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【摘要】 The mammalian kidney bumetanide-sensitive Na + -K + -2Cl - and thiazide-sensitive Na + -Cl - cotransporters are the major pathways for salt reabsorption in the thick ascending limb of Henle's loop and distal convoluted tubule, respectively. These cotransporters serve as receptors for the loop- and thiazide-type diuretics, and inactivating mutations of corresponding genes are associated with development of Bartter's syndrome type I and Gitleman's disease, respectively. Structural requirements for ion translocation and diuretic binding specificity are unknown. As an initial approach for analyzing structural determinants conferring ion or diuretic preferences in these cotransporters, we exploited functional differences and structural similarities between Na + -K + -2Cl - and Na + -Cl - cotransporters to design and study chimeric proteins in which the NH 2 -terminal and/or COOH-terminal domains were switched between each other. Thus six chimeric proteins were produced. Using the heterologous expression system of Xenopus laevis oocytes, we observed that four chimeras exhibited functional activity. Our results revealed that, in the Na + -K + -2Cl - cotransporter, ion translocation and diuretic binding specificity are determined by the central hydrophobic domain. Thus NH 2 -terminal and COOH-terminal domains do not play a role in defining these properties. A similar conclusion can be suggested for the Na + -Cl - cotransporter.
【关键词】 bumetanide thiazide cotransporter chimeras salt reabsorption
APICAL BUMETANIDE - SENSITIVE Na + -K + -2Cl - cotransporter (BSC1) and thiazide-sensitive Na + -Cl - cotransporter (TSC) are the major pathways for salt reabsorption in the luminal membrane of thick ascending limb of Henle's loop (TALH) and distal convoluted tubule, respectively ( 8, 13, 14, 24, 36 ). These cotransporters also serve as targets for loop diuretics and thiazide-type diuretics, which are among the most commonly prescribed drugs in the world ( 5, 7 ). Inactivating mutations of BSC1 are the cause of Bartter's syndrome type I ( 25, 39 ) and those in TSC produce Gitelman's disease ( 26, 40 ). These are the so-called inherited hypokalemic metabolic alkalosis syndromes that feature arterial hypotension. In addition, loss of TSC regulation by WNK4 kinase is implicated in the pathophysiology of pseudohypoaldosteronism type II ( 45, 47 ), an inherited hypertension syndrome that is the result of mutations in the WNK4 gene ( 44 ). Therefore, BSC1 and TSC are genes involved in determining blood pressure ( 37 ).
Structural requirements for ion translocation and diuretic binding in the TSC are completely unknown. A recent characterization of single nucleotide polymorphisms in TSC revealed that a glycine in the transmembrane segment 4 plays a role in defining affinity for extracellular chloride and metolazone ( 29 ). In the basolateral isoform of the Na + -K + -2Cl - cotransporter (BSC2), Isenring and Forbush (for a review, see Ref. 17 ) took advantage of the kinetic differences in apparent affinity for ions and bumetanide between shark and human isoforms to reveal that transmembrane segments define ion transport kinetics ( 20 ) and implicated TM2 in Na +, K +, and bumetanide affinity ( 16, 19 ), TM4 in K + and Cl - affinity ( 18 ), and TM7 in Na +, K +, and Cl - affinity ( 18 ). The role of TM2 has also been studied in three alternatively spliced isoforms of the apical BSC1 cotransporter that exhibit distinct bumetanide affinity and ion transport kinetic properties ( 12, 34, 35 ). Because these studies were performed using chimeras between orthologs of BSC2 ( 17 ) or spliced isoforms of BSC1 ( 35 ), all constructs were expected to behave as Na + -K + -2Cl - cotransporters. Thus, although valuable information is available regarding structural requirements to define kinetic properties in the Na + -K + -2Cl - cotransporter, little information was obtained concerning the specificity for ions or diuretics. Moreover, in other membrane transporters, it has been shown that domains or amino acid residues that are crucial to define kinetic properties are not necessarily the same as those defining the specificity of transport process or the binding of a particular inhibitor ( 1, 31 ). In this regard, consistent with physiological studies by Sun et al. ( 41 ) and Eveloff and Calamia ( 9 ), which demonstrated switching between Na + -K + -2Cl - and Na + -Cl - transport mode in the apical membrane of the TALH from mouse and rabbit, respectively, we have shown that a shorter COOH-terminal domain spliced isoform of murine apical BSC1 cotransporter ( 30 ) encodes a K + -independent, but nevertheless furosemide-sensitive, Na + -Cl - cotransporter that is activated by hypotonicity and inhibited by cAMP ( 33 ), suggesting that, in BSC1, sequences within the COOH-terminal domain could be critical to define the specificity for K + transport and some regulatory properties.
