doi:10.1152/ajprenal.00412.2010 299:1111-1119, 2010. First published Aug 18, 2010;Am J Physiol Renal Physiol

Cariño-Cortés, Erika Moreno, Norma A. Bobadilla and Gerardo Gamba Hernández, Erika Rodríguez-Lobato, Diana Pacheco-Alvarez, Raquel María Castañeda-Bueno, Norma Vázquez, Ismael Bustos-Jaimes, Damian

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A single residue in transmembrane domain 11 defines the different affinityfor thiazides between the mammalian and flounder NaCl transporters

María Castañeda-Bueno,1 Norma Vázquez,1 Ismael Bustos-Jaimes,2 Damian Hernández,1

Erika Rodríguez-Lobato,1 Diana Pacheco-Alvarez,3 Raquel Cariño-Cortés,4 Erika Moreno,4

Norma A. Bobadilla,1 and Gerardo Gamba1

1Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Instituto Nacional deCardiología Ignacio Chávez, and Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México;2Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Coyoacan; 3Escuela deMedicina, Universidad Panamericana, Mexico City; and 4Instituto de Ciencias de la Salud, Universidad Autónoma delEstado de Hidalgo, Pachuca, Hidalgo, México

Submitted 13 July 2010; accepted in final form 17 August 2010

Castañeda-Bueno M, Vázquez N, Bustos-Jaimes I, HernándezD, Rodríguez-Lobato E, Pacheco-Alvarez D, Cariño-Cortés R,Moreno E, Bobadilla NA, Gamba G. A single residue in transmem-brane domain 11 defines the different affinity for thiazides betweenthe mammalian and flounder NaCl transporters. Am J Physiol Endo-crinol Metab 299: F1111–F1119, 2010. First published August 18,2010; doi:10.1152/ajprenal.00412.2010.—Little is known about theresidues that control the binding and affinity of thiazide-type diureticsfor their protein target, the renal Na�-Cl� cotransporter (NCC).Previous studies from our group have shown that affinity for thiazidesis higher in rat (rNCC) than in flounder (flNCC) and that the trans-membrane region (TM) 8–12 contains the residues that produce thisdifference. Here, an alignment analysis of TM 8–12 revealed thatthere are only six nonconservative variations between flNCC andmammalian NCC. Two are located in TM9, three in TM11, and onein TM12. We used site-directed mutagenesis to generate rNCC con-taining flNCC residues, and thiazide affinity was assessed usingXenopus laevis oocytes. Wild-type or mutant NCC activity wasmeasured using 22Na� uptake in the presence of increasing concen-trations of metolazone. Mutations in TM11 conferred rNCC anflNCC-like affinity, which was caused mostly by the substitution of asingle residue, S575C. Supporting this observation, the substitutionC576S conferred to flNCC an rNCC-like affinity. Interestingly, theS575C mutation also rendered rNCC more active. Substitution ofS575 in rNCC for other residues, such as alanine, aspartate, andlysine, did not alter metolazone affinity, suggesting that reducedaffinity in flNCC is due specifically to the presence of a cysteine. Weconclude that the difference in metolazone affinity between rat andflounder NCC is caused mainly by a single residue and that thisposition in the protein is important for determining its functionalproperties.

diuretics; distal convoluted tubule; salt transport

THE RENAL Na�-Cl

� COTRANSPORTER (NCC) is the major transportprotein that is expressed in the apical membrane of the distalconvoluted tubule, which is located beyond the macula densa.The macula densa is where intratubular fluid chloride concen-tration is sensed to adjust the glomerular filtration rate by thetubuloglomerular feedback mechanism. Thus, salt handling byNCC escapes this regulatory mechanism that affects the uri-nary salt excretion and thereby the mean circulatory fillingpressure (19). Inactivating mutations of NCC in Gitelman’s

disease or increased activity of NCC due to dysregulation ofthe cotransporter by the mutant with no lysine kinases (WNK1or WNK4) in Gordon’s syndrome lead to arterial blood pres-sure decreases or increases, respectively, demonstrating theimportance of NCC activity in blood pressure regulation (8).This role of NCC has been hypothesized for many yearsbecause this cotransporter is the target of the thiazide-typediuretics that were introduced into clinical medicine in 1957(17). Fifty years later, thiazide diuretics are still recommendedby the Joint National Committee for the Prevention, Detection,Evaluation, and Treatment of High Blood Pressure as the firstline pharmacological therapy for patients with arterial hyper-tension (3). Consequently, thiazides are one of the most fre-quently prescribed drugs in the world. Little is known, how-ever, about the residues or domains that control the kineticproperties or specificity for thiazide binding to NCC.

