A Single Nucleotide Polymorphism Alters the Activity of the Renal Na+:Cl- Cotransporter and Reveals a Role for Transmembrane Segment 4 in Chloride and Thiazide Affinity

The thiazide-sensitive Na+:Cl- cotransporter is the major salt transport pathway in the distal convoluted tubule of the kidney, and a role of this cotransporter in blood pressure homeostasis has been defined by physiological studies on pressure natriuresis and by its involvement in monogenic diseases that feature arterial hypotension or hypertension. Data base analysis revealed that 135 single nucleotide polymorphisms along the human SLC12A3 gene that encodes the Na+:Cl- cotransporter have been reported. Eight are located within the coding region, and one results in a single amino acid change; the residue glycine at the position 264 is changed to alanine (G264A). This residue is located within the fourth transmembrane domain of the predicted structure. Because Gly-264 is a highly conserved residue, we studied the functional properties of this polymorphism by using in vitro mutagenesis and the heterologous expression system in Xenopus laevis oocytes. G264A resulted in a significant and reproducible reduction (∼50%) in 22Na+ uptake when compared with the wild type cotransporter. The affinity for extracellular Cl- and for thiazide diuretics was increased in G264A. Western blot analysis showed similar immunoreactive bands between the wild type and the G264A cotransporters, and confocal images of oocytes injected with enhanced green fluorescent protein-tagged wild type and G264A cotransporter showed no differences in the protein surface expression level. These observations suggest that the G264A polymorphism is associated with reduction in the substrate translocation rate of the cotransporter, due to a decrease in the intrinsic activity. Our study also reveals a role of the transmembrane segment 4 in defining the affinity for extracellular Cl- and thiazide diuretics. The thiazide-sensitive Na+:Cl- cotransporter is the major salt transport pathway in the distal convoluted tubule of the kidney, and a role of this cotransporter in blood pressure homeostasis has been defined by physiological studies on pressure natriuresis and by its involvement in monogenic diseases that feature arterial hypotension or hypertension. Data base analysis revealed that 135 single nucleotide polymorphisms along the human SLC12A3 gene that encodes the Na+:Cl- cotransporter have been reported. Eight are located within the coding region, and one results in a single amino acid change; the residue glycine at the position 264 is changed to alanine (G264A). This residue is located within the fourth transmembrane domain of the predicted structure. Because Gly-264 is a highly conserved residue, we studied the functional properties of this polymorphism by using in vitro mutagenesis and the heterologous expression system in Xenopus laevis oocytes. G264A resulted in a significant and reproducible reduction (∼50%) in 22Na+ uptake when compared with the wild type cotransporter. The affinity for extracellular Cl- and for thiazide diuretics was increased in G264A. Western blot analysis showed similar immunoreactive bands between the wild type and the G264A cotransporters, and confocal images of oocytes injected with enhanced green fluorescent protein-tagged wild type and G264A cotransporter showed no differences in the protein surface expression level. These observations suggest that the G264A polymorphism is associated with reduction in the substrate translocation rate of the cotransporter, due to a decrease in the intrinsic activity. Our study also reveals a role of the transmembrane segment 4 in defining