The major goal of the present study was to determine the role of NH 2 -terminal, central TM, and COOH-terminal domains of renal-specific apical sodium- and chloride-coupled cotransporters in defining the specificity of the transport process and diuretic interaction. We exploited the fact that BSC1 and TSC share extensive protein sequence similarity but display differences with respect to the type and stoichiometry of transported ions and diuretic inhibition. Accordingly, we constructed chimeric proteins by exchanging NH 2 -terminal and/or COOH-terminal domains between rat BSC1 and TSC. Our results show that ion transport process and diuretic specificity reside within the central TM domain.
METHODS
Silent mutagenesis and construction of chimeric cotransporters. We used BSC1 and TSC cDNAs that we previously cloned from rat kidney ( 10 ) as a source for constructing chimeric cotransporters. Thus all constructs are inserted in plasmid pSPORT1 (GIBCO-BRL), and BSC1 isoform used was the F isoform. Wild-type TSC cDNA contains a unique Nsi I site on base pairs 444-449, which encodes residues located at the middle of the first transmembrane domain. To exchange NH 2 -terminal domains between BSC1 and TSC, a silent Nsi I site was introduced in exactly the same region of BSC1, by a G-A substitution at base pair 761. No restriction sites were available in wild-type clones to exchange COOH-terminal domains. Therefore, a unique silent Hpa I site was introduced into BSC1 by a G-T change at base 2150 and in TSC by an A-T change at base 1836. The mutated bases are part of the codon that encode valine-645 in BSC1 and valine-608 in TSC, which are located two residues after the end of predicted TM12. Next, the unique Eco RI (in the 5' side of the polylinker), together with the unique Nsi I and Hpa I sites, was used to exchange the NH 2 - and/or COOH-terminal domains between BSC1 and TSC by gel purification and ligation of the appropriate cDNA bands. All silent mutations were introduced by using the Quickchange site-directed mutagenesis system (Stratagene) following the manufacturer's recommendations. Restriction analysis and automatic DNA sequencing was used to corroborate all mutations and switching place of each chimera. All primers used for mutagenesis were custom made (Sigma Genosys).
In vitro cRNA translation. To prepare cRNA for microinjection, each wild-type or chimeric cDNA was digested at the 3'-end using Not I from Invitrogen (Carlsba, CA), and cRNA was transcribed in vitro using the T7 RNA polymerase mMESSAGE mMACHINE (Ambion) transcription system. cRNA product integrity was confirmed on agarose gels, and concentration was determined by absorbance reading at 260 nm (DU 640; Beckman, Fullerton, CA). cRNA was stored frozen in aliquots at -80°C until used.
