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Amino acids involved in sodium interaction of murine type II Na + ‐P i cotransporters expressed in Xenopus oocytes

2001; Wiley; Volume: 531; Issue: 2 Linguagem: Inglês

10.1111/j.1469-7793.2001.0383i.x

1469-7793

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

Pancreatic function and diabetes

The Journal of PhysiologyVolume 531, Issue 2 p. 383-391 Free Access Amino acids involved in sodium interaction of murine type II Na+-Pi cotransporters expressed in Xenopus oocytes Carmen de la Horra, Carmen de la Horra Institute of Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057, SwitzerlandSearch for more papers by this authorNati Hernando, Nati Hernando Institute of Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057, SwitzerlandSearch for more papers by this authorIan Forster, Ian Forster Institute of Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057, SwitzerlandSearch for more papers by this authorJürg Biber, Jürg Biber Institute of Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057, SwitzerlandSearch for more papers by this authorHeini Murer, Heini Murer Institute of Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057, SwitzerlandSearch for more papers by this author Carmen de la Horra, Carmen de la Horra Institute of Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057, SwitzerlandSearch for more papers by this authorNati Hernando, Nati Hernando Institute of Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057, SwitzerlandSearch for more papers by this authorIan Forster, Ian Forster Institute of Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057, SwitzerlandSearch for more papers by this authorJürg Biber, Jürg Biber Institute of Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057, SwitzerlandSearch for more papers by this authorHeini Murer, Heini Murer Institute of Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057, SwitzerlandSearch for more papers by this author First published: 01 March 2001 https://doi.org/10.1111/j.1469-7793.2001.0383i.xCitations: 14 Corresponding author H. Murer: Institute of Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057, Switzerland. E-mail address: hmurer@access.unizh.ch AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract 1 Type IIa and IIb Na+-Pi cotransporters are highly conserved proteins expressed in brush border membranes of proximal tubules and small intestine, respectively. The kinetics of IIa and IIb differ significantly: type IIb is saturated at lower concentrations of Na+ and Pi. 2 To define the domain responsible for the difference in Na+ affinity we constructed several mouse IIa-IIb chimeras as well as site-directed mutagenized cotransporters. Pi uptake activity was determined after injection of cRNAs into Xenopus laevis oocytes. 3 From the chimera experiments we concluded that the domain containing part of the second intracellular loop, the fifth transmembrane domain (TD) and part of the third extracellular loop determines the specific Na+ activation properties for both types of cotransporter. Within this domain only a few residues located in the fifth TD are not conserved between type IIa and IIb. 4 Site-directed mutagenesis on non-conserved residues was performed. Substitution of F402 of IIa by the corresponding L418 from IIb yielded a cotransporter that behaved like the IIb. On the other hand, substitution of the specific L418 of IIb by the corresponding F402 of IIa produced a cotransporter with a Na+ activation similar to IIa. (Single letter amino acid nomenclature is used throughout the paper.) 5 These data suggest that the specific Na+ activation properties exhibited by type IIa and type IIb Na+-Pi cotransporters are at least in part due to the presence of a specific amino acid (F402 in IIa, and L418 in IIb) within the fifth TD of the protein. Kidney and small intestine are key tissues in the control of inorganic phosphate (Pi) homeostasis. The mechanisms of Pi transport were functionally characterized in brush border membrane vesicles (BBMVs) isolated from kidney and small intestine (Cheng & Sacktor, 1981; Amstutz et al. 1985; Cross et al. 1990; Danisi & Murer, 1991; Nakagawa & Ghishan, 1993). These studies showed that in both tissues the transport of Pi was Na+ dependent (Cheng & Sacktor, 1981; Amstutz et al. 1985; Bèliveau & Ibnoul-Khatib, 1988; Cross et al. 1990; Nakagawa & Ghishan, 1993). More recently, the molecular nature of such transporters was identified. They both belong to the type II family of Na+-Pi cotransporters, which couple the transport of Pi to the movement of Na+ (Magagnin et al. 1993; Hartman et al. 1995; Hilfiker et al. 