NCC is a protein of 1,002 to 1,028 amino acid residuescomposed of a central hydrophobic domain that contains 12putative transmembrane-spanning segments [transmembraneregions (TMs) 1–12]. These segments are interconnected bysix extracellular and five intracellular hydrophilic loops. Thelonger interconnecting segment between TMs 7 and 8 isglycosylated (12) and thus faces the extracellular side. Thecentral hydrophobic domain is flanked by a short amino-terminal domain and a long carboxyl-terminal hydrophilicdomain, presumably located within the cell (6) (Fig. 1). Tovar-Palacio et al. (21) determined that the critical residues definingthe specificity for thiazide inhibition reside within the centralhydrophobic domain by studying a chimeric protein. Thechimeric protein contained the transmembrane segments ofNCC flanked by the hydrophilic loops of the renal Na�-K�-2Cl� cotransporter (NKCC), which is sensitive to loop diuret-ics and not to thiazides and behaves in a similar fashion asNCC.

The functional properties of rNCCs and flNCCs, such as iontransport kinetics and sensitivity to thiazide-type diuretics, aresignificantly different; the rNCC exhibits higher affinity forions and diuretics (15, 22). These differences were exploitedrecently in a study in which multiple chimeric proteins betweenrNCCs and flNCCs were produced and analyzed at the func-tional level (16). In that study, it was observed that thedifference in thiazide affinity between cotransporters fromdifferent species was conferred by the region containing theTM8 to TM12 segments because a chimeric protein in whichthis segment from flounder was inserted into the rNCC exhib-

Address for reprint requests and other correspondence: G. Gamba, Molec-ular Physiology Unit, Vasco de Quiroga No. 15, Tlalpan 14000, Mexico City,Mexico (e-mail: gamba@biomedicas.unam.mx).

Am J Physiol Renal Physiol 299: F1111–F1119, 2010.First published August 18, 2010; doi:10.1152/ajprenal.00412.2010.

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ited a flounder-like affinity for metolazone and vice versa.Thus, it was proposed that the affinity-defining residues forthiazides are located within the TM8 to TM12 segments of thecotransporter.

The major goal of the present study was to determine thespecific amino acid residues within the TM8 to TM12 seg-ments of rat NCC (rNCC) that are responsible for the differentaffinity for thiazide between the mammalian and flounderorthologues. To do this, we constructed several mutant NCCsand changed key residues within specific TM segments. Thefunctional properties of the resulting proteins were determinedby functional expression in Xenopus laevis oocytes. Our resultsshow that a single amino acid residue, the serine at position575 of the rat NCC, corresponding to a cysteine at position 576in flounder NCC (flNCC), explains the difference in thiazideaffinity between the mammalian and flounder cotransporters.

METHODS

Xenopus laevis oocyte preparation. Oocytes were harvested surgi-cally from adult female Xenopus laevis frogs (Nasco) under 0.17%tricaine anesthesia and incubated in ND96 (in mM: 96 NaCl, 2 KCl,1.8 CaCl2, 1 MgCl, and 5 HEPES-Tris, pH 7.4) in the presence ofcollagenase B (2 mg/ml) for 1 h. After four washes in ND96, theoocytes were manually defolliculated and incubated at 18°C in ND96supplemented with 2.5 mM sodium pyruvate and 5 mg/100 mlgentamicin overnight. The next day, stage V-VI oocytes (5) wereinjected with 50 nl of water or 20 ng of cRNA/oocyte. Then, the oocyteswere incubated for 2 or 3 days in ND96 with sodium pyruvate andgentamicin, which were changed every 24 h. Two hours before theuptake experiments were performed, oocytes were incubated in Cl�-freeND96 (96 mM Na� isethionate, 2 mM K� gluconate, 1.8 mM Ca2�

gluconate, 1.0 mM Mg2� gluconate, 5 mM HEPES, 2.5 mM sodiumpyruvate, and 5 mg/100 ml gentamicin, pH 7.4) (7).

Site-directed mutagenesis. The NCC cDNAs used in this studywere the rat and flounder orthologs that we previously isolated fromrat kidney (rNCC) (6) and flounder urinary bladder (flNCC) (7). ThesecDNAs were previously engineered to contain unique silent restrictionsites for SacII and HpaI at the beginning of the TM8 segment and atthe end of the TM12 segment, respectively (16). Single, double, ortriple mutants for TMs 9, 11, and 12 were constructed for thesecDNAs using the Quickchange site-directed mutagenesis system(Stratagene), following the manufacturer’s recommendations. All mu-tations were confirmed by automatic DNA sequencing from TM8 toTM12 to avoid unwanted mutations elsewhere, and the SacII to HpaIfragment from each mutant was subcloned into wild-type NCC by gelpurification and ligation of the appropriate cDNA band. All primersused for mutagenesis were custom made (Sigma).