Xenopus laevis oocytes preparation. Oocytes were harvested by surgery from adult female X. laevis frogs (Nasco) under 0.17% tricaine anesthesia. After 1 h incubation in frog Ringer ND-96 (in mM: 96 NaCl, 2 KCl, 1.8 CaCl 2, 1 MgCl, and 5 HEPES/Tris, pH 7.4) in presence of collagenase B (2 mg/ml), oocytes were washed four times in ND-96, manually defolliculated, and incubated overnight at 18°C in ND-96 supplemented with 2.5 mM sodium pyruvate and 5 mg/100 ml gentamicin. Stage V-VI oocytes ( 6 ) were injected with 50 nl water alone or containing cRNA at 0.5 µg/µl (25 ng cRNA/oocyte). Next, oocytes were incubated 4 days in ND-96 with sodium pyruvate and gentamicin. The incubation medium was changed every 24 h. The night before the uptake experiments were performed, oocytes were incubated in Cl - -free ND-96 (in mM: 96 sodium isethionate, 2 potassium gluconate, 1.8 calcium gluconate, 1.0 magnesium gluconate, 5 mM HEPES, 2.5 sodium pyruvate, and 5 mg/mg gentamicin, pH 7.4; see Ref. 11 ).
Transport assays. Function of Na + -K + -2Cl - cotransporter was assessed by measuring tracer 86 Rb + uptake (New England Nuclear) in groups of at least 12 oocytes following our general protocol ( 35 ) as follows: 30-min incubation in K + - and Cl - -free medium (in mM: 96 sodium gluconate, 6.0 calcium gluconate, 1.0 magnesium gluconate, and 5 HEPES/Tris, pH 7.4) with 1 mM ouabain, followed by 60-min uptake period in the presence of Na +, K +, and Cl -. For most experiments, the isotonic medium contained (in mM) 96 NaCl, 10 KCl, 1.8 CaCl 2, 1 MgCl 2, and 5 HEPES, pH 7.4, supplemented with 1 mM ouabain and 2.0 µCi 86 Rb +. Because X. laevis oocytes express an endogenous Na + -K + -2Cl - cotransporter ( 10 ), every experiment included appropriate groups of water-injected oocytes.
Function of Na + -Cl - cotransporter was determined by assessing tracer 22 Na + uptake (New England Nuclear) in groups of 12 oocytes with the following protocol ( 28 ): 30-min incubation in a Cl - -free ND-96 medium containing 1 mM ouabain, 0.1 mM amiloride, and 0.1 mM bumetanide followed by a 60-min uptake period in a K + -free, NaCl medium (in mM: 40.0 NaCl, 56.0 sodium gluconate, 1.8 CaCl 2, 1.0 MgCl 2, and 5.0 HEPES/Tris, pH 7.4) containing ouabain, amiloride, bumetanide, and 2 µCi 22 Na + /ml. To determine the Cl - -dependent fraction of 22 Na + uptake, paired groups of oocytes were incubated in uptake media without Cl - (in mM: 40.0 sodium gluconate, 56 N -methyl- D -glucamine-gluconate, 6.0 calcium gluconate, 1.0 magnesium gluconate, and 5.0 HEPES/Tris, pH 7.4).
All uptakes were performed at 30°C. At the end of the uptake period, oocytes were washed five times in ice-cold uptake solution without isotope to remove extracellular fluid tracer. After the oocytes were dissolved in 10% SDS, tracer activity was determined for each oocyte by -scintillation counting.
Western blotting. Western blot analysis was used to analyze TSC and TTB protein (see Nomenclature and construction of chimeric proteins ) in corresponding cRNA-injected oocytes following our standard protocol ( 27 ). In brief, proteins extracted from four oocytes per lane were heated in sample buffer containing 6% SDS, 15% glycerol, 0.3% bromphenol blue, 150 mM Tris, pH 7.6, and 2% -mercaptoethanol, resolved by Laemmli SDS-polyacrylamide (7.5%) gel electrophoresis, and transferred to a polyvinylidene difluoride membrane. Immunodetection was performed using a rabbit polyclonal anti-rat TSC antibody generously provided by Wang et al. ( 43 ), diluted 1:1,000. Membranes were exposed to anti-TSC antibody overnight at 4°C and incubated for 60 min at room temperature with alkaline phosphatase-conjugated secondary (anti-rabbit) antibody (Bio-Rad) diluted 1:2,000 in blocking buffer and washed again. Bands were detected by using the Immun-Star Chemiluminescent Protein Detection Systems (Bio-Rad).