1998). Two different isoforms of the type II Na+-Pi cotransporter, type IIa and IIb, have been identified. Type IIa cotransporters have been detected to date only in the proximal tubule (Custer et al. 1994; Murer & Biber 1996, 1998); they mediate Pi-reabsorption in kidney (Oberbauer et al. 1996; Murer & Biber 1997; Beck et al. 1998). Type IIb cotransporters are expressed in small intestine and other tissues but are not expressed in kidney; they are involved in intestinal Pi absorption (Hilfiker et al. 1998; Traebert et al. 1999). The homology between mouse type IIa and type IIb cotransporters is between 55 and 75 %. Most of the differences are located in the N- and C-terminal tails; in contrast, the putative transmembrane domains (TDs) show a higher homology (Magagnin et al. 1993; Hilfiker et al. 1998). New studies with cysteine scanning and epitope tagging on type IIa (Lambert et al. 1999a,b) have confirmed preliminary hydropathy plots (Magagnin et al. 1993). Both type II cotransporters are predicted to contain at least eight TDs, with cytoplasmic N- and C-terminal tails; and a large hydrophilic loop between the third and fourth TDs contains two N-glycosylation sites, though glycosylation does not seem critical for the transporters' activity (Hayes et al. 1994). The kinetic features of both type II cotransporters have been analysed by electrophysiological and isotope flux techniques after expression in Xenopus laevis oocytes (Magagnin et al. 1993; Hartman et al. 1995; Hilfiker et al. 1998; Forster et al. 1998). This system confirmed that the properties of the IIa and IIb cotransporters (including their distinct pH dependency) are similar to those of the Na+-Pi cotransport activity previously characterized in BBMVs isolated respectively from proximal tubules and small intestine (Gmaj & Murer 1986; Cross et al. 1990; Murer, 1992; Nakagawa & Ghishan, 1993; Hartman et al. 1995; Hilfiker et al. 1998). Both types of cotransporters also have different affinities for their substrates: type IIa has a lower affinity for Na+ and Pi (Magagnin et al. 1993; Hartman et al. 1995) compared to type IIb (Hilfiker et al. 1998). The transport stoichiometry is most likely 3 Na+: 1 Pi (divalent) for both isoforms (Magagnin et al. 1993; Hartman et al. 1995; Hilfiker et al. 1998; Forster et al. 1999). Differences in functional characteristics of structurally related proteins offer the possibility of identifying functional/structural relevant domains. Thus, chimera studies together with directed mutagenesis led to the identification of residues involved in cation selectivity of the Na+,K+- and H+,K+-ATPase (Mense et al. 2000). Based on a similar approach, we have recently reported that a cluster of three charged amino acids (REK) present in type IIa but not in type IIb is involved in the pH dependency of the IIa-mediated cotransport (de la Horra et al. 2000). In the present study a chimera approach was used to localize the region involved in the different Na+ affinity of IIa and IIb Na+-Pi cotransporters. The chimeras were expressed in Xenopus laevis oocytes, and the Na+ kinetics were studied by Pi flux techniques using different extracellular Na+ concentrations. We found that the domain containing the last amino acids of the second intracellular loop (IL2), the fifth TD (TD5) and the first residue of the third extracellular loop (EL3) confers the different Na+ activation characteristics on both types of cotransporters. Within this domain, F402 from IIa and the corresponding L418 from IIb are crucially involved. These amino acids are located in the proximity of the EL3, a domain that is critical for the activity and regulation of the cotransporter (Lambert et al. 1999a; de la Horra et al. 2000). METHODS Materials 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 from Pharmacia or Gibco BRL. All chemicals were purchased from Fluka. All constructs were cloned in pSPORT-1 (Gibco BRL). Construction of chimeras (IIa-IIb) To obtain the partial IIa and IIb fragments used to construct the different chimeras, we selectively amplified by PCR wild-type (WT) mouse IIa or IIb cDNA using the oligonucleotides indicated in Table 1 as primer. When indicated, oligonucleotides were modified to introduce new restriction sites. Although we tried to minimize the number of point mutations necessary to introduce the new sites, in most cases they resulted in single amino acid substitutions. These changes were always at the connection between type IIa and type IIb cDNAs; in addition they were always located either on intracellular or extracellular sequences, and based on our experience substitutions in loops are more tolerated than those located in