Portal de Periódicos da CAPES

Molecular Determinants of pH Sensitivity of the Type IIa Na/Pi Cotransporter

2000; Elsevier BV; Volume: 275; Issue: 9 Linguagem: Inglês

10.1074/jbc.275.9.6284

1083-351X

Carmen de la Horra, Nati Hernando, Georg Lambert, Ian C. Forster, Jürg Biber, Heini Murer,

Plant Stress Responses and Tolerance

Type II Na/Pi cotransporters play key roles in epithelial Pi transport and thereby contribute to overall Pi homeostasis. Renal proximal tubular brush border membrane expresses the IIa isoform, whereas the IIb isoform is preferentially expressed in small intestinal brush border membrane of mammals. IIa and IIb proteins are predicted to contain eight transmembrane domains with the N- and C-terminal tails facing the cytoplasm. They differ in their pH dependences: the activity of IIa increases at higher pH, whereas the IIb shows no or a slightly opposite pH dependence. To determine the structural domains responsible for the difference in pH sensitivity, mouse IIa and IIb chimeras were constructed, and their pH dependence was characterized. A region between the fourth and fifth transmembrane domains was required for conferring pH sensitivity to the IIa-mediated Na/Picotransport. Sequence comparison (IIa versus IIb) of the third extracellular loops revealed a stretch of three charged amino acids in IIa (REK) replaced by uncharged residues in IIb (GNT). Introduction of the uncharged GNT sequence (by REK) in IIa abolished its pH dependence, whereas introduction of the charged REK stretch in IIb (by GNT) led to a pH dependence similar to IIa. These findings suggest that charged residues within the third extracellular loop are involved in the pH sensitivity of IIa Na/Pi cotransporter. Type II Na/Pi cotransporters play key roles in epithelial Pi transport and thereby contribute to overall Pi homeostasis. Renal proximal tubular brush border membrane expresses the IIa isoform, whereas the IIb isoform is preferentially expressed in small intestinal brush border membrane of mammals. IIa and IIb proteins are predicted to contain eight transmembrane domains with the N- and C-terminal tails facing the cytoplasm. They differ in their pH dependences: the activity of IIa increases at higher pH, whereas the IIb shows no or a slightly opposite pH dependence. To determine the structural domains responsible for the difference in pH sensitivity, mouse IIa and IIb chimeras were constructed, and their pH dependence was characterized. A region between the fourth and fifth transmembrane domains was required for conferring pH sensitivity to the IIa-mediated Na/Picotransport. Sequence comparison (IIa versus IIb) of the third extracellular loops revealed a stretch of three charged amino acids in IIa (REK) replaced by uncharged residues in IIb (GNT). Introduction of the uncharged GNT sequence (by REK) in IIa abolished its pH dependence, whereas introduction of the charged REK stretch in IIb (by GNT) led to a pH dependence similar to IIa. These findings suggest that charged residues within the third extracellular loop are involved in the pH sensitivity of IIa Na/Pi cotransporter. polymerase chain reaction wild type kilobase transmembrane domain The kidney and the small intestine are involved in maintaining overall phosphate (Pi) homeostasis. Renal tubular Pi reabsorption contributes to "acute" and "chronic" regulatory control, whereas only slow "adaptive" changes occur in small intestinal Pi absorption (1.Dennis V. Windhager E.E. Renal Physiology Handbook of Physiology. 2nd Ed. American Physiological Society, Bethesda, MD1992: 1785-1815Google Scholar, 2.Berndt T. Knox F. Seldin D.W. Giebish G. The Kidney: Physiology and Pathophysiology. Raven Press, New York1992: 2511-2532Google Scholar, 3.Danisi G. Murer H. Field M. Frizzel R.A. Handbook of Physiology. 2nd Ed. Oxford University Press, New York1991: 232-336Google Scholar, 4.Murer H. Biber J. Seldin D.W. Giebish G. The Kidney: Physiology and Pathophysiology. Raven Press, New York1992: 2481-2590Google Scholar). Na/Pi cotransporters located in renal proximal tubular and small intestinal brush border membrane are the rate-limiting and physiologically controlled steps (4.Murer H. Biber J. Seldin D.W. Giebish G. The Kidney: Physiology and Pathophysiology. Raven Press, New York1992: 2481-2590Google Scholar, 5.Cross H. Debiec H. Peterlik M. Miner. Electrolyte Metab. 