Assessment of enhanced green fluorescent protein-TSC and enhanced green fluorescent protein-TTB expression in the oocyte plasma membrane. Surface expression of TSC and TTB chimera (see Nomenclature and construction of chimeric proteins ) was assessed by fluorescence using enhanced green fluorescent protein (EGFP)-TSC and EGFP-TTB fusion constructs, a strategy that we have reported previously ( 15, 27, 29, 45 ). Oocytes were microinjected with water or with EGFP-TSC or EGFP-TTB cRNA and 4 days later were monitored for EGFP fluorescence using a Zeiss laser-scanning confocal microscope (objective lens x 10; Nikon). The light of 488-nm excitation wavelength and 515- to 565-nm emission was used to visualize green fluorescent protein fluorescence. Plasma membrane fluorescence was quantified by determining pixel intensity around the entire oocyte circumference using SigmaScan Pro image analysis software.
Data analysis. All results presented are based on a minimum three different experiments with at least 12 oocytes/group in each experiment. Significance of the differences between groups was evaluated by one-way ANOVA, with multiple comparisons using Bonferroni correction. Results are presented as means ± SE.
RESULTS
Nomenclature and construction of chimeric proteins. As shown in Fig. 1, hydropathy analysis of BSC1 and TSC amino acid sequence proposed a similar secondary structure featuring a central hydrophobic TM domain, flanked by a short NH 2 - and long COOH-terminal hydrophilic loops, presumably located within the cell. The hydrophobic TM domain contains 12 putative transmembrane-spanning segments, with a hydrophilic loop between TM7 and TM8 that is glycosylated. We generated six chimeras by exchanging NH 2 - and/or COOH-terminal domains between BSC1 and TSC. Chimeras are denoted by three letters, the first signifying NH 2 -terminal, second the central TM domain, and third the COOH-terminal domain. The letters B or T denote when the domain belongs to BSC or TSC, respectively. For example, the construct containing NH 2 -terminal domain from rat TSC and central TM and COOH-terminal domains from BSC1 is referred to as TBB. Therefore, the six chimeras constructed were TBB, BBT, TBT, BTT, TTB, and BTB. Functional expression of the first four chimeras was obtained. Chimeras in which BSC1 COOH-terminal domain was fused into TSC central TM domain (TTB and BTB) were not functional.
Fig. 1. Topological models for bumetanide-sensitive cotransporter (BSC1), thiazide-sensitive cotransporter (TSC), and 6 constructed chimeras. BSC1 is shown in red, and TSC is shown in blue. Black regions in BSC1 represent site for splicing in TM2 and a unique fragment of 70 residues in COOH-terminal domain that is not present in TSC.
Ion transport specificity in chimeric clones with BSC1 TM domain. As we have shown previously ( 10, 35 ), injection of BSC1 cRNA in X. laevis oocytes resulted in an increased 86 Rb + uptake, over the water-injected oocytes, that was Cl - dependent, bumetanide sensitive, and resistant to thiazides ( Fig. 2 A ). The functional expression of the chimeric clones containing BSC1 central TM domain revealed a similar behavior to BSC1-injected oocytes. Injection of X. laevis oocytes with TBB cRNA ( Fig. 2 B ) resulted in an increased 86 Rb + uptake (TBB 4.8 ± 0.31 vs. H 2 O-injected oocytes 1.0 ± 0.07 nmol·oocyte -1 ·h -1, P < 0.01) that was Cl - dependent (0.33 ± 0.049 nmol·oocyte -1 ·h -1 ) and