transmembrane domains. PCR reactions were performed using Pfu DNA polymerase, and 30 thermal cycles of 95 °C for 1 min, 55 °C for 1 min and 72 °C for 2 min kb−1 of PCR target. The different chimeric constructs are schematized in Fig. 1. Figure 1Open in figure viewerPowerPoint Schematic representation of the wild-type (WT) and chimeric IIa and IIb cotransporters WT IIa (or IIa domains) is represented by filled cylinders and lanes, whereas WT IIb (or IIb domains) is indicated by open cylinders and lanes. Cylinders represent transmembrane domains (TDs) and lane extracellular (top) or intracellular (bottom) segments. The first set of chimeras (IIa-IIb and IIb-IIa) contained approximately half of each type of cotransporter. The second set of chimeras (IIaN-IIbC and IIbN-IIaC) had only interchanged cytoplasmic tails. The third set of chimeras (IIaN5-IIb and IIaN4-IIb) contained either 5 or 4 TDs from IIa. 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). The IIa portion was obtained by amplification of the WT IIa with primers V-SphI-S and IIa-BglII304-AS in which Sph I and Bgl II restriction sites were introduced. The PCR fragment and the WT IIb were then digested with Bgl II and Sph I. The double digestion of WT IIb released 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 PCR product and the 7 kb fragment were ligated overnight with T4 ligase at 16 °C. The complementary 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 (Fig. 1). The IIa fragment was amplified using the primers IIa-BglII304-S and V-SalI-AS, in which Bgl II and Sal I restriction sites were introduced. Then, the PCR product and WT IIb were digested with Sal I and Bgl II. Digestion of WT IIb released two fragments; a 4.5 kb fragment containing the whole pSPORT plus the N-terminal tail and three first TD of the cotransporter was ligated to the PCR product as described before. As a consequence of the introduction of a Bgl II site in the IIa cDNA, the last IIa residue of the IIa-IIb chimera was Q instead of R. It is most likely that this change, located on the second extracellular loop, did not affect the properties of the chimera, since Q is also present at the corresponding position of the human IIa cotransporter. In the IIb-IIa chimera this position remained unchanged, occupied by a K also present in the WT IIb. 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 (Fig. 1). The IIa fragment was amplified with primers V-SphI-S and IIa-NheI596-AS in which the indicated restriction sites were introduced; the C-terminal tail of the IIb was amplified with primers IIb-NheI612-S and V-AS (upstream a Sal I site located in the polylinker). PCR products were digested with the Sph I plus Nhe I, and Nhe I plus Sal I, respectively. In addition, pSPORT was digested with Sph I and Sal I. After purification in agarose gel, a three-fragment ligation was carried out overnight. The complementary IIbN-IIaC chimera contained the N-terminal tail plus the eight TDs of IIb, and only the C-terminal cytoplasmic tail of IIa (Fig. 1). The IIb fragment was amplified using primers V-S (upstream a Sph I site located in the polylinker) and IIb-NheI612-AS; for amplification of the IIa cytoplasmic tail primers, IIa-NheI596-S and V-SalI-AS were used. A three-fragment ligation was carried out as described above. Introduction of Nhe I sites on the C-terminal cytoplasmic tails of both WT cDNAs involved the replacement of a T residue present in IIa and IIb by S, both in the IIaN-IIbC and the IIbN-IIaC chimera. This mutation is not expected to have a significant effect on the properties of the chimeras since (1) it is a conserved change and (2) a S is also present at the corresponding position of the Xenopus laevis type IIa cotransporter. The IIaN5-IIb chimera contained the N-terminal cytoplasmic tail plus the first five TDs from IIa, and the last three TDs plus the C-terminal cytoplasmic tail from IIb (Fig. 1). To obtain the IIa fragment, WT IIa was amplified using the primers V-SphI-S and IIa-NheI423-AS in which the indicated restriction sites were introduced; to amplify the IIb fragment, we used the primers IIb-NheI423-S and V-AS. A three-fragment ligation was carried out overnight as described above. Introduction of Nhe I sites on the third extracellular loop of both WT cDNAs led to the substitution by S of a position occupied in the WT cotransporters by either I (IIa) or M (IIb). This mutation, although non-conserved, is located on an extracellular loop. The IIaN4-IIb chimera contained the N-terminal tail plus the first four TDs from IIa, and the last four TDs plus the cytoplasmic tail from IIb cotransporter (Fig. 1). We used the primers V-SphI-S and IIa-NheI380-AS to amplify