In vitro cRNA translation. cRNA for microinjection was synthe-sized from wild-type, mutant, or chimeric cDNAs previously digestedat their 3= end using NotI from New England Biolabs, using the T7RNA polymerase mMESSAGE mMACHINE (Ambion) transcriptionsystem. cRNA integrity was confirmed using agarose gels, and theconcentration was assessed by absorbance reading at 260 nm (DU640; Beckman, Fullerton, CA). cRNA was stored frozen in aliquots at�80°C until use.

Transport assays. The activity of the NCC was determined byassessing 22Na� tracer uptake (New England Nuclear) in groups of10–15 oocytes, following our protocol (15). Briefly, a 30-min incu-bation in a Cl�-free ND96 medium containing 1 mM ouabain, 0.1mM amiloride, and 0.1 mM bumetanide was followed by a 60-minuptake period in a K�-free NaCl medium (40 mM NaCl, 56 mMNa-gluconate, 4.0 mM CaCl2, 1.0 mM MgCl2, and 5.0 mM HEPES-Tris, pH 7.4) containing ouabain, amiloride, bumetanide, and 2 �Ci of22Na�/ml. Because Xenopus laevis oocytes do not express a thiazide-

Fig. 1. General topology and alignment anal-ysis of transmembrane regions (TM) 8–12 inmammalian and flounder Na�-Cl� cotrans-porters (NCC). The proposed topology forNCC includes a central hydrophobic domainmade up of 12 putative TM segments di-vided into 2 fragments by a large extracel-lular glycosylated loop between segments 7and 8. The central hydrophobic domain isflanked by intracellular short and long aminoand carboxyl-terminal domains. Alignmentanalysis of TM segments 8–12 is shown.Amino acid residues in boxes are those lessconserved between flounder and all mamma-lian sequences and were thus the residueschosen for study.

F1112 COMPARATIVE ANALYSIS OF RAT AND FLOUNDER NCC

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sensitive NCC (7, 15), only one group of water-injected oocytes wasincluded in every experiment to determine the basal, nonspecific trace22Na� uptake. The affinity for thiazide diuretics was assessed byexposing groups of cRNA-injected oocytes to increasing concentra-tions of drug (from 10�9 to 10�4 M). The desired concentration of thediuretic was present in both the incubation and uptake periods. Allmetolazone dependency curves were assessed at least twice for eachclone. Ion transport kinetics were determined by performing experi-ments with varying concentrations of Na� and Cl�. Isethionate wasused as a Cl� substitute and N-methyl-D-glucamine as a Na� substi-tute to preserve osmolarity and ionic strength.

All uptake experiments were performed at 32°C. At the end of theuptake period, oocytes were washed five times in ice-cold uptakesolution without isotope to remove tracer in the extracellular fluid.After oocytes were dissolved in 10% sodium dodecyl sulfate, thetracer activity was determined for each oocyte by �-scintillationcounting.

Western blotting. Western blot analysis was used to assess theamount of protein expression in wild-type or mutant NCC-injectedoocytes. Expression of NCC was determined as FLAG-NCC becauseour cDNA contains a FLAG epitope after the first methionine (16).Proteins extracted from 50 oocytes injected with wild-type or mutantNCCs were quantified by Bradford’s technique, and 50 �g of eachprotein per lane was run using sample buffer containing 6% SDS, 15%glycerol, 0.3% bromophenol blue, 150 mM Tris, pH 7.6, and �-mer-captoethanol (when indicated), resolved by Laemmli SDS-polyacryl-amide (7.5%) gel electrophoresis, and transferred to a polyvinylidenedifluoride membrane. Immunoblotting was performed using anti-FLAG monoclonal antibody (Sigma). Membranes were exposed toanti-FLAG antibody overnight at 4°C, washed and incubated for 60min at room temperature with alkaline phosphatase-conjugated sec-ondary (anti-mouse) antibody (Bio-Rad) diluted 1:2,000 in blockingbuffer, and washed again. Bands were detected by using Immun-StarChemiluminescent Protein Detection Systems (Bio-Rad).

Data analysis. All results presented are based on a minimum ofthree different experiments with �12 oocytes/group in each experi-ment. Results are presented as means of uptake values withingroups � SE unless stated otherwise. Prism 5.0 was used to fit thekinetic data. Chloride and sodium km values were determined byfitting the uptake data with the Michaelis-Menten equation. Forthiazide, nonlinear fittings were done using the Hill equation, fromwhich the IC50 values shown in the text were obtained. In all cases, aHill coefficient close to unity was observed.