bumetanide sensitive (0.18 ± 0.013 nmol·oocyte -1 ·h -1 ) but unaffected by the 10 -4 M concentration of the thiazide-type diuretic metolazone (5.1 ± 0.45 nmol·oocyte -1 ·h -1 ). A similar pattern of function was observed in BBT cRNA-injected oocytes ( Fig. 2 C ). The 86 Rb + uptake of 7.1 ± 0.35 nmol·oocyte -1 ·h -1 was significantly different from the 2.4 ± 0.13 nmol·oocyte -1 ·h -1 observed in water-injected oocytes ( P < 0.01). Increased uptake in BBT-injected oocytes was Cl - dependent (0.53 ± 0.10 nmol·oocyte -1 ·h -1 ), bumetanide sensitive (1.7 ± 0.16 nmol·oocyte -1 ·h -1 ), and metolazone resistant (7.3 ± 0.34 nmol·oocyte -1 ·h -1 ). Finally, as shown in Fig. 2 D, TBT-injected oocytes exhibited the same behavior. The increased uptake in TBT-injected oocytes (4.5 ± 0.25 nmol·oocyte -1 ·h -1 ) was Cl - dependent (0.13 ± 0.15 nmol·oocyte -1 ·h -1 ), bumetanide sensitive (0.80 ± 0.97 nmol·oocyte -1 ·h -1 ), and metolazone resistant (5.5 ± 0.37 nmol·oocyte -1 ·h -1 ). Chimeras TBB, BBT, and TBT also increased 22 Na + uptake when injected in X. laevis oocytes. As shown in Fig. 3, chimera TBT induced a significant increase in 22 Na + uptake that was Cl - dependent, bumetanide sensitive, and metolazone resistant. TBB and BBT chimeras exhibited a similar behavior (data not shown). Therefore, chimeras containing the BSC1 central hydrophobic domain exhibited a BSC1-like behavior.
Fig. 2. 86 Rb + uptake in Xenopus laevis oocytes that were injected with water or with 25 ng cRNA from BSC1 ( A ), TBB ( B ), BBT ( C ), and TBT ( D ), as indicated. Uptake was assessed in control conditions (open bars), in absence of extracellular Cl - (filled bars), or in presence of 10 -4 M concentration of bumetanide (gray bars) or metolazone (hatched bars). Each bar represents a mean of 25 oocytes from 2 different frogs. * P < 0.01 vs. water control group. ** P < 0.01 vs. corresponding cRNA oocytes in control conditions.
Fig. 3. 22 Na + uptake in X. laevis oocytes that were injected with water or with 25 ng cRNA from TBT, as indicated. Uptake was assessed in control conditions (open bars), in absence of extracellular Cl - (filled bars), or in presence of 10 -4 M concentration of bumetanide (gray bars) or metolazone (hatched bars). Each bar represents a mean of 20 oocytes. * P < 0.01 vs. water control group. ** P < 0.01 vs. corresponding cRNA oocytes in control conditions.
Functional characterization of chimeric clones with TSC TM domain. Of the three chimeras in which NH 2 - and/or COOH-terminal domains of BSC1 were fused in the TSC central TM domain, only BTT was functional. As shown in Fig. 4, injection of TSC cRNA in X. laevis oocytes resulted in an increased 22 Na + uptake over the water-injected oocytes that was Cl - dependent, metolazone sensitive, K + independent, and resistant to bumetanide ( Fig. 4 A ). Functional expression of BTT chimera revealed a similar behavior. Injection of X. laevis oocytes with BTT cRNA ( Fig. 2 B ) resulted in an increased 22 Na + uptake (BTT 8.7 ± 0.75 vs. H 2 O-injected oocytes 1.1 ± 0.2 nmol·oocyte -1 ·h -1, P < 0.01) that was Cl - dependent (0.41 ± 0.017 nmol·oocyte -1 ·h -1 ) and metolazone sensitive (0.51 ± 0.03 nmol·oocyte -1 ·h -1 ) but it was K + independent (9.9 ± 0.69 nmol·oocyte -1 ·h -1 ) and unaffected by a 10 -4 M concentration of bumetanide (9.7 ± 1.21 nmol·oocyte -1 ·h -1 ). Moreover, 86 Rb + uptake in water- or BTT-injected oocytes was similar (data not shown).
