Inhibition of Na+-K+-2Clminus cotransport by mercury

Steven C. Jacoby1, Edith Gagnon2, Luc Caron2, John Chang1, and Paul Isenring2

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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-697right-arrowLeu) 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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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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Fig. 1.   Models of Squalus acanthias Na+-K+-Cl- cotransporter type 1 (sNKCC1), of 2 chimeras, and of 8 mutations based on proposed structure (42). A: cysteine residues (black dots) as they occur in sNKCC1 sequence. B: magnification of central sNKCC1 domain showing locations of cysteine residues. Circled minus signs, sites of substitution (in all cases, Cysright-arrowGly vs. Leu). C and D: chimeras hsh0.9/13.1 and hsh10.5/13.1, respectively. Dark symbols, human residues; light symbols, shark residues.

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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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 cysteineright-arrowglycine or cysteineright-arrowleucine substitutions. A number in parentheses indicates the site of the mutations following the nomenclature above. In Cysright-arrowLeu (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 Cysright-arrowLeu (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 Cysright-arrowLeu (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 Cysright-arrowLeu (11.8) transfected cells expressed comparable quantities of carriers (results not shown). The levels of functional expression were also comparable, about five- to sixfold above the endogenous cotransport activity of mock-transfected HEK-293 cells.

Flux 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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INTRODUCTION
MATERIALS AND METHODS
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-1 · s-1 (first-order kinetics). In this cell line, most of the 86Rb+ flux is bumetanide sensitive (20) and is severalfold above the endogenous cotransport activity of HEK-293 cells.


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Fig. 2.   A: Hg2+ inhibition time course of 86Rb+ influx in sNKCC1-transfected HEK-293 cells. Cells were preincubated with 100 µM Hg2+ (see MATERIALS AND METHODS) for 0.5, 1, 2, 5, and 12 min and assayed for 86Rb+ uptake in regular flux medium. In each experiment (1-4 per time point), individual uptakes were normalized to value of uninhibited flux and averaged (n = 3-19). B: effect of 250 µM Hg2+ on cotransporter activity for sNKCC1-transfected HEK-293 cells and for untransfected HEK-293 cells. Bars, means ± SE of 19-22 determinations from 4-5 experiments.

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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Fig. 3.   A: Hg2+ inhibition curves of 86Rb+ influx in human NKCC1- (hNKCC1) and sNKCC1-transfected HEK-293 cells. After preincubation in 0, 6.25, 12.5, 37.5, 62.5, 100, and 250 µM Hg2+, cells were assayed for 86Rb+ influx in a regular flux medium. In each experiment (n = 6-8), individual flux rows were normalized to counts measured at 0 µM Hg2+. These normalized counts were averaged (27-39 flux rows) and fitted by using the Michaelis-Menten equation and Hill coefficient. B: inhibitor constant (Ki) values for Hg2+ and Hill coefficients derived from data in A. Data are shown as averages of individual flux rows in all experiments.

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 SH- in either the extracellular, transmembranous, or intracellular domains of NKCC1 could interact with Hg2+. To determine which of these residues or domains are involved in this interaction, different approaches were used.

Role 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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Fig. 4.   Hg2+ inhibition curves of 86Rb+ influx in hsh0.9/13.1-, hsh10.5/13.1-, and Cysright-arrowLeu (11.8)-transfected HEK-293 cells. Methods are similar to those used for Fig. 3. Data are from 7-24 flux rows in 2-5 experiments.

To next determine the contribution of tms 10-12 relative to that of tms 1-9 to Hg2+ binding by the NKCC1 protein, we examined the human-shark chimera hsh10.5/13.1 (Fig. 1D). This chimera is identical to hNKCC1 in amino acid sequence except for having tms 10-12 replaced by the corresponding shark tms. For this construct, the [Hg2+]-vs.-86Rb+ activity curve (Fig. 4B) yields a Ki(Hg2+) that is also identical to that of the wild-type sNKCC1. Thus residues included in the distal portion of the central domain determine the difference between sNKCC1 and hNKCC1 in apparent Hg2+ binding.

Within the tm10-tm12 region, the secretory NKCCs harbor a conserved pair of contiguous cysteine residues (Cys-696 and Cys-697 in sNKCC1 and Cys-723 and Cys-724 in hNKCC1). These residues are localized in the putative outer portion of tm11. Presumably, Cys-696 and Cys-723 and/or Cys-697 and Cys-724 are involved in Hg2+ binding, but these residues may be less accessible in hNKCC1, as suggested by the higher sensitivity of sNKCC1 to Hg2+ inhibition.

To test these hypotheses, the two cysteine residues in the tm11 of sNKCC1 were mutated, either individually (Cysright-arrowGly at position 696 and Cysright-arrowLeu at position 697) or as a pair. The Cysright-arrowGly (11.8) and Cys-Cysright-arrowGly-Leu (11.8) mutants were nonfunctional, indicating that Cys-696 may play an important structural role through disulfide bonding. The effect of [Hg2+] on 86Rb+ influx by the sNKCC1 Cysright-arrowLeu (11.8) mutant is shown in Fig. 4C. Interestingly, this tm11 substitution changes the Ki(Hg2+) and the (best) Hill coefficient to values that are similar to those reported above for hNKCC1. The substitution, however, has no effect on the kinetics of cation and anion transport; the Michaelis-Menten constants for Na+, Rb+, and Cl- [Km(Na+), Km(Rb+), and Km(Cl-), respectively] for the Cys-697right-arrowLeu mutation (results not shown) are similar to those previously reported for sNKCC1 (9, 11, 15, 34). Thus replacement of a single cysteine residue (Cys-697) in the shark sequence results in a phenotype of Hg2+ inhibition that is indistinguishable from that for hNKCC1. This finding is consistent with the hypothesis that one of the cysteine residues in tm11 is a binding site for Hg2+ and that this site is more accessible in sNKCC1.