the affinity for extracellular Cl- and thiazide diuretics. The thiazide-sensitive Na+:Cl- cotransporter (TSC, 1The abbreviations used are: TSC, thiazide-sensitive Na+:Cl- cotransporter; EGFP, enhanced green fluorescent protein; SNPs, single nucleotide polymorphisms; WT, wild type. 1The abbreviations used are: TSC, thiazide-sensitive Na+:Cl- cotransporter; EGFP, enhanced green fluorescent protein; SNPs, single nucleotide polymorphisms; WT, wild type. gene symbol, SLC12A3; locus ID 6559) is the major NaCl transport pathway in the apical membrane of the mammalian distal convoluted tubule and the teleost urinary bladder (1Stokes J.B. Lee I. D'Amico M. J. Clin. Investig. 1984; 74: 7-16Crossref PubMed Scopus (100) Google Scholar, 2Gamba G. Saltzberg S.N. Lombardi M. Miyanoshita A. Lytton J. Hediger M.A. Brenner B.M. Hebert S.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2749-2753Crossref PubMed Scopus (338) Google Scholar, 3Kunau R.T. Weller D.R. Webb H.L. J. Clin. Investig. 1975; 56: 401-407Crossref PubMed Scopus (87) Google Scholar, 4Velazquez H. Good D.W. Wright F.S. Am. J. Physiol. 1984; 247: F904-F911PubMed Google Scholar, 5Costanzo L.S. Am. J. Physiol. 1985; 248: F527-F535Crossref PubMed Google Scholar, 6Ellison D.H. Velazquez H. Wright F.S. Am. J. Physiol. 1987; 253: F546-F554PubMed Google Scholar, 7Plotkin M.D. Kaplan M.R. Verlander J.M. Lee W.-S. Brown D. Poch E. Gullans S.R. Hebert S.C. Kidney Int. 1996; 50: 174-183Abstract Full Text PDF PubMed Scopus (173) Google Scholar). The fundamental role of the Na+:Cl- cotransporter encoded by the SLC12A3 gene in preserving the extracellular fluid volume and divalent cation homeostasis has been firmly established by the identification of inactivating mutations of this gene as the cause of Gitelman's disease (8Simon D.B. Nelson-Williams C. Johnson-Bia M. Ellison D. Karet F.E. Morey-Molina A. Vaara I. Iwata F. Cushner H.M. Koolen M. Gainza F.J. Gitelman H.J. Lifton R.P. Nat. Genet. 1996; 12: 24-30Crossref PubMed Scopus (1033) Google Scholar, 9Mastroianni N. DeFusco M. Zollo M. Arrigo G. Zuffardi O. Bettinelli A. Ballabio A. Casari G. Genomics. 1996; 35: 486-493Crossref PubMed Scopus (112) Google Scholar, 10Mastroianni N. Bettinelli A. Bianchetti M. Colussi G. de Fusco M. Sereni F. Ballabio A. Casari G. Am. J. Hum. Genet. 1996; 59: 1019-1026PubMed Google Scholar), an inherited disorder featuring arterial hypotension, hypokalemic metabolic alkalosis with hypocalciuria, hypomagnesemia, and renal salt wasting. TSC also serves as the target for the thiazide-type diuretics that are currently recommended as the drug of choice for treatment of hypertension (11Chobanian A.V. Bakris G.L. Black H.R. Cushman W.C. Green L.A. Izzo Jr., J.L. Jones D.W. Materson B.J. Oparil S. Wright Jr., J.T. Roccella E.J. J. Am. Med. Assoc. 2003; 289: 2560-2571Crossref PubMed Scopus (16367) Google Scholar). Finally, a defect in TSC regulation by the WNK1 and WNK4 kinases has been implicated in the pathogenesis of a salt-dependent form of human hypertension known as pseudohyopaldosteronism type II (12Wilson F.H. Kahle K.T. Sabath E. Lalioti M.D. Rapson A.K. Hoover R.S. Hebert S.C. Gamba G. Lifton R.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 680-684Crossref PubMed Scopus (356) Google Scholar, 13Yang C.L. Angell J. Mitchell R. Ellison D.H. J. Clin. Investig. 2003; 111: 1039-1045Crossref PubMed Scopus (397) Google Scholar), which features marked sensitivity to hydrochlorothiazide and a clinical picture that is a mirror image of Gitelman's disease (hypertension, hyperkalemia, and metabolic acidosis) (14Mayan H. Vered I. Mouallem M. Tzadok-Witkon M. Pauzner R. Farfel Z. J. Clin. Endocrinol. Metab. 