RESULTS

Single amino acid residue differences between mammalianand flNCC. We have shown previously that the difference inthiazide affinity between rat and flNCC is encoded within theTM segments 8 to 12 (16). In addition, we have also observedthat thiazide affinity is similar between rat and mouse NCCs(18). Thus, we reasoned that the difference in thiazide affinitybetween mammalian and flNCCs could be due to residuedifferences between them. We performed an alignment analy-sis of NCC TMs 8 to 12, using all of the available NCCsequences to find the residues that are identical in mammalianorthologs but different in the flounder, with a change betweenmammalian and flounder that is not conservative. As shown inFig. 1, we found only six amino acid residues with thesecharacteristics. The two located in TM segment 9 are thealanine residues 510 and 516, which correspond to threonineand cysteine, respectively, in flounder. The three located in TMsegment 11 are alanine, isoleucine, and serine at positions 568,574, and 575, which correspond to serine, cysteine, and cys-teine, respectively, in the flNCC. Finally, a valine at position

601 in the rNCC TM12 is substituted by a threonine in theflNCC. No such differences were found in TM8 or TM10,which are almost identical between the mammalian andflNCCs. Thus, by site-directed mutagenesis, we introduced thesingle-point mutations to change one TM segment at a timefrom rNCC to flNCC. Three mutant clones were generated:mutant TM9 (A510T and A516C), mutant TM11 (A568S,I574C, and S575C), and mutant TM12 (V601T). As shown inFig. 2, all three mutant cotransporters were functional, sincethey exhibited a thiazide-sensitive 22Na� uptake that wassimilar to that observed for rNCC. Of note, functional expres-sion observed in the TM11 mutant was significantly higherthan in oocytes injected with any of the other clones.

The difference in thiazide affinity between rNCC and flNCCresides in TM11. rNCCs and flNCCs exhibit a difference inthiazide affinity of approximately one order of magnitude (15,16, 22). Thus, in the present study, we assessed the thiazidedose-response curves in oocytes injected with wild-type rNCCor flNCC cRNA as well as oocytes injected with mutant NCCcRNAs at TM9, TM11, or TM12. The thiazide affinity for allclones was assessed in the same experiment to assure that theoocytes, solutions, and metolazone concentration were identi-cal among groups. Figure 3 shows the compilation of fivedifferent experiments. As shown previously (16), rNCC (inblue) and flNCC (in red) display a significant difference inaffinity for metolazone, which is of about one order of mag-nitude. Interestingly, the dose-response curves for the rNCC-TM9 mutant (orange) and for the rNCC-TM12 mutant (pink)were similar to that of rNCC. In contrast, the affinity formetolazone in oocytes injected with the rNCC-TM11 mutantcRNA (green) was identical to that observed in oocytes in-jected with flNCC cRNA. These observations strongly sug-gested that the difference in metolazone affinity betweenrNCCs and flNCCs is encoded in TM11 and that one to threeamino acid residues could be responsible.

rNCCs and flNCCs also exhibit a significant difference inaffinity for Na� and Cl� ions. Our recent observations sug-gested that the difference in chloride affinity is encoded within

Fig. 2. Mutant constructs for TM9, TM11, and TM12 are functional. Percent-age of rat NCC activity in wild-type and mutant NCC. The thiazide-sensitive22Na� uptake in wild-type NCC was set as 100%, and the values observed inmutant clones were normalized accordingly. At least 5 different 10-oocyteexperiments were done. Normalization of data was done for each experimentbefore the groups were compared. The data shown represent the mean � SEof 5 different experiments. *P � 0.05 vs. wild-type NCC.

F1113COMPARATIVE ANALYSIS OF RAT AND FLOUNDER NCC

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TM segments 1–7, whereas no specific domain seems to beresponsible for the difference in sodium affinity (15, 16, 22).We thus analyzed the effect of TM9, TM11, and TM12mutations for affinity to cotransported ions. The results areshown in Table 1. As expected, the sodium and chloridetransport kinetics of all three mutants are similar to each otherand to that observed for wild-type rNCC, indicating that theseresidues play no role in defining transport ion kinetics.