the IIa fragment, and primers IIb-NheI380-S and V-AS to amplify the indicated portion of the IIb. Then we proceeded as described above. The introduction of a Nhe I site on the second intracellular loop of both WT cDNAs led to the replacement of a M (IIa) or T (IIb) residue by S. This can be considered as a conserved change regarding the type IIb cotransporter and therefore is not expected to have a significant effect on the properties of the chimeras. Plasmids encoding the chimeric constructs were introduced into E. Coli competent cells. After plasmid purification sequences were verified by sequencing (Microsynth, Balgach, Switzerland). Construction of IIa and IIb point mutations Point mutations of the specific residues of WT IIa (F402 and S409) to the equivalent residues of IIb (L418 and G425) and vice versa were done by site-directed mutagenesis (Fig. 5). Briefly 20 ng of the plasmids containing the WT mouse IIa or mouse IIb cDNAs were amplified with 2.5 U of Pfu DNA polymerase in the presence of 250 nm overlapping primers. The primers (IIa-L-S, IIa-L-AS and IIa-G-S, IIa-G-AS for IIa; IIb-F-S, IIb-F-AS and IIb-S-S, IIb-S-AS for IIb; Table 1) had in the middle of the sequence the mutated codon (shown in bold). PCR amplification was performed with 18 thermal cycles of 95 °C for 30 s, 55 °C for 1 min and 68 °C for 16 min. Then, the parental DNA was digested for 1 h at 37 °C with 10 U of Dpn I. 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 sequencing as indicated above. Figure 5Open in figure viewerPowerPoint Na+-dependent Pi uptake mediated by WT and point mutated proteins Oocytes were injected with cRNAs encoding the WT (IIa, IIb) and the different point mutated cotransporters (IIaL, IIaG, IIbF, IIbS). Na+-dependent 32Pi uptake was assayed as mentioned in Fig. 2. The bars represent the means ±s.e.m. obtained from 8-10 oocytes per group of a representative experiment. At least 3 independent experiments were performed. Xenopus laevis oocyte expression and transport assay All the techniques for oocyte preparation, cRNA injection and the 32Pi uptake assay have been described already (Werner et al. 1990). Female Xenopus laevis frogs were anaesthetized with 0.5 % methanesulfonate salt (Sigma) and placed on ice, and oocytes were removed from the ovaries and prepared according to standard procedures. Frogs were allowed to recover postoperatively before being returned to the aquarium facilities. Briefly, in vitro synthesis and capping of cRNAs were done by incubating previously linearized cDNAs in the presence of Cap Analog (New England Biolabs, Inc) and 40 U of T7 RNA Polymerase (for WT IIa, IIaL and IIaG) or Sp6 RNA Polymerase (for WT IIb, IIbF, IIbS 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 (Werner et al. 1990) using external solutions with Na+ concentration adjusted to 10, 25, 50, 75 and 100 mm. The osmolarity of the solutions was corrected with choline chloride. The solutions were buffered at pH 7.4. For the pH dependency assays, Pi uptake was measure at three pH values, 6.2, 7.4 and 8 (at 100 mm Na+ and 0.5 mm Pi). Data analysis For each set of experiments, qualitatively similar data were obtained in at least three different batches of oocytes. All figures show the means ±s.e.m. obtained with 8-10 oocytes from a representative experiment. Each set of data was collected from the same batch of oocytes. Values are plotted as the ratio of Na+-dependent Pi uptake (% activity) at the indicated extracellular Na+ concentration versus the uptake at 100 mm Na+ (maximal activity). DISCUSSION Mouse type IIa-type IIb chimeras To identify the regions responsible for the different Na+ activation properties exhibited by 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: in the IIa-IIb chimera the first 304 residues (N-terminal cytoplasmic tail plus the three first TD) corresponded to IIa and the last 395 (five TDs plus the C-terminal cytoplasmic tail) to IIb, whereas in the complementary IIb-IIa chimera the first 301 residues (N-terminal cytoplasmic tail plus the three first TD) corresponded to IIb and the last 332 (five TDs plus the C-terminal cytoplasmic tail) to IIa. The second set of constructs had only interchanged cytoplasmic tails: the IIaN-IIbC chimera contained the first 596 residues (N-terminal cytoplasmic tail plus all the TDs) of IIa and only the last 84 (C-terminal tail) from IIb, whereas the complementary IIbN-IIaC chimera contained the first 612 residues (N-terminal tail plus all the TDs) from IIb and only the last 41 (C-terminal tail) from IIa. The IIa5N-IIb chimera was constructed with the first 423 residues (N-terminal cytoplasmic tail plus first five TDs) from IIa and the last 258