1990; 16: 115-124PubMed Google Scholar, 6.Murer H. Lotscher M. Kaissling B. Levi M. Kempson S.A. Biber J. Kidney Int. 1996; 49: 1769-1773Abstract Full Text PDF PubMed Scopus (57) Google Scholar). Recently they have been structurally identified as type II Na/Pi cotransporters (7.Magagnin S. Werner A. Markovich D. Sorribas V. Stange G. Biber J. Murer H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5979-5983Crossref PubMed Scopus (334) Google Scholar, 8.Murer H. Biber J. Annu. Rev. Physiol. 1996; 58: 607-618Crossref PubMed Scopus (96) Google Scholar, 9.Murer H. Biber J. Pfluegers Arch. Eur. J. Physiol. 1997; 433: 379-389Crossref PubMed Scopus (90) Google Scholar, 10.Biber J. Murer H. Curr. Opin. Cell Biol. 1998; 10: 429-434Crossref PubMed Scopus (10) Google Scholar, 11.Hilfiker H. Hattenhauer O. Traebert M. Forster I. Murer H. Biber J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14564-14569Crossref PubMed Scopus (321) Google Scholar): the proximal tubule expresses the type IIa isoform (7.Magagnin S. Werner A. Markovich D. Sorribas V. Stange G. Biber J. Murer H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5979-5983Crossref PubMed Scopus (334) Google Scholar, 12.Custer M. Lotscher M. Biber J. Murer H. Kaissling B. Am. J. Physiol. 1994; 266: F767-F774PubMed Google Scholar), whereas the type IIb isoform in mammals is preferentially expressed in small intestine and in other tissues such as lung, colon, liver, and testis but not in kidney (11.Hilfiker H. Hattenhauer O. Traebert M. Forster I. Murer H. Biber J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14564-14569Crossref PubMed Scopus (321) Google Scholar). Mouse IIa and IIb cotransporters show an overall identity of 57%, which increases to 75% in the predicted transmembrane domains (11.Hilfiker H. Hattenhauer O. Traebert M. Forster I. Murer H. Biber J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14564-14569Crossref PubMed Scopus (321) Google Scholar). Most of the differences are found in the N- and C-terminal regions (11.Hilfiker H. Hattenhauer O. Traebert M. Forster I. Murer H. Biber J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14564-14569Crossref PubMed Scopus (321) Google Scholar). A topology model of both cotransporters, based on hydropathy analysis and epitope tagging, predicts eight transmembrane segments, with the N- and C-terminal tails facing the cytoplasm and a large hydrophilic loop between the third and fourth transmembrane domains (7.Magagnin S. Werner A. Markovich D. Sorribas V. Stange G. Biber J. Murer H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5979-5983Crossref PubMed Scopus (334) Google Scholar,11.Hilfiker H. Hattenhauer O. Traebert M. Forster I. Murer H. Biber J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14564-14569Crossref PubMed Scopus (321) Google Scholar, 13.Lambert G. Traebert M. Hernando N. Biber J. Murer H. Pfluegers Arch. Eur. J. Physiol. 1999; 437: 972-978Crossref PubMed Scopus (76) Google Scholar). This large loop is glycosylated (14.Hayes G. Busch A. Lotscher M. Waldegger S. Lang F. Verrey F. Biber J. Murer H. J. Biol. Chem. 1994; 269: 24143-24149Abstract Full Text PDF PubMed Google Scholar). The kinetic properties of mouse IIa and IIb cotransporters have been characterized after expression in Xenopus laevis oocytes. There are some differences in the affinities for the substrates, Na+ and Pi (11.Hilfiker H. Hattenhauer O. Traebert M. Forster I. Murer H. Biber J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14564-14569Crossref PubMed Scopus (321) Google Scholar). Moreover, IIa and IIb show different pH dependence of both 32Pi uptake and Pi-induced electrogenic responses (11.Hilfiker H. Hattenhauer O. Traebert M. Forster I. Murer H. Biber J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14564-14569Crossref PubMed Scopus (321) Google Scholar, 15.Hartmann C.M. Wagner C.A. Busch A.E. Markovich D. Biber J. Lang F. Murer H. Pfluegers Arch. Eur. J. Physiol. 