Fig. 4. 22 Na + uptake in X. laevis oocytes that were injected with water or with 25 ng cRNA from TSC ( A ), BTT ( B ), TTB ( C ), and BTB ( D ), as indicated. Uptake was assessed in control conditions (open bars), in absence of extracellular K + (filled bars) or Cl - (gray bars), or in presence of 10 -4 M concentration of bumetanide (hatched bars) or metolazone (horizontal bars). Each bar represents a mean of 25 oocytes from 2 different frogs. * P < 0.01 vs. water control group. ** P < 0.01 vs. corresponding cRNA oocytes in control conditions.
In three different experiments, although BSC1, TSC, and BTT exhibited robust activity, TTB and BTB chimeras did not induce tracer 22 Na + or 86 Rb + uptake above values observed in water-injected oocytes. The absence of activity in oocytes injected with TTB or BTB chimeras could result from a low concentration of transported protein within oocytes, secondary to a decreased synthesis or increased degradation of chimera protein, trafficking defect that prevents the cotransporter from reaching the plasma membrane, and/or a lack of function in an otherwise normally processed protein.
Because BTT was functional, it is likely that the exchange of BSC1 COOH-terminal domain in TSC was the reason for the absence of activity in TTB and BTB chimeras. Thus quality and amount of TTB chimera was analyzed by Western blot analysis, whereas arrival to the plasma membrane was determined by assessing the fluorescence in the surface of oocytes injected with wild-type TSC or TTB chimera to which the EGFP was attached to NH 2 -terminal domain, as we have previously described ( 15 ). As shown in Fig. 5 A, Western blot analysis revealed that oocytes injected with an equal amount of either rat TSC or TTB cRNA produced a similar amount of the expected molecular weight proteins. Both bands appear to be glycosylated. These observations suggest that the absence of activity in TTB-injected oocytes is not the result of a major defect in protein synthesis. Figure 5 B shows confocal image analysis of 24 X. laevis oocytes, 12 of which were injected with wild-type EGFP-TSC cRNA and 12 with EGFP-TTB cRNA. Green fluorescence is observed in the surface of each oocyte. This strategy allows the assessment of the amount of EGFP-tagged cotransporter that reaches the plasma membrane because EGFP is attached to the cotransporter ( 3, 4, 23, 32 ). We have previously shown a 99% surface colocalization of EGFP-TSC and the lipophilic fluorophore FM 4-64 ( 15 ). This compound has been used to measure surface expression of other membrane proteins because it possess optimal properties for a fluorescence membrane marker ( 21, 22 ). In addition, EGFP-TSC cRNA injection in X. laevis oocytes induced the appearance of a thiazide-sensitive 22 Na + uptake mechanism, indicating that the protein is located in plasma membrane ( 15 ). Figure 5 C depicts fluorescence analysis of oocytes in Fig. 5 B, revealing that plasma membrane fluorescence was significantly higher in EGFP-TTB- than in EGFP-TSC-injected oocytes. In spite of the presence of EGFP-TTB in the plasma membrane, no functional activity was observed. Figure 5 D depicts the 22 Na + uptake assessed in the oocytes from Fig. 5, B and C. Although oocytes injected with EGFP-TSC cRNA exhibited increased 22 Na + uptake that was sensitive to thiazides, the EGFP-TTB exhibited no activity as a cotransporter. All these observations suggest that, when injected in X. laevis oocytes, TTB chimera is synthesized and inserted in the plasma membrane, but it is not functional.