Role of the extracellular domains compared with that of the transmembranous and intracellular domains. The Cysright-arrowLeu (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 (Cysright-arrowGly) 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.

To circumvent this problem, we took advantage of differences in the liposolubilities of two reducing agents, DTT and GSH (see Ref. 27), and determined their effects on Hg2+ inhibition. Hypothetically, the less liposoluble of the two reducing agents, GSH, should have limited access to SH- radicals that are localized in the hydrophobic environment of the lipid bilayer or that are in the cell. Accordingly, GSH should only reverse the Hg2+ effect if extracellular cysteinyl SH- groups are available.

The effect of DTT on Hg2+ inhibition is shown in Fig. 5, A and B, in which the sNKCC1-transfected cells are used for illustration. In these experiments, three 5-min incubations in NMG-gluconate were used (first, with or without DTT; second, with Hg2+ or NaCl; third, with or without DTT). It is seen that the Hg2+ effect is partially reversed when DTT is added to the cells either before or after the Hg2+ preincubation. In control experiments, three incubations in NMG-gluconate without DTT (without DTT, with NaCl, and then without DTT) or with DTT (with DTT, with NaCl, and then with DTT) had no effect on sNKCC1-mediated 86Rb+ activity (results not shown). Thus the data indicate that DTT can prevent the formation of Hg2+-SH- bonds by directly interacting with SH- radicals in the cotransporter sequence.


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Fig. 5.   Effect of dithiothreitol (DTT) and GSH on Hg2+ inhibition of 86Rb+ influx in sNKCC1-transfected HEK-293 cells. Flux procedures are described in materials and methods. HEK-293 cells were treated with reducing agents before (A and C) or after (B and D) incubation with Hg2+. Data were processed as for Fig. 3 and are from 3-6 flux rows in 1-2 experiments.

In similar experiments using the impermeant GSH agent at a concentration of 1 mM (Fig. 5, C and D), the effect of Hg2+ on cotransporter activity is not modified, suggesting that reactive SH- groups are poorly available at extracellular sites. Thus the Cysright-arrowLeu (11.8) mutant retained partial Hg2+ sensitivity because of additional Hg2+-SH- interactions, probably occurring in the lipid bilayer of the sNKCC1 (Cys-267, Cys-603, or Cys-696) or in intracellular domains.

The above experiment, in which we studied the effect of GSH or DTT on cotransporter inhibition by Hg2+, represents an indirect method of assessing the availability of extracellular cysteinyl SH-. Differences in the effects of GSH and DTT could also be due to differences in the accessibility of extracellular sites to the reducing agents. To identify available SH- groups by alternative means, we used the compounds MTSEA and MTSET, which are known to form mixed disulfides with cysteinyl SH- (40). MTSEA (C3H9NO2S2 · HBr) diffuses freely across cellular membranes, whereas MTSET (C6H16NO2S2 · Br) is an impermeant (charged) thiol-reacting compound. The effects of both compounds on 86Rb+ influx in the sNKCC1-, hNKCC1-, and Leuright-arrowCys (11.8)-transfected HEK-293 cells were determined. The procedures and conditions were similar to those used for assessing the Hg2+ effect on cotransporter activity, that is, a 5-min preincubation with MTSEA or MTSET diluted in a NMG-gluconate solution, followed by a 1-min incubation with the 86Rb+ tracer.

As illustrated in Fig. 6, A-C, MTSET has a very weak inhibitory effect on the activity of both mutant and wild-type transporters, confirming that extracellular SH- groups are unavailable to form mixed disulfides with thiol-reacting compounds. The permeant agent MTSEA, on the other hand, has a pronounced inhibitory effect on 86Rb+ influx. In addition, the activity-vs.-ligand concentration curves show a clear sigmoidal progression with Hill coefficients approx  3 (Fig. 6). These results further suggest that permeant SH--reacting agents bind to at least two sites localized in the transmembrane segments or in the intracellular domains of NKCC1.


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Fig. 6.   2-Aminoethyl methanethiosulfonate hydrobromide (MTSEA) and 2-trimethylammonium ethyl methanethiosulfonate bromide (MTSET) inhibition curves of 86Rb+ influx in sNKCC1-, hNKCC1-, and Cysright-arrowLeu (11.8)-transfected HEK-293 cells. After preincubation in 0.0125, 0.75, 1.25, 2, 2.5, 5, and 10 µM MTSEA or in 0, 10, and 25 µM MTSET, cells were assayed for 86Rb+ influx in a regular flux medium. Data for MTSEA (3-11 flux rows in 1-3 experiments) were fitted with the Michaelis-Menten equation and Hill coefficient. Data for MTSET are from 6-18 flux rows in 3-4 experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-697right-arrowLeu substitution in sNKCC1 and the human right-arrowshark 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 Cysright-arrowLeu 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 Cysright-arrowLeu (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 Cysright-arrowLeu (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 Cysright-arrowLeu (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-696right-arrowGly mutation (as described above) and a Cys-696right-arrowSer 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 Cysright-arrowLeu substitution decreases Hg2+ sensitivity but does not prevent inhibition of ion transport. It is also apparent that the effect of Hg2+ on hNKCC1- and Cysright-arrowLeu (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 ~10-2 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).


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 277(4):C684-C692
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