2002; 87: 3248-3254Crossref PubMed Scopus (150) Google Scholar). Taken together, all these observations suggest that TSC molecular variants, resulting from single nucleotide polymorphisms (SNPs), could contribute to the normal variations in blood pressure in the population at large, to the inherited predisposition toward essential hypertension, and/or to the differential response to diuretic therapy.Despite the important role of TSC in cardiovascular physiology, pharmacology, and pathophysiology, little is currently known about the structure-function relationships in this cotransporter. By using [3H]metolazone binding to membrane preparations from rat renal cortex, Tran et al. (15Tran J.M. Farrell M.A. Fanestil D.D. Am. J. Physiol. 1990; 258: F908-F915PubMed Google Scholar) proposed that thiazides and Cl- share the same binding site. Recent studies in which the functional properties of the cloned cotransporter were determined, however, provided evidence that metolazone competes with both Na+ and Cl- ions (16Monroy A. Plata C. Hebert S.C. Gamba G. Am. J. Physiol. 2000; 279: F161-F169Crossref PubMed Google Scholar), suggesting that the thiazide-binding site may be shared by both ions and not only by Cl-, as suggested by Tran et al. (15Tran J.M. Farrell M.A. Fanestil D.D. Am. J. Physiol. 1990; 258: F908-F915PubMed Google Scholar). In addition, nothing is known regarding domains or amino acid residues defining the TSC ion transport kinetics or thiazide affinity. So far, within the family of electroneutral cotransporters, some aspects of the structure-function relationships have been investigated only in the two isoforms of the Na+:K+:2Cl- cotransporter, BSC1/NKCC2 and BSC2/NKCC1. Results between both isoforms, however, have shown important differences suggesting that conclusions reached in one member of the family cannot be extended to the other members. For example, in BSC2/NKCC1, Isenring et al. (17Isenring P. Forbush III, B. J. Biol. Chem. 1997; 272: 24556-24562Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 18Isenring P. Jacoby S.C. Chang J. Forbush III, B. J. Gen. Physiol. 1998; 112: 549-558Crossref PubMed Scopus (77) Google Scholar) have implicated transmembrane domains 4 and 7 in defining Cl- transport affinity, whereas recent studies (19Plata C. Meade P. Vazquez N. Hebert S.C. Gamba G. J. Biol. Chem. 2002; 277: 11004-11012Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 20Gimenez I. Isenring P. Forbush III, B. J. Biol. Chem. 2002; 277: 8767-8770Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 21Gagnon E. Bergeron M.J. Brunet G.M. Daigle N.D. Simard C.F. Isenring P. J. Biol. Chem. 2003; 278: 5648-5654Google Scholar) in BSC1/NKCC2 clearly showed that transmembrane domain 2 contains affinity modifier residues for extracellular Cl-.In the present study we show that an SNP that changes one amino acid residue in TSC results in a dramatic decrease in TSC function, apparently secondary to a decrease in the intrinsic activity of the cotransporter, and reveals a role of transmembrane segment 4 in TSC affinity for extracellular Cl- and for thiazide diuretics.MATERIALS AND METHODSAn extensive search of genome data bases (lpgws.nci.nih.gov/cgi-bin/GeneViewer.cgi; ncbi.nlm.nih.gov/SNP) was performed to find the SNPs that have been informed within the SLC12A3 gene. The SNPs within the coding regions that were considered as potentially important were incorporated into the rat TSC cDNA by using the QuickChange site-directed mutagenesis system (Stratagene) following the manufacturer's recommendations. Automatic DNA sequencing was used to corroborate all the mutations. All primers used for mutagenesis were custom-made (Sigma).Genotyping of the G264A Polymorphism—A restriction fragment length polymorphism method was created for the G264A polymorphism to confirm it and to simplify its detection in 200 normal subjects. Total genomic DNA was extracted from whole blood according to standard procedures. PCR was conducted using 125 ng of genomic DNA using the primer pair sense 5′-AGACCGTGCGGGACCTGCTC-3′ and antisense 5′-CCTCCTCCATGGCCTCCTCACCTT-3′. PCR was conducted for 34 cycles with denaturation at 96 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s, with a final extension step at 72 °C for 5 min. The G264A variant was recognized by restriction fragment length polymorphism by using Btg1 (New England Biolabs), and the restriction fragments were separated on 7.5% PAGE, and visualized under ultraviolet light after staining with ethidium bromide. The polymorphism was confirmed by automatic sequencing (AbiPrism®) in all positive cases.Assessment of the Na+:Cl-Cotransporter Function—Oocytes were harvested from anesthetized adult female Xenopus laevis frogs, defolliculated, and prepared for microinjection following our standard procedure (16Monroy A. Plata C. Hebert S.C. Gamba G. Am. J. Physiol. 2000; 279: F161-F169Crossref PubMed Google Scholar, 19Plata C. Meade P. Vazquez N. Hebert S.C. Gamba G. J. Biol. Chem. 2002; 277: 11004-11012Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The next day mature oocytes were injected with 50 nl of water or cRNA transcribed in vitro, using the T7 RNA polymerase mMESSAGE kit (Ambion), at a concentration of 0.5 μg/μl. Oocytes were then incubated for 3 days in ND96 with sodium pyruvate and gentamicin and 1 day in Cl--free ND96 (16Monroy A. Plata C. Hebert S.C. Gamba G. Am. J. Physiol. 2000; 279: F161-F169Crossref PubMed Google Scholar). The function of the Na+:Cl- cotransporter was determined by assessing tracer 22Na+ uptake (PerkinElmer Life Sciences) in groups of at least 15 oocytes following our standard protocol (16Monroy A. Plata C. Hebert S.C. Gamba G. Am. J. Physiol. 2000; 279: F161-F169Crossref PubMed Google Scholar): 30 min of incubation in a Cl--free ND96 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 containing the same drugs plus 2 μCi of 22Na+ per ml. Gluconate was used as a Cl- substitute and N-methyl-d-glucamine as a Na+ substitute. At the end of the uptake period tracer activity was determined for each dissolved oocyte by β-scintillation counting.Western Blotting—Western blot analysis was used to compare WT with mutant protein in cRNA-injected oocytes following our standard protocol (22Meade P. Hoover R.S. Plata C. Vazquez N. Bobadilla N.A. Gamba G. Hebert S.C. Am. J. Physiol. 2003; 284: F1145-F1154Crossref PubMed Scopus (59) Google Scholar). In brief, groups of 15 oocytes injected with water or cRNA were homogenized in 2 μl/oocyte of homogenization buffer and centrifuged twice at 100 × g for 10 min at 4 °C, and the supernatant was collected. Protein extracts from oocytes (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% β-mercaptoethanol, resolved by Laemmli SDS-7.5% PAGE, and transferred to a polyvinylidene difluoride membrane. For immunodetection we used a rabbit polyclonal anti-rat TSC antibody (generously provided by Dr. Mark Knepper, National Institutes of Health), diluted 1:1000 (23Wang X.Y. Masilamani S. Nielsen J. Kwon T.H. Brooks H.L. Nielsen S. Knepper M.A. J. Clin. Investig. 