A serine to cysteine change in TM11 is responsible for thedifference in thiazide affinity between rNCCs and flNCCs.Because thiazide affinity between wild-type rNCC and rNCC-TM9 or rNCC-TM12 mutants was similar, no additional anal-ysis was done for these clones. In contrast, rNCC harboringthree mutations in TM11, which correspond to the flNCCsequence, exhibited an affinity for metolazone that was iden-tical to that in flNCC. Therefore, we further explored the roleof each of these amino acid residues. Two of these mutationsin TM11 switched the original amino acid for a cysteineresidue (I574C and S575C). Cysteines are special residuesbecause they can establish strong covalent interactions that canaffect the structural and functional properties of proteins;therefore, we decided to study these two residues first. We usedsite-directed mutagenesis to create a double mutant rNCC thatharbors both the I574C and S575C mutations. In addition, wemade two rNCC single mutants containing either the I574C orS575C mutation alone. The effect of these mutations on me-tolazone affinity was determined. Figure 4 depicts the mean ofthe IC50 values obtained from five different dose-responsecurves that were assessed simultaneously in oocytes injectedwith rNCC-TM11, the double mutant I574C S575C, or thesingle mutants I574C or S575C. Similar to the dose-responsecurves shown in Fig. 3, rNCC-TM11 shifted the metolazoneaffinity to the right by about one order of magnitude (P � 0.05vs. rNCC). A similar shift was observed in oocytes injectedwith double mutant or the single mutant S575C. In contrast, theI574C mutation had no effect on thiazide affinity because theIC50 in oocytes injected with this mutant cRNA was identicalto that shown for wild-type rNCC.

If the serine vs. cysteine substitution at position 575 in rNCCor 576 in flNCC is responsible for the difference in affinity for

Fig. 3. The effect of TM mutations on rat NCC (rNCC) affinity for metolazone.Metolazone dose response analysis was assessed in 5 different experiments, inall of which the 5 different clones were tested simultaneously, using the sameuptake solutions and metolazone dilutions. A: a compilation of the dose-response curves from the 5 experiments is shown. Wild-type rNCC (blue),wild-type flounder NCC (flNCC; red), TM9 mutant (orange), TM12 mutant(pink), and TM11 mutant (green). Data were fit to the Hill equation, and in allcases Hill coefficients close to unity were obtained. All group data are shownas means � SE of %activity. The coefficient of determination (r2) was between0.95 and 0.99 for the fits shown. B: metolazone IC50 for rNCC and mutantscalculated from the nonlinear regression fit of uptake data to the Hill equation.Data are shown as mean IC50 � SE of 5 different experiments. *P � 0.05 vs.rNCC.

Table 1. Ion transport affinities in rNCC, flNCC, and mutant clones

rNCC TM9 TM11 TM12 flNCC

Na� km, mM 6.95 � 1.8 9.2 � 1.5 7.7 � 1.3 8.6 � 1.3 31 � 4.3Cl� km, mM 5.25 � 2.0 4.1 � 1.3 4.8 � 1.0 3.3 � 1.2 14 � 1.3

Values are means � SE. rNCC, rat Na�-Cl� cotransporter; flNCC, flounder Na�-Cl� cotransporter; TM, transmembrane region.

Fig. 4. The effect of individual mutations in the rNCC TM11 segment on theaffinity for metolazone. Methods are similar to those explained in Fig. 3, and5 different experiments were performed. The bars represent mean IC50 � SEfor each tested clone, as stated. For each experiment, all Hill slopes were �1and coefficients of determination (r2) were between 0.92 and 0.99. *P � 0.05vs. rNCC.

F1114 COMPARATIVE ANALYSIS OF RAT AND FLOUNDER NCC

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metolazone between species, one would expect that this mu-tation should be enough to switch the affinity for metolazone inflounder to an IC50 similar to that observed in wild-type rNCC.We thus introduced by site-directed mutagenesis the C576Smutation into flNCC cDNA. Metolazone dose-response curveswere performed in three different experiments. A representa-tive experiment is shown in Fig. 5A, and the mean IC50 valuesfrom three different experiments are shown in Fig. 5B. Asexpected, the affinity for metolazone was increased signifi-cantly by the C576S substitution in flNCC.

We have observed previously that, whereas rNCCs andflNCCs exhibit a difference in affinity for thiazide-type diuret-ics, the profile of inhibition among different thiazides is similar(15, 22). The observed inhibition is greatest for polythiazide,followed by metolazone, bendroflumethiazide, trichlorome-thaizide, hydrochlorothiazide, and chlorthalidone. This inhibi-

tory profile is similar to thiazide potencies in clinical medicineas well as their potency to block the [3H]metolazone binding torenal cortical membranes (1). We assessed the thiazide inhib-itory profile of mutant S575C. For all thiazides tested, the IC50

in S575C was shifted to the right compared with rNCC (datanot shown). In addition, the inhibitory profile observed wassimilar to that previously seen for rNCC (15) or to thatobserved in simultaneous experiments. Thus, substitution ofserine 575 to cysteine in rNCC similarly affected the affinityfor all thiazide-type diuretics tested.