1995; 430: 830-836Crossref PubMed Scopus (73) Google Scholar). For the IIa, a decrease in external pH results in a significant decrease in transport activity (up to 80%) (7.Magagnin S. Werner A. Markovich D. Sorribas V. Stange G. Biber J. Murer H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5979-5983Crossref PubMed Scopus (334) Google Scholar, 15.Hartmann C.M. Wagner C.A. Busch A.E. Markovich D. Biber J. Lang F. Murer H. Pfluegers Arch. Eur. J. Physiol. 1995; 430: 830-836Crossref PubMed Scopus (73) Google Scholar), whereas IIb shows no or a slightly opposite pH dependence (11.Hilfiker H. Hattenhauer O. Traebert M. Forster I. Murer H. Biber J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14564-14569Crossref PubMed Scopus (321) Google Scholar). The characteristics of the type IIa cotransporter determine, to a large extent, Na+-dependent Pireabsorption at the proximal tubular brush border membrane (16.Beck L. Karapalis A.C. Amikuza N. Hewson A.S. Ozawa H. Tenhouse H.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5372-5377Crossref PubMed Scopus (524) Google Scholar, 17.Oberbauer R. Scheneider G.F. Biber J. Murer H. Meyer T.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4903-4906Crossref PubMed Scopus (46) Google Scholar). The pH dependence of this activity has been extensively characterized by vesicle uptake studies (18.Cheng L. Sacktor B. J. Biol. Chem. 1981; 256: 1556-1564Abstract Full Text PDF PubMed Google Scholar, 19.Amstutz M. Mohrmann M. Gmaj P. Murer H. Am. J. Physiol. 1985; 248: F705-F710PubMed Google Scholar) and by electrophysiology applied to the IIa cotransporter expressed in X. laevis oocytes (7.Magagnin S. Werner A. Markovich D. Sorribas V. Stange G. Biber J. Murer H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5979-5983Crossref PubMed Scopus (334) Google Scholar, 15.Hartmann C.M. Wagner C.A. Busch A.E. Markovich D. Biber J. Lang F. Murer H. Pfluegers Arch. Eur. J. Physiol. 1995; 430: 830-836Crossref PubMed Scopus (73) Google Scholar,20.Busch A. Waldegger S. Herzer T. Biber J. Markovich D. Hayes G. Murer H. Lang F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8205-8208Crossref PubMed Scopus (74) Google Scholar, 21.Forster I.C. Loo D.D. Eskandari S. Am. J. Physiol. 1999; 276: F644-F649PubMed Google Scholar). These data indicate that the pH sensitivity of renal brush border Na/Pi cotransport (IIa-mediated) is not explained by a titration of divalent Pi, as a preferred species transported (18.Cheng L. Sacktor B. J. Biol. Chem. 1981; 256: 1556-1564Abstract Full Text PDF PubMed Google Scholar, 21.Forster I.C. Loo D.D. Eskandari S. Am. J. Physiol. 1999; 276: F644-F649PubMed Google Scholar). Thus, the differences in pH sensitivity between IIa- and IIb-mediated Na/Pi cotransporter activities may be attributable to differences within the two proteins. The aim of the present work was to identify the structural domains that confer pH sensitivity to the type IIa Na/Pi cotransporter. For this purpose, we constructed several mouse IIa-mouse IIb chimeras as well as mutated Na/Pi cotransporters and compared their pH dependences with those of the wild type proteins. These studies were done by expression of the various constructs in X. laevisoocytes and measurement of the Na+-dependent Pi uptake over an extracellular pH range from 6.2 to 8. Our data suggest that the third extracellular loop is involved in conferring the pH sensitivity of the IIa-mediated Pitransport. Within this loop, a cluster of three charged amino acids (REK), present in IIa but not in IIb Na/Pi cotransporters, are necessary to provide the pH dependence to IIa. Oligonucleotide primers were obtained from Microsynth (Balgach, Switzerland). The site-directed mutagenesis kit containing Pfu DNA polymerase was purchased from Stratagene, and the restriction and modifying enzymes were from Amersham Pharmacia Biotech or Life Technologies, Inc. All chemicals were purchased from Fluka. All constructs