2001; 108: 215-222Crossref PubMed Scopus (107) Google Scholar). The membrane was exposed to anti-TSC antibody diluted in blocking buffer (TTBS, 0.2% Tween 20) overnight at 4 °C, subsequently washed in TTBS, and incubated for 60 min at room temperature with alkaline phosphatase-conjugated secondary (anti-rabbit) antibody (Bio-Rad) diluted 1:2000 in blocking buffer and washed again. Immunoreactive species were detected using Immun-Star Chemiluminescent Protein Detection Systems (Bio-Rad).Assessment of the TSC Expression at the Oocytes Plasma Membrane—The surface expression of wild type or mutant TSC (see below) was determined by assessing the fluorescence in the Xenopus oocytes using a TSC fusion construct that we have previously validated (12Wilson F.H. Kahle K.T. Sabath E. Lalioti M.D. Rapson A.K. Hoover R.S. Hebert S.C. Gamba G. Lifton R.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 680-684Crossref PubMed Scopus (356) Google Scholar, 24Hoover R.S. Poch E. Monroy A. Vazquez N. Nishio T. Gamba G. Hebert S.C. J. Am. Soc. Nephrol. 2003; 14: 271-282Crossref PubMed Scopus (84) Google Scholar). In this construct, the EGFP was fused to the amino-terminal domain of TSC. Xenopus oocytes were then microinjected with water as control or with EGFP-WT-TSC or EGFP-mutant-TSC cRNA. After 4 days of incubation, oocytes were monitored for EGFP fluorescence in the oocytes surface using a Zeiss laser-scanning confocal microscope (objective lens ×10, Nikon). Excitation and emission wavelengths used to visualize EGFP fluorescence were 488 and 515–565 nm, respectively. We have shown previously that EGFP-TSC fluorescence in the oocytes surface colocalizes with the F-404 specific plasma membrane dye and that oocytes injected with EGFP-TSC exhibit significant thiazide-sensitive 22Na+ uptake, indicating the EGFP-TSC fluorescence is located in the plasma membrane (24Hoover R.S. Poch E. Monroy A. Vazquez N. Nishio T. Gamba G. Hebert S.C. J. Am. Soc. Nephrol. 2003; 14: 271-282Crossref PubMed Scopus (84) Google Scholar). For densitometry analysis, the plasma membrane fluorescence was quantified by determining the pixel intensity around the entire oocytes circumference using SigmaScan Pro image analysis software.Statistical Analysis—Statistical significance is defined as two-tailed p < 0.05, and the results are presented as mean ± S.E. The significance of the differences between means was tested with the Student's t test.RESULTSSingle Nucleotide Polymorphisms in the SLC12A3 Gene—Up to 135 SNPs have been informed within the SLC12A3 gene. 127 SNPs are located within intronic sequences and only eight are within exonic sequences. Fig. 1 depicts the proposed TSC topology (25Gamba G. Miyanoshita A. Lombardi M. Lytton J. Lee W.S. Hediger M.A. Hebert S.C. J. Biol. Chem. 1994; 269: 17713-17722Abstract Full Text PDF PubMed Google Scholar) and the localization of the eight SNPs within the coding sequence. Six SNPs result in no change of the amino acid sequence. These are the SNPs A122A, T465T, S628S, A714A, G875G, and I1017I corresponding to the NCBI SNP cluster IDs rs2304479, rs5801, rs55802, rs5803, rs5804, and rs2289113, respectively. Two SNPs result in a single amino acid change. One is the R863K SNP (cluster ID rs8060046) that was considered as irrelevant because this SNP located within the carboxyl-terminal domain results in a conserved substitution of the positively charged amino acid arginine, which is present in the human cotransporter (8Simon D.B. Nelson-Williams C. Johnson-Bia M. Ellison D. Karet F.E. Morey-Molina A. Vaara I. Iwata F. Cushner H.M. Koolen M. Gainza F.J. Gitelman H.J. Lifton R.P. Nat. Genet. 1996; 12: 24-30Crossref PubMed Scopus (1033) Google Scholar), for the positively charged residue lysine, which is present in the TSC from rabbit, rat, mouse, and fish (2Gamba G. Saltzberg S.N. Lombardi M. Miyanoshita A. Lytton J. Hediger M.A. Brenner B.M. Hebert S.