The serine-to-cysteine substitution affects the NCC basalactivity. In the experiments described above, we noticed thatfunctional expression of the rNCC-TM11 mutant was alwayshigher than wild-type NCC by �20%. Thus, we assessed the22Na� uptake in oocytes injected with the same amount ofcRNA for wild-type NCC, rNCC-TM11 mutant, or singlemutants I574C and S575C rNCC, as determined by densitom-etry of the cRNA bands in agarose gels. A representativeexperiment is shown in Fig. 6A. The 22Na� uptake in oocytesinjected with the rNCC-TM11 mutant or S575C mutant rNCCcRNA was significantly higher than that observed in wild-typerNCC. In contrast, the activity of the I574C cotransporter wassimilar to wild-type rNCC. Proteins from the same oocytesused in the experiment shown in Fig. 6A were extracted andused for Western blot analysis. The results of this experimentand the corresponding densitometric analysis are shown in Fig.6, B and C. No increase in protein expression of TM11 orS575C that would explain the higher 22Na� uptake in thesemutants was observed. Together, these observations suggestthat the difference in basal activity between the rNCC-TM11 orS575C mutants and wild-type NCC cannot be explained bychanges in protein expression. Therefore, S575C increases thetonic activity of the cotransporter by improving either itssurface expression or its translocation rate. Testing these pos-sibilities is beyond the scope of this study.

The decrease of thiazide affinity in rNCC is specific for acysteine substitution. We have shown that substituting serinefor cysteine in rNCC or cysteine for serine in flNCC switchedthe cotransporter affinity from rat to flounder like and viceversa. We wanted to know whether the decreased thiazideaffinity in rNCC-S575C is specifically due to the introductionof the cysteine and, thus, probably related to its capacity toestablish disulfide bonds. Another possibility is that this phe-nomenon could be due to a change in the local environment ofthis residue produced by the different physical and chemicalproperties of serine and cysteine. Sulfur and oxygen atomshave different electronegativities, so the polarity of thesechemical groups is not identical, with hydroxyls having ahigher electronegativity. This produces a weak H-bondingpropensity in the sulfhydryl groups of cysteines. In addition,the proton in the sulfhydryl groups of cysteines is much moreacidic (pKa 8.18) than the hydroxylic proton of serine. All ofthese different properties could explain the difference in affin-ity if, for example, a hydrogen bond established with thethiazide or another residue in the protein is stronger in thepresence of a serine. With this in mind, we constructed threemore mutants in which serine 575 of rNCC was substituted fora positive, a negative, or a neutral amino acid residue, namelylysine, aspartate, or alanine, respectively. We reasoned thatpositive and negative charges could establish an electrostaticinteraction that would resemble a hydrogen bond formed by the

Fig. 5. The effect of TM mutations in flNCC on the affinity for metolazone.Metolazone dose-response analysis was assessed as explained in Fig. 3.Similar observations were done in 3 different experiments. A: a representativeexperiment is shown. Dose-response curves are shown for wild-type flNCC (�)and C576S-flNCC (‘). The coefficient of determination (r2) for wild-typeflNCC and C576S-flNCC was 0.96 and 0.98, respectively. B: metolazone IC50

for flNCC and C576S mutant was calculated from the nonlinear regression fitof uptake data to the Hill equation. Data are shown as mean IC50 � SE of 3different experiments. *P � 0.05 vs. flNCC.

F1115COMPARATIVE ANALYSIS OF RAT AND FLOUNDER NCC

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cysteine/serine and that the alanine would prevent the forma-tion of any interaction. However, none of the introducedresidues would favor the formation of disulfide bonds. Asshown in Fig. 7A, substitution of serine for lysine resulted in anonfunctional cotransporter. In contrast, substitution of serine575 with alanine or aspartate produced rNCC mutants that

exhibited enough 22Na� uptake to determine a dose-responsecurve for metolazone. As shown in Fig. 7B, affinity for meto-lazone was similar in oocytes injected with wild-type rNCC,rNCC-S575A, and rNCC-S575D but different from that ob-served in rNCC-S575C. These observations suggest that it isspecifically a change for cysteine in position 575 that makesrNCC less sensitive to metolazone.

It has been suggested that NCC functions as a homodimer,but it is not clear whether the interaction between monomersinvolves the formation of disulfide bonds (4). If this type ofbond is not present between NCC homodimers, then a cysteinedifference between rNCC and flNCC could be allowing theflounder cotransporter to form a disulfide bond between mono-mers that is not present in the mammalian cotransporter. Todetermine whether disulfide bonds are present in NCC dimersand to find out whether this is affected by S575C substitutionin rNCC, we performed a Western blot analysis of proteinsextracted from Xenopus laevis oocytes microinjected withwater, wild-type rNCC, or S575C-rNCC cRNA. Protein ex-tracts were prepared for standard SDS-PAGE with Laemmlibuffer with or without reducing agent (5% �-mercaptoethanol).As shown in Fig. 8, in the absence of reducing agent, bands ofexpected size for NCC dimers and monomers are observed inboth wild-type NCC and S575C-NCC. When a reducing agent

Fig. 7. Functional expression and metolazone affinity of mutant rNCCs S575C,S575A, S575D, and S575K. Oocytes were injected with cRNA from eachmutant clone. A: 3 days later, the 22Na� uptake was assessed in the absence(open bars) or presence (closed bars) of 100 �M metolazone. Data aremeans � SE. B: metolazone IC50 values for rNCC and mutants were calculatedfrom the nonlinear regression fit of uptake data to the Hill equation. Data areshown as mean IC50 � SE of 3 different experiments. *P � 0.05 vs. rNCC.

