-558,
1998
22.
Isenring, P.,
S. C. Jacoby,
and
B. Forbush.
Characterization of the renal absorptive Na+-K+-Cl (NKCC2): comparative studies including hNKCC1, sNKCC1 and the HEK-293 NKCC1.
J. Biol. Chem.
273:
11295-11301,
1998
23.
Isenring, P.,
S. C. Jacoby,
and
B. Forbush.
The importance of transmembrane domain 2 in cation transport by the Na+-K+-Cl transporter.
Proc. Natl. Acad. Sci. USA
95:
7179-7184,
1998
24.
Jennings, M. L.,
and
R. K. Schulz.
Okadaic acid inhibition of KCl cotransport. Evidence that protein dephosphorylation is necessary for activation of transport by either cell swelling or N-ethylmaleimide.
J. Gen. Physiol.
97:
799-817,
1991[Abstract].
25.
Kaplan, M. R.,
M. D. Plotkin,
W. S. Lee,
Z. C. Xu,
J. Lytton,
and
S. C. Hebert.
Apical localization of the Na+-K+-Cl cotransporter, rBSC1, on rat thick ascending limbs.
Kidney Int.
49:
40-47,
1996[Medline].
26.
Kinne-Safran, E.,
and
R. K. H. Kinne.
Sidedness and reversibility of mercury inhibition of Na+-K+-Cl cotransport in intact T84 cells, a human colon cancer cell line.
Bull. Mount Desert Island. Biol. Lab.
36:
19-20,
1997.
27.
Kone, B. C.,
R. M. Brenner,
and
S. R. Gullans.
Sulfhydryl-reactive heavy metals increase cell membrane K+ and Ca2+ transport in renal proximal tubule.
J. Membr. Biol.
113:
1-12,
1990[Medline].
28.
Lin, J. L.,
and
P. S. Lim.
Massive oral ingestion of elemental mercury.
J. Toxicol. Clin. Toxicol.
31:
487-492,
1993[Medline].
29.
Mueckler, M.,
and
C. Makepeace.
Transmembrane segment 5 of the Glut1 glucose transporter is an amphipathic helix that forms part of the sugar permeation pathway.
J. Biol. Chem.
274:
10923-10926,
1999
30.
Naidu, K. A.,
K. A. Naidu,
and
R. Ramamurthi.
Histological observations in gills of the teleost Sarotherodon mossambicus with reference to mercury toxicity.
Ecotoxicol. Environ. Saf.
7:
455-462,
1983[Medline].
31.
Payne, J. A.,
and
B. Forbush.
Alternatively spliced isoforms of the putative renal Na+-K+-Cl cotransporter are differentially distributed within the rabbit kidney.
Proc. Natl. Acad. Sci. USA
91:
4544-4548,
1994[Abstract].
32.
Payne, J. A.,
J. C. Xu,
M. Haas,
C. Y. Lytle,
D. Ward,
and
B. Forbush.
Primary structure, functional expression, and chromosomal localization of the bumetanide-sensitive Na+-K+-Cl cotransporter.
J. Biol. Chem.
270:
17977-17985,
1995
33.
Prasad, M. S.
Effect of short-term exposure to mercuric chloride on the air-breathing catfish, Heteropneustes fossilis. II. Scanning electron microscopic study of the gill.
Biomed. Environ. Sci.
7:
337-345,
1994[Medline].
34.
Prasad, M. S.
Effect of short-term exposure to mercuric chloride on the air-breathing catfish, Heteropneustes fossilis. I. Light microscopic study of the gill.
Biomed. Environ. Sci.
7:
327-336,
1994[Medline].
35.
Riestenpatt, S.,
H. Onken,
and
D. Siebers.
Active absorption of Na+ and Cl across the gill epithelium of the shore crab Carcinus maenas: voltage-clamp and ion-flux studies.
J. Exp. Biol.
199:
1545-1554,
1996
36.
Riordan, J. R.,
B. Forbush,
and
J. W. Hanrahan.
The molecular basis of chloride transport in shark rectal gland.
J. Exp. Biol.
196:
405-418,
1994
37.
Silva, P.,
J. A. Epstein,
M. A. Myers,
A. Stevens,
P. Silva,
and
F. H. Epstein.
Inhibition of chloride secretion by BaCl2 in the rectal gland of the spiny dogfish, Squalus acanthias.
Life Sci.
38:
547-552,
1986[Medline].
38.
Silva, P.,
F. H. Epstein,
and
R. J. Solomon.
The effect of mercury on chloride secretion in the shark (Squalus acanthias) rectal gland.
Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol.
103:
569-575,
1992.
39.
Silva, P.,
J. Stoff,
M. Field,
L. Fine,
J. N. Forrest,
and
F. H. Epstein.
Mechanism of active chloride secretion by shark rectal gland: role of Na+-K+-ATPase in chloride transport.
Am. J. Physiol.
233 (Renal Fluid Electrolyte Physiol. 2):
F298-F306,
1977[Medline].
40.
Sine, S. M.
Identification of equivalent residues in the gamma, delta, and epsilon subunits of the nicotinic receptor that contribute to alpha-bungarotoxin binding.
J. Biol. Chem.
272:
23521-23527,
1997
41.
Wang, X.,
and
J. D. Horisberger.
Mercury binding site on Na+/K+-ATPase: a cysteine in the first transmembrane segment.
Mol. Pharmacol.
50:
687-691,
1996[Abstract].
42.
Xu, J. C.,
C. Lytle,
T. T. Zhu,
J. A. Payne,
E. Benz,
and
B. Forbush.
Molecular cloning and functional expression of the bumetanide-sensitive Na+-K+-Cl cotransporter.
Proc. Natl. Acad. Sci. USA
91:
2201-2205,
1994[Abstract].