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2749-2753Crossref PubMed Scopus (338) Google Scholar, 25Gamba G. Miyanoshita A. Lombardi M. Lytton J. Lee W.S. Hediger M.A. Hebert S.C. J. Biol. Chem. 1994; 269: 17713-17722Abstract Full Text PDF PubMed Google Scholar, 26Velazquez H. Naray-Fejes-Toth A. Silva T. Andujar E. Reilly R.F. Desir G.V. Ellison D.H. Kidney Int. 1998; 54: 464-472Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 27Kunchaparty S. Palcso M. Berkman J. Zquez H. Desir G.V. Bernstein P. Reilly R.F. Ellison D.H. Am. J. Physiol. 1999; 277: F643-F649PubMed Google Scholar). In contrast, the other SNP that alter the primary sequence of human TSC (ID number rs1529927) predicts a change of the nonpolar amino acid glycine at the position 264 for the residue alanine. The residue glycine is located within the fourth transmembrane domain and is a conserved amino acid residue, not only in the available TSC sequences from rat (25Gamba G. Miyanoshita A. Lombardi M. Lytton J. Lee W.S. Hediger M.A. Hebert S.C. J. Biol. Chem. 1994; 269: 17713-17722Abstract Full Text PDF PubMed Google Scholar), mouse (27Kunchaparty S. Palcso M. Berkman J. Zquez H. Desir G.V. Bernstein P. Reilly R.F. Ellison D.H. Am. J. Physiol. 1999; 277: F643-F649PubMed Google Scholar), rabbit (26Velazquez H. Naray-Fejes-Toth A. Silva T. Andujar E. Reilly R.F. Desir G.V. Ellison D.H. Kidney Int. 1998; 54: 464-472Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), human (8Simon D.B. Nelson-Williams C. Johnson-Bia M. Ellison D. Karet F.E. Morey-Molina A. Vaara I. Iwata F. Cushner H.M. Koolen M. Gainza F.J. Gitelman H.J. Lifton R.P. Nat. Genet. 1996; 12: 24-30Crossref PubMed Scopus (1033) Google Scholar), and flounder (2Gamba G. Saltzberg S.N. Lombardi M. Miyanoshita A. Lytton J. Hediger M.A. Brenner B.M. Hebert S.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2749-2753Crossref PubMed Scopus (338) Google Scholar), but also in all members of SLC12 family that include two genes encoding Na+:K+:2Cl- cotransporters (25Gamba G. Miyanoshita A. Lombardi M. Lytton J. Lee W.S. Hediger M.A. Hebert S.C. J. Biol. Chem. 1994; 269: 17713-17722Abstract Full Text PDF PubMed Google Scholar, 28Delpire E. Rauchman M.I. Beier D.R. Hebert S.C. Gullans S.R. J. Biol. Chem. 1994; 269: 25677-25683Abstract Full Text PDF PubMed Google Scholar) and four genes encoding K+:Cl- cotransporters (29Gillen C.M. Brill S. Payne J.A. Forbush III, B. J. Biol. Chem. 1996; 271: 16237-16244Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 30Payne J.A. Stevenson T.J. Donaldson L.F. J. Biol. Chem. 1996; 271: 16245-16252Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar, 31Mount D.B. Mercado A. Song L. Xu J. Geroge Jr., A.L. Delpire E. Gamba G. J. Biol. Chem. 1999; 274: 16355-16362Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Thus, the G264A SNP was considered as potentially important and therefore was introduced into TSC by in vitro mutagenesis.Allele Frequency of the G264A Polymorphism—A restriction fragment length polymorphism strategy was used to assess the presence of G264A SNP by PCR. The PCR product (510 bp) contained a constant Btg1 restriction site, and therefore, when digested, two bands of 461 and 49 bp, respectively, are observed in GG genotype (encoding glycine at position 264 in both alleles). In contrast, in GA genotype heterozygotes (that is one allele encoding glycine and the other encoding alanine at position 264), a new specific Btg1 restriction site was used, and then four bands of 461, 390, 71, and 49 bp were observed. To test the allele frequency of the G264A polymorphism, 200 Caucasian subjects were genotyped. The sample included 119 males and 81 females with the following characteristics (mean ± S.D.): age 52 ± 16 years, systolic