were cloned in pSPORT-1 (Life Technologies, Inc.). The partial cDNA fragments used to construct the chimeras were amplified by PCR,1 using as template the wild type (WT) mouse IIa or IIb cDNAs subcloned into pSPORT and the indicated nucleotides as primers (see TableI). PCR reactions were performed usingPfu DNA Polymerase, and 30 thermal cycles of 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min/kb of PCR target. The different chimera constructs used in this study are schematized in Fig.1.Table IPCR primers sequence used to make several constructsNamePrimer1-SphI5′-GAAAGCTGGTACGCATGCAGGTACCGGTCC-3′2-BglII5′-GTCTGGGTGACACAGATCTGAATGAGACTGTG-3′3-SalI5′-GAGCTATGACGTCGACTGCACGCGTACG-3′4-BglII5′-CACAGTCTGATTCAGATCTGGTGTCACCCAG-3′5-NheI5′-CCAGGCATAACACAAGCTAGCTCTTGTGATAAGGCC-3′6-NheI5′-GCCCTTATCACAAGAGCTAGCTTGTGTGTTATGCCAGG-3′75′-GAGCTATGACGTCGCATGCACGCGTACG-3′8-NheI5′-CTGGAAGCAGGTGCTAGCCAGGGAGATGAC-3′95′-GAAAGCTGGTACGCTTGCAGGTACCG-3′10NheI5′-GTCATCTCCCTGGCTAGCACCTGCTTCCAG-3′11-NheI5′-GACAAGAAAGGTCATGCTAGCACCCACCAC-3′12-NheI5′-GCAGGGCAGCGGCTAGCACAGTAGGATGCC-3′13-NheI5′-CTTGTGGGGGCTAGCATGACCTTC-3′14-NheI5′-GGAATATTGCTTTGCTACGCCAATCCCATTC-3′GNT-s5′-GCCATCCTGGCAGCCGTTGCCAGCCCCGCCAACACCCTATCCAGCTCATTTCAGATTGCCCTC-3′GNT-as5′-GAGGGCCAATCTAAATGAGCTGGATAGGGTGTTGGCGGGGCTGGCAACGGCTGCCAGCATGCC-3′REK-s5′-GCCATTCTGGCTGCTTTAGCCAGCCCAAGGGAGAAGTTGAGGAGTTCTCTCCAGATTGCCCTG-3′REK-as5′-CAGGGCAATCTGGAGAGAACTCCTCAACTTCTCCCTTGGGCTGGCTAAAGCAGCCAGAATGGC-3′ Open table in a new tab The IIa-IIb chimera contained the N-terminal cytoplasmic tail plus the first three transmembrane domains (TDs) of the IIa and the last five TDs plus the C-terminal cytoplasmic tail of IIb (Fig. 1). To obtain the IIa fragment, WT IIa was amplified using the primers 1-SphI and 2-BglII in which SphI and BglII restriction sites were introduced (shown in bold in Table I). Then, the PCR fragments as well as the WT IIb were digested with BglII and SphI. The double digestion of the WT IIb cotransporter produced two fragments: one of about 1 kb containing the N-terminal portion, and one other of about 7 kb containing the last five TDs plus the C-terminal cytoplasmic tail and the whole pSPORT sequence. After purification in 1% agarose gel, the appropriate fragments were ligated over night with T4 Ligase at 16 °C. The IIb-IIa chimera contained the N-terminal cytoplasmic tail plus the first three TDs of IIb, and the last five TDs plus the C-terminal cytoplasmic tail of IIa (see Fig. 1). The IIa fragment was amplified using the primers 3-SalI and 4-BglII in which the indicated restriction sites were introduced (shown in bold in Table I). Then, the WT IIb and the IIa-PCR product were digested withSalI and BglII. This double digestion released two fragments from the WT IIb cotransporter: one of about 4.5 kb containing the three N-terminal TDs and the whole pSPORT sequence, and another of about 3.5 kb containing the C-terminal portion. After purification in agarose gel, the appropriate fragments (see Fig. 1) were ligated as described before. The IIaN-IIbC chimera contained the N-terminal tail plus the eight TDs from IIa and only the C-terminal cytoplasmic tail from IIb cotransporter (see Fig. 1). We used the primers 1-SphI and 5-NheI to amplify the IIa fragment and primers 10-NheI and 9 (downstream of a SalI site located in the polylinker) and 10-NheI to amplify the C-terminal tail of the IIb. Both PCR fragments were digested with the corresponding restriction enzymes. In addition, pSPORT was digested with SphI and SalI. After purification in agarose gel, a three-fragments ligation was carried out overnight. The IIbN-IIaC chimera contained the N-terminal tail plus the eight TDs of IIb and only the C-terminal cytoplasmic tail of IIa (see Fig. 1). We amplified by PCR the IIb fragment using the primers 7 (upstream of aSphI site located in the polylinker) and 8-NheI; for amplification of the IIa cytoplasmic tail, the primers 3-SalI and 6-NheI were used. Then, we followed a strategy similar to that described for the IIaN-IIbC chimera. The IIaN6-IIb chimera contained the N-terminal tail plus the