Fig. 6. TM11 mutant rNCC and the single mutant S575C-rNCC exhibitedhigher activity than wild-type NCC or I574C-rNCC. Oocytes were injectedwith the same amount of cRNA of each clone, as explained in the text. A: 3days later the 22Na� uptake was assessed in the absence (open bars) orpresence (filled bars) of metolazone. Data presented are means � SE of theuptake observed for each oocyte. Similar observations were done in at least 5experiments. *P � 0.05 vs. control wild-type rNCC. B: proteins were extractedfor a Western blot analysis using anti-FLAG or anti-actin antibody. A repre-sentative Western blot is shown in B. C: densitometric analysis of bandsobserved in the Western blot. OD, optical density.

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was used, single bands of the expected size for the NCCmonomer were present, whereas the bands corresponding tothe dimer size were absent. This finding suggests that disulfidebonds are involved in NCC dimerization. However, no differ-ence was observed between wild-type rNCC and the S575CNCC mutant. As shown in Fig. 8, to rule out the possibility thatmutant rNCC becomes slightly resistant to the reducing agentbecause of the presence of an extra disulfide bond, we repeatedthe experiment using �-mercaptoethanol at different concen-trations. No difference between wild-type and mutant NCCwas observed at any point.

DISCUSSION

In the present study, we show that substituting serine 575 inrNCC for the cysteine present in flNCC at the same position issufficient to render rNCC more resistant to thiazides, exhibit-ing a flounder-like affinity. Supporting these observations,substituting the cysteine 576 in the flNCC for the correspond-ing serine in the rNCC increased the affinity of flNCC to amammalian-like IC50. Therefore, a single residue difference inTM11 segment between rNCC and flNCC explains the differ-ence in affinity for thiazides between species. In addition, weobserved that substituting the serine 575 in rNCC with residuesother than cysteine, such as alanine or aspartate, had no effectupon affinity for thiazides, suggesting that it is not the presenceof the serine in this position that increases affinity, but insteadit is the absence of a cysteine residue that is present in flNCC.Although it is unknown whether loop diuretics or thiazidesbind to a similar region in NKCCs and NCC, our observationsare supported by previous work on chimeric proteins betweenshark and human NKCC1 that suggests roles for transmem-brane segments 11 and 12 (13).

Two different possible mechanisms can potentially explain thedecrease in affinity for metolazone observed in the NCC-S575C

mutant. The first mechanism is that this residue may be part of thethiazide-binding site on the cotransporter, and the second mech-anism is that substitution of S575C may modify the tridimensionalconfiguration of the protein, indirectly affecting the interaction ofthiazides with their binding site or the protein’s response upondrug binding. Our results favor the second possibility because, ifserine 575 were part of the thiazide-binding site, it would beexpected that substituting the serine with different amino acidresidues would result in altered affinity for thiazide. However, ourdata show (Fig. 7) that changes in affinity occur only when serineis substituted with cysteine, suggesting that it is a unique propertyof cysteines that decreases the thiazide affinity in mutant rNCC-S575C.

Cysteines are very important residues in the tridimensionalstructure of proteins because they form disulfide bonds. Twotypes of interactions can be established by the sulfhydryl groupin their side chain, hydrogen bonds and disulfide bonds (10). InNCC, the establishment of a hydrogen bond by this cysteineprobably would not generate a significant difference in terms ofstructure, compared with the structure of the wild-type cotrans-porter, because the serine originally present in rNCC alsopossesses the ability to form hydrogen bonds. Thus, a plausiblepossibility is that the substitution of serine 575 for cysteinemay introduce a new covalent bond, changing the tertiarystructure of NCC. This substitution could reduce the access ofthiazides to their binding site located elsewhere, introduce astructural change that allosterically affects the binding site forthe inhibitor, or affect the protein’s response upon drug bind-ing. According to the hydropathy predictions (6), serine 575lies approximately in the middle of TM11 segment. A modelfor the putative configuration of the �-helix formed by TM11predicts that serine 575 is located in a hydrophilic face of thehelix (Supplemental Fig. S1; Supplemental Material for thisarticle can be found on the AJP-Renal Physiology web site),