Fig. 5. Biochemical, functional, and plasma membrane fluorescence of enhanced green fluorescent protein EGFP-TTB chimera. A : Western blot analysis of rat TSC and TTB chimera using polyclonal antibodies directed against TSC NH 2 -terminal domain. B : color confocal images of 24 X. laevis oocytes. Before confocal microscope analysis (4 days) was conducted, oocytes on right were injected with EGFP-TSC cRNA, and oocytes in left were injected with identical amount of EGFP-TTB cRNA. C : densitometry of surface fluorescence in oocytes depicted in B, quantified at equatorial focal sections of oocytes using Sigma-Scan Pro (Jandel Scientific) image-analysis software. Results are expressed as means ± SE of arbitrary units (AU) in 12 oocytes/group. D : thiazide-sensitive 22 Na + uptake was assessed in oocytes from B and C and expressed as pmol·oocytes -1 ·h -1. In C and D, results from EGFP-TSC-injected oocytes are shown in white bars and from EGFP-TTB-injected oocytes are shown in black bars. * P < 0.05 vs. EGFP-TTB.
DISCUSSION
The present work describes the construction of chimeras between the rat renal apical Na + -K + -2Cl - and Na + -Cl - cotransporters, in which extensive structural similarities and functional differences between these two cotransporters were exploited. Four out of six chimeras were functional, and their characterization provides new insights into structural determinants for ion and diuretic specificity.
Amino acid residues or domains that are critical to define the ion and/or diuretic preferences in renal Na + -coupled Cl - cotransporters are not known. Previous research in this area indicated that, in BSC2, the basolateral Na + -K + -2Cl - cotransporter, residues within the central hydrophobic domain are involved in defining the kinetic properties for Na +, K +, and Cl - uptake, as well as for bumetanide inhibition of uptake ( 16 - 20 ). However, two lines of evidence suggested that these observations cannot be extended to ion and/or diuretic specificity of BSC1 or TSC. On one hand, an alternatively spliced COOH-terminal truncated isoform of BSC1 exhibited a dramatic change in K + affinity, since the shorter isoform encodes a K + -independent, Na + -Cl - cotransporter ( 33 ), suggesting that the COOH-terminal domain could be critical in defining specificity for K + transport. On the other hand, it has been shown in other membrane transporters that residues with no effect in defining transport kinetic properties of a particular substrate can be critical to determine specificity of other substrates. For instance, although NH 2 -terminal domain of glucose transporters GLUT1, GLUT2, and GLUT4 plays no role in determining transport kinetics of 2-deoxyglucose and 3- O -methylglucose influx, it is critical to define specificity, since this domain contains information that allows GLUT2, but not GLUT1 or GLUT4, to transport other sugars, such as fructose, arabinose, and streptozotocin ( 31 ). We now present clear evidence that, in apical Na + -K + -2Cl - cotransporter BSC1, central hydrophobic domain is the protein region that defines the specificity for the transported ions, as well as the sensitivity to loop diuretics. These conclusions are based on the observation that three chimeras containing the BSC1 central domain exhibited functional properties compatible with the bumetanide-sensitive Na + -K + -2Cl - cotransporter, that is, they all exhibited a bumetanide-sensitive, thiazide-resistant, and Cl - -dependent 22 Na + and 86 Rb + uptake. Thus information contained within BSC1 central transmembrane domains endow this membrane protein with Na + -K + -2Cl - transport capacity, as well as with sensitivity to loop diuretics. Therefore, the binding site for bumetanide should be located within the central hydrophobic domain. Interestingly, we have previously observed that a BSC1 COOH-terminal truncated spliced isoform behaves as a K + -independent, but nevertheless bumetanide-sensitive, Na + -Cl - cotransporter ( 33 ). Thus the large isoform of 1095 residues encodes the Na + -K + -2Cl - cotransporter ( 10, 34 ), whereas the shorter spliced isoform encodes the Na + -Cl - cotransporter ( 33 ). These two proteins are identical in the first 715 residues and differ in the COOH-terminal domain, 74 residues after the end of the transmembrane segment 12. From this point, there are 380 and 55 unique residues in the long and short isoforms, respectively ( 30 ). Because these two isoforms share the same central hydrophobic domain, according to our present study, both isoforms must contain the information for K + transport. Yet, shorter isoform requires no K + for Na + -Cl - translocation ( 33 ), suggesting the intriguing hypothesis that the 55 unique piece could be responsible for preventing K + translocation in the cotransporter by a ball-and-chain mechanism similar to what has been shown to occur with K + channels ( 2, 42, 46 ). Supporting this idea, there are 11 positively charged residues in the last 30 amino acids of the 55 residue unique piece. Further studies will be required to reveal the mechanisms by which the shorter isoform of the SLC12A1 gene functions as a K + -independent, Na + -Cl - cotransporter.