blood pressure 117 ± 11 mmHg, diastolic blood pressure 69 ± 7 mmHg, and body mass index of 24 ± 4 kg/m2. None of the subjects had present or past cardiovascular conditions including hypertension, coronary heart disease, stroke, or diabetes. The frequency of the GA genotype was 2% in the sample studied. No subjects with AA phenotype were detected (that is homozygotes encoding alanine at position 264 in both alleles). Shown in Fig. 2A is a representative gel containing the Btg1-digested PCR fragment from a normal subject and one heterozygous for the GA genotype. Fig. 2, B and C, illustrates sequencing of this region in a GG and a GA phenotype (codons GGC and GCC, respectively). Thus, our results suggest that G264A is a true SNP.Fig. 2A, example of a genotyping result for the Gly-264 → Ala (G264A) polymorphism. The polymorphism consists in a G to C transversion at codon 264 that changed the glycine-encoding codon GGC to the alanine-encoding codon, GCC. The PCR products were digested with Btg1 and resolved on SDS-PAGE: lane 1, molecular weight marker, lane 2, GA heterozygous (codon GCC); lane 3, GG homozygous (codon GGC), and lane 4 (undigested PCR product). B, sequence of wild type and C, polymorphic variant G264A PCR products were excised from the gel and fully sequenced.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Functional Consequences of the TSC G264A Polymorphism— The functional consequences of the G264A SNP were assessed using a heterologous expression system in X. laevis oocytes. This expression system has been shown to be an excellent tool for a robust and reproducible expression of TSC in our hands (2Gamba G. Saltzberg S.N. Lombardi M. Miyanoshita A. Lytton J. Hediger M.A. Brenner B.M. Hebert S.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2749-2753Crossref PubMed Scopus (338) Google Scholar, 12Wilson F.H. Kahle K.T. Sabath E. Lalioti M.D. Rapson A.K. Hoover R.S. Hebert S.C. Gamba G. Lifton R.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 680-684Crossref PubMed Scopus (356) Google Scholar, 16Monroy A. Plata C. Hebert S.C. Gamba G. Am. J. Physiol. 2000; 279: F161-F169Crossref PubMed Google Scholar, 24Hoover R.S. Poch E. Monroy A. Vazquez N. Nishio T. Gamba G. Hebert S.C. J. Am. Soc. Nephrol. 2003; 14: 271-282Crossref PubMed Scopus (84) Google Scholar, 25Gamba G. Miyanoshita A. Lombardi M. Lytton J. Lee W.S. Hediger M.A. Hebert S.C. J. Biol. Chem. 1994; 269: 17713-17722Abstract Full Text PDF PubMed Google Scholar, 32Vazquez N. Monroy A. Dorantes E. Munoz-Clares R.A. Gamba G. Am. J. Physiol. 2002; 282: F599-F607Crossref PubMed Scopus (33) Google Scholar) and also in other laboratories (13Yang C.L. Angell J. Mitchell R. Ellison D.H. J. Clin. Investig. 2003; 111: 1039-1045Crossref PubMed Scopus (397) Google Scholar, 27Kunchaparty S. Palcso M. Berkman J. Zquez H. Desir G.V. Bernstein P. Reilly R.F. Ellison D.H. Am. J. Physiol. 1999; 277: F643-F649PubMed Google Scholar, 33De Jong J.C. Willems P.H. Mooren F.J. van den Heuvel L.P. Knoers N.V. Bindels R.J. J. Biol. Chem. 2003; 278: 24302-24307Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 34De Jong J.C. Van Der Vliet W.A. van den Heuvel L.P. Willems P.H. Knoers N.V. Bindels R.J. J. Am. Soc. Nephrol. 2002; 13: 1442-1448Crossref PubMed Scopus (132) Google Scholar). In contrast, TSC expression in transfected eukaryotic cells has not been successful in many laboratories, including our own. The best expression so far observed in stably transfected eukaryotic cells (MDCK cells) with human TSC cDNA consists of small increase (∼25%) over background (35De Jong J.C. Willems P.H. van den Heuvel L.P. Knoers N.V. Bindels R.J. J. Am. Soc. Nephrol. 