Fig. 8. Electrophoretic behavior of rNCC andS575C-rNCC in the absence or presence of areducing agent. Protein extracts from oocytesinjected with water, rNCC cRNA, or rNCC-S575C cRNA were diluted for standard SDS-PAGE in Laemmli buffer without reducingagent (A) or containing either 0.5 (B), 2.5 (C),or 5% �-mercaptoethanol (D). In the absenceof reducing agent (A), bands of expected sizefor monomeric and dimeric forms of the co-transporter were observed, and in both cases adiffused signal of slightly higher molecularweight was detected, which probably corre-sponds to glycosylated forms of the protein.This was seen for the wild-type and mutantform of rNCC. In the presence of reducingagent, the bands corresponding to the dimericform of the cotransporter disappeared even atthe lowest concentration of reducing agentused. At this concentration, we were able toobserve a slight signal corresponding to thedimers, but no difference in intensity was ob-served between the wild type and the mutant.

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implying that this face could be involved in contacts with otherprotein domains and not facing the membrane, thus making itpossible that changing the serine for cysteine creates thepossibility of a disulfide bond formation with another residuein a membrane domain of the same polypeptide or of anotherNCC monomer. There is evidence that NCC forms ho-modimers that may be the functional form of the cotransporter;however, the TM segments involved in establishing the inter-face between monomers are unknown. Although no crystalstructure of any member of the SLC12 family is available, thecrystal structures of transporters of the APC (amino acids,polyamine, organocation) transporter superfamily, in which theSLC12 family is included, have been reported (9, 20). Two ofthese transporters, as detailed in the legend of SupplementalFig. S1, present a generally similar folding pattern, withsegments 1–10 forming the core structure of the cotrans-porter that comprises the ion translocation pathway andTM11 and -12 lying outside as accessory segments. Thisgeneral folding pattern is not only conserved in APC trans-porters but is also present in a more distantly relatedbacterial leucine cotransporter, LeuT (2, 11, 23). Thus,expecting similarities, at least at a general level of structure,between NCC and these transporters is sound. This findingis supported by a recent thorough analysis of the structuralconservation among sodium transporters (14). Continuingthis line of reasoning, the residue that was found to alterNCC affinity for thiazides would be located in an accessoryTM segment. However, it is not possible to speculate on itsexact position within this segment. AdiC, one of the crys-tallized APC transporters, has a dimeric structure in whichTM segments 11 and 12 lie outside the core structure andform the interface between the dimers. LeuT also formsdimers, and there is evidence that, in the eukaryotic ho-mologs of this cotransporter, TM11 participates in dimer-ization. Therefore, the cysteine in position 576 of flNCCcould be forming an interaction either with another residuewithin the same polypeptide chain or located in the secondmonomer of the NCC homodimer.

Considering the possibility that substituting a serine forcysteine at position 575 in rNCC could be mediating aninteraction between NCC monomers, we performed a westernblot analysis of wild-type and mutant rNCC-S575C in theabsence or presence of a reducing agent. The existence of NCChomodimers has been demonstrated previously (4), but thatdisulfide bonds are involved in the interaction between NCCmonomers has never been explored. Thus, we reasoned that thenew cysteine introduced could perhaps be creating a newcovalent bond between subunits that was originally nonexist-ent. If this were the case, and if there are no disulfide bondsbetween wild-type rat NCC monomers, we would expect to seebands corresponding to the homodimers in the absence of areducing agent only in the mutant protein. However, the resultshown in Fig. 8 suggests that wild-type rNCC forms ho-modimers in which disulfide bonds are involved, and nodifference is observed between the wild-type and mutantNCCs. Also, no difference in resistance to the effects of thereducing agent was observed between the wild-type and themutant clones when different concentrations of �-mercapto-ethanol were used. This finding suggests that no additionalintermolecular bonds are formed when serine 575 is switchedfor a cysteine. However, this observation does not rule out the

possibility that this cysteine may be forming a new disulfidebond, perhaps altering the original pattern of disulfide bondsformed between NCC subunits.

In summary, we propose that the difference in thiazideaffinity observed between mammalian and flNCCs is duemainly to a single amino acid residue difference betweencotransporters. This residue is located within the transmem-brane segment 11. Further investigation will be required todefine the mechanism by which substitution of a cysteine inflNCC for a serine in rNCC increases the affinity for thiazides.

ACKNOWLEDGMENTS

We are grateful to all members of the Molecular Physiology Unit forsuggestions and stimulating discussion.

GRANTS

This work was supported by National Institute of Diabetes and Digestiveand Kidney Diseases Grant DK-64635 to G. Gamba and Mexican Council ofScience and Technology Grants 59992 to G. Gamba and 104451 to E. Moreno.

DISCLOSURES

No conflicts of interest are declared by the authors.

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