first six TDs from IIa and the last two C-terminal TDs plus the C-terminal tail from IIb cotransporter (see Fig. 1). The IIa fragment was amplified using primers 1-SphI and 12-NheI and the IIb portion with primers 14-NheI and 9. We then followed an approach similar to that described for the IIaN-IIbC chimera. Plasmids encoding the chimeric constructs were introduced intoEscherichia coli competent cells. After plasmid purification sequences were verified by automatic sequencing (Microsynth, Balgach, Switzerland). Mutations of the REK residues of IIa to GNT, and GNT of IIb to REK, were done by site-directed mutagenesis. Briefly 20 ng of the plasmids containing the WT mouse IIa or mouse IIb cDNAs were amplified with 2.5 units ofPfu DNA polymerase in the presence of 250 nmoverleaping primers containing in the middle of their sequence the three mutated codons (GNT-s and GNT-as for IIa, and REK-s and REK-as for IIb; see Table I). PCR amplification was performed with 25 thermal cycles of 95 °C for 30 s, 55 °C for 1 min, and 68 °C for 16 min. Then, 10 units of DpnI were added directly to the amplification reaction, and the sample was incubated for 1 h at 37 °C to digest the parental, methylated DNA. XL1-blue supercompetent cells were finally transformed with 1 μl of the reaction mixture and plated onto LB-ampicillin plates. After plasmid purification sequences were verified by automatic sequencing (Microsynth, Balgach, Switzerland). The procedures for oocytes preparation and cRNA injection, as well as for the 32Pi uptake assay have been described in detail elsewhere (22.Werner A. Biber J. Forgo J. Palacin M. Murer H. J. Biol. Chem. 1990; 265: 12331-12336Abstract Full Text PDF PubMed Google Scholar). Briefly, the cDNAs encoding the two WT and the constructs were first linearized. In vitro synthesis and capping of cRNAs were done by incubating the linearized cDNAs in the presence of Cap Analog (New England Biolabs, Inc.) and 40 units of T7 RNA polymerase (for WT IIa and IIa-GNT) or Sp6 RNA polymerase (for WT IIb, IIb-REK, and the chimeric constructs). Oocytes were injected with either 50 nl of water or 50 nl of water containing 5 ng of cRNA.32Pi uptake was measured 4 days after cRNA injection as already described (22.Werner A. Biber J. Forgo J. Palacin M. Murer H. J. Biol. Chem. 1990; 265: 12331-12336Abstract Full Text PDF PubMed Google Scholar), using external solutions with pH adjusted to 6.2, 7.4, and 8. Throughout the study, we have shown experiments obtained with single batches of oocytes, with 8–10 oocytes measured for each experimental condition. All studies have been performed with qualitatively similar data on at least three batches of oocytes. To identify the regions responsible for the different pH dependence of the IIa and IIb Na/Pi cotransporters, we have constructed several chimeras (Fig. 1). The first set of constructs contained approximately half of each type of cotransporter: the IIa-IIb chimera comprised the first 304 amino acids (N-terminal cytoplasmic tail and the three first TDs) from IIa, and the last 395 amino acids (last five TDs and the C-terminal cytoplasmic tail) from IIb. In the complementary IIb-IIa chimera, the first 301 amino acids correspond to IIb and the last 332 amino acids to the IIa. Because most of the differences between IIa and IIb reside in the C-terminal cytoplasmic tails, we constructed a second set of chimeras with only interchanged cytoplasmic tails. Thus, the IIaN-IIbC chimera comprised the first 596 amino acids (N-terminal cytoplasmic tail plus eight TDs) of IIa and the last 84 amino acids (C-terminal tail) from IIb. To obtain the complementary IIbN-IIaC chimera, we fused the first 612 residues of IIb to the last 41 amino acids of IIa. Finally, the IIaN6-IIb chimera was constructed by fusing the first 487 residues (N-terminal cytoplasmic tail plus six TDs) of IIa to the last 193 amino acids (last two TDs plus the C-terminal tail) of IIb. Based on Western blot analysis using IIa- and IIb-specific antibodies (11.Hilfiker H. Hattenhauer O. Traebert M. Forster I. Murer H. Biber J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14564-14569Crossref PubMed Scopus (321) Google Scholar, 12.Custer M. Lotscher M. Biber J. Murer H. Kaissling B. Am. J. Physiol. 