2003; 14: 2428-2435Crossref PubMed Scopus (23) Google Scholar). Thus, X. laevis oocytes were microinjected with cRNA transcribed from wild type TSC (WT) or from TSC harboring the G264A SNP (G264A). As shown in Fig. 3, WT or G264A cRNA injection induced a significant increase in 22Na+ uptake in X. laevis oocytes. However, the increase in G264A-injected oocytes was of a significantly lower magnitude than the increase observed in WT oocytes. Uptake in WT-injected oocytes was 3448 ± 234 pmol oocyte-1 h-1, whereas in G264A-injected oocytes was 1712 ± 366 pmol oocyte-1 h-1 (p < 0.01, n = 20). As shown in Fig. 3, the uptake was due to the TSC activity because a complete inhibition of uptake was observed in the absence of extracellular chloride or in the presence of the thiazide-type diuretic, metolazone. A reduction of a similar magnitude in the G264A activity was consistently observed in every single experiment. In order to ensure that the reduction in 22Na+ uptake was not due to differences in the amount of cRNA injected or in the transcription rate of the protein, cRNA concentration was determined by densitometry of the corresponding bands in the ethidium bromide-stained agarose gel, and the transcribed protein was assessed by Western blot analysis of the injected oocytes. No differences were found between WT and G264A in the amount of cRNA injected (Fig. 3B), as well as in the TSC protein produced by the oocytes (Fig. 3C).Fig. 3Functional expression of WT and G264A cotransporters in X. laevis oocytes.A, 22Na+ uptake in oocytes that were injected with water, with 25 ng of cRNA from WT, or from G264A. Uptake was assessed in control conditions (open bars), in the absence of extracellular Cl- (hatched bars), or in the presence of 10-4m of the inhibitor metolazone (closed bars). The absence of endogenous thiazide-inhibitable 22Na+ uptake in X. laevis oocytes has been shown before (2Gamba G. Saltzberg S.N. Lombardi M. Miyanoshita A. Lytton J. Hediger M.A. Brenner B.M. Hebert S.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2749-2753Crossref PubMed Scopus (338) Google Scholar, 16Monroy A. Plata C. Hebert S.C. Gamba G. Am. J. Physiol. 2000; 279: F161-F169Crossref PubMed Google Scholar, 25Gamba G. Miyanoshita A. Lombardi M. Lytton J. Lee W.S. Hediger M.A. Hebert S.C. J. Biol. Chem. 1994; 269: 17713-17722Abstract Full Text PDF PubMed Google Scholar). *, p < 0.01 versus WT cRNA oocytes in control conditions. n = 20 oocytes per bar. B, ethidium bromide-stained agarose gel showing 2 μl of 0.5 μg/μl of WT and G264A cRNA as stated. C, autoradiograms of Western blot analysis of proteins extracted from WT or G264A cRNA-injected oocytes, as stated. The analysis was performed using rabbit polyclonal anti-TSC antibodies. Comparable immunoreactivities are observed in both lanes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Surface Expression Analysis in EGFP-WT or EGFP-G264A— Because X. laevis oocytes injected with WT or G264A exhibited similar immunoreactive proteins in the Western blot, we reasoned that potential explanations for the reduction in functional expression of the TSC containing the G264A could be a decrease in the amount of the transporter that reaches the plasma membrane, a decrease in the affinity for the cotransported ions, or a decrease in the intrinsic activity of the cotransporter. To study the first possibility, we assessed the surface expression of the WT and G264A proteins by injecting X. laevis oocytes with the cRNA encoding the WT or G264A cotransporters that had been tagged previously with the EGFP. To perform these experiments, we used the EGFP-tagged TSC construct that we have characterized previously (24Hoover R.S. Poch E. Mo