The chimeras containing the BSC1 COOH-terminal domain attached to TSC central hydrophobic domain (TTB and BTB) were not functional. However, we believe that results in the present study also suggest that TSC central membrane domain endows this cotransporter with the K + -independent, Na + -Cl - cotransport capacity, as well as sensitivity to thiazides diuretics. This conclusion is supported by two lines of evidence. First, chimera BTT exhibited a TSC-like behavior, indicating that NH 2 -terminal domain of TSC is not required for thiazide inhibition and, second, the fact that 22 Na + and 86 Rb + uptake in chimeras TBB, BBT, and TBT were not inhibited by metolazone, suggesting that sensitivity to thiazides does not seem to be encoded by TSC NH 2 - or COOH-terminal domains. Interestingly, although TTB chimera is synthesized and reaches the plasma membrane in oocytes, it was not functional, whereas chimeras containing BSC1 central hydrophobic domain and BTT chimera were active. Thus it is possible that, in TSC, specific residues of COOH-terminal domain are required to build a functional cotransporter. In this regard, a recent study from our laboratory ( 38 ) revealed that a Gitelman-type mutation in the glycine residue 610 that is located five residues after the end of transmembrane domain 12 results in a reduction of cotransporter activity by 90%, supporting the possibility that, in TSC, specific sequences at the beginning of the COOH-terminal are critical to allow the cotransporter to be active.
Results obtained in the present study show that it is feasible to exchange big fragments between BSC1 and TSC to obtain functional cotransporters with a level of activity that allows definition of major properties in resulting chimeras, that is, ion preferences and diuretic specificity. This observation suggests that further construction of chimeras between BSC1 and TSC could be of help to define the functional role of smaller fragments of the cotransporters. The results in this study that neither the NH 2 -terminal or COOH-terminal domains is necessary to define ion translocation and diuretic specificity, together with high identity of at least one-half of the central hydrophobic domain between BSC1 and TSC, reduce potential domains responsible for specificity differences. In this regard, as shown in Fig. 6, overall identity at the amino acid level between rat BSC1 and TSC is 52% ( 10 ), but identity degree varies along the central TM domain. The highest degree 85%; Fig. 6, black) is observed in transmembrane helixes 1, 2, 3, 6, 8, and 10. Helixes 4 and 9, and interconnecting segments facing the intracellular side of the protein, exhibit an identity degree between 55 and 75% ( Fig. 6, blue). In contrast, identity is <50% in transmembrane domains 5, 7, 11, and 12, as well as interconnecting segments facing the extracellular side of the cotransporters ( Fig. 6, red), pointing out these regions as the more divergent sequences within the central TM domain between BSC1 and TSC. Further studies will be required to narrow down the domains or residues, until the minimum required are defined.
Fig. 6. Structural differences between rat BSC1 and TSC. Shown is proposed topology of the cotransporters to highlight identities and differences between both cotransporters, as stated.
GRANTS
This work was supported by research grants 36124 and G34511M from the Mexican Council of Science and Technology, The Wellcome Trust (GR070159MA), and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-064635 (to G. Gamba).
DISCLOSURES
Preliminary results of this study were presented at the Experimental Biology 2003 Meeting of the Federation of American Societies for Experimental Biology in San Diego, CA.
ACKNOWLEDGMENTS
We are grateful to members of the Molecular Physiology Unit for suggestions and to Guillermo Soriano for assistance with frog care.
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作者单位：Molecular Physiology Unit, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México and Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Tlalpan 1400 Mexico City, Mexico