1994; 266: F767-F774PubMed Google Scholar), we found that all chimeras were synthesized in oocytes after cRNA-injection although their level of expression was only about 25–30% compared with the wild types (data not shown). In a previous study we have shown that, in oocytes, transport activity correlates with the transporter present in the surface membrane but not with the total amount of protein (23.Forster I. Traebert M. Jankowski M. Stange G. Biber J. Murer H. J. Physiol (London). 1999; 517: 327-340Crossref Scopus (42) Google Scholar). Here, we have measured Na+-dependent Pi uptake and its pH dependence for all the different constructs. Because we did not quantify the transporter at the surface, we cannot make conclusions about the influence of the different chimeric constructions on overall transport characteristics (e.g. transport rates) but only on the effect on pH dependence. As shown in Fig. 2, the Pitransport activities of all chimeric constructions were significantly higher than that of water-injected oocytes in the presence of 100 mm Na+ and at pH 7.4. The Pi uptake activities were entirely dependent on the presence of Na+(data not shown). Thus, all chimeric proteins are present at the oocyte surface and mediate Na/Pi cotransport. This was a prerequisite for our analysis of the pH dependence of Pitransport activity mediated by these chimeric proteins (see below). The pH dependence of the WT IIa and IIb as well as the chimeric cotransporters was analyzed in oocytes by measuring the Na+-dependent 32Piuptake at three external pH values: 6.2, 7.4, and 8. As shown in Fig.3 A, the Na/Picotransport mediated by the WT IIa increased with higher pH values. This increase is a characteristic feature of IIa cotransporters (7.Magagnin S. Werner A. Markovich D. Sorribas V. Stange G. Biber J. Murer H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5979-5983Crossref PubMed Scopus (334) Google Scholar) that most likely reflects an interaction of protons with the unloaded carrier and the last Na+ binding step. 2I. Forster, J. Biber, and H. Murer, submitted for publication. In contrast, IIb-mediated Na/Pi cotransport was largely independent of the external pH and was even slightly higher at more acidic pH (11.Hilfiker H. Hattenhauer O. Traebert M. Forster I. Murer H. Biber J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14564-14569Crossref PubMed Scopus (321) Google Scholar). As shown in Fig. 3 B, the IIb-IIa chimera behaved similarly to the WT IIa, with higher activity at higher pH values. In contrast, the IIa-IIb chimeras showed no pH dependence, similarly to the WT IIb. Therefore, the pH dependence can be attributed to sequences within the C-terminal half of IIa. We next analyzed the chimeras in which only the C-tails were interchanged. As shown in Fig. 3 C, the C-terminal tail does not seem to be involved in the pH dependence of IIa since replacement with the IIb tail did not abolish the pH dependence of IIa, and conversely the IIa tail did not restore the pH dependence of the IIb. These experiments suggested that the pH-dependent site is located within the last five TDs of IIa. To locate more specifically the pH-sensitive region, we tested a chimera containing the first six TDs of IIa and only the last two TDs and the C-terminal tail of IIb. Fig. 3 D shows that this chimera (IIaN6-IIb) has pH sensitivity similar to the WT IIa. Thus, the domain responsible for the pH sensitivity of IIa is located between the fourth and sixth TD, i.e. within the third extracellular loop. In a cysteine-scanning study on to the rat IIa cotransporter, the third extracellular loop was found to be of functional importance (25.Lambert G. Forster I. Stange G. Biber J. Murer H. J. Gen. Physiol. 1999; 114: 1-16Crossref PubMed Scopus (38) Google Scholar). A sequence comparison of this loop revealed a high degree of homology between the mouse IIa and IIb; however, a cluster and single residues differ (Fig. 4). Among them, three charged amino acids (REK) present in IIa correspond to three neutral amino acids (GNT) in IIb. Single amino acid substitution in the rat IIa cotransporter indicated that replacement of charged amino acid residues at sites 462 (R) and 464 (K) by cysteines, reduced the pH sensitivity of the mutants as compared with the WT. 3G. Lambert, G. Stange, I. Forster, J. Biber, and H. Murer, unpublished observations. Moreover, these charged amino acids are conserved in IIa proteins identified from various species, all of which show the same characteristic increase in transport activity with increasing pH (7.Magagnin S. Werner A. Markovich D. Sorribas V. Stange G. Biber J. Murer H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5979-5983Crossref PubMed Scopus (334) Google Scholar, 11.Hilfiker H. Hattenhauer O. Traebert M. Forster I. Murer H. Biber J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14564-14569Crossref PubMed Scopus (321) Google Scholar). Therefore, we investigated the role of these three residues in determining pH sensitivity of IIa. Replacement of the charged amino acids in the IIa protein by the IIb neutral residues (construct IIa-GNT) led to the loss of the characteristic IIa pH dependence, such that the cotransporter behaved similarly to the WT IIb (Fig. 5,A and B). Furthermore, pH sensitivity could be introduced into the IIb protein by replacing the neutral residues with the IIa charged amino acids residues (IIb-REK) so that this construct now mimicked the strong pH dependence of the WT IIa (Fig. 5,A and B). The behavior of these two constructs strongly suggested that the cluster of three charged amino acids (REK) located on the third extracellular loop is responsible for the pH dependence of IIa.Figure 5Site-directed mutagenesis in the third extracellular loop. A, pH dependence of WT IIa and IIa-GNT mutant. B, pH dependence of WT IIb compared with IIb-REK construct. We used the same conditions as described in Fig.3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The role of specific domains and charged amino acids in conferring pH sensitivity has been described for other transport proteins. For example, for the erythrocyte K-Cl-cotransporter, internal histidine residues are involved in the inhibition at acidic pH (26.Lauf P.K. Adragna N.C. Cell. Physiol. Biochem. 1998; 8: 46-60Crossref PubMed Scopus (18) Google Scholar). A charged residue (Glu-419) has been reported to play a role in the pH sensitivity of a chloride channel (ClC-2G419) (27.Stroffekova K. Kupert E.Y. Malinowska D.H. Cuppoletti J. Am. J. Physiol. 1998; 275: C1113-C1123Crossref PubMed Google Scholar). For the bacterial Na+/H+ antiporter (NhaA), His-225 has been proposed as part of the pH sensor (28.Gerchman Y. Olami Y. Rimon A. Taglicht D. Schuldiner S. Padan E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1212-1216Crossref PubMed Scopus (131) Google Scholar); furthermore, Leu-73 and/or its vicinity may contribute to the pH sensitivity of the antiporter (29.Noumi T. Inoue H. Sakurai T. Tsuchiya T. Kanazawa H. J. Biochem. 1997; 121: 661-670Crossref PubMed Scopus (47) Google Scholar). In the mammalian Na+/H+ antiporter (NHE1 and related isoforms), a pH-modifier site seems to be located in the N-terminal TD although its precise sequence has not been identified (30.Wakabayashi S. Fafournoux P. Sardet C. Pouyssegur J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2424-2428Crossref PubMed Scopus (237) Google Scholar). Also for the mammalian AE2 anion exchanger, a chimera approach led to the identification of the region involved in the pH sensitivity of anion exchanger (24.Zhang Y. Chernova M. Stuart-Tilley A.K. Jiang L. Alper S. J. Biol. Chem. 1996; 271: 5741-5749Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In conclusion, we have identified a "molecular determinant" of the pH sensitivity of the IIa Na/Pi cotransporter, namely three charged amino acids located in the third extracellular loop (REK) which play a critical role in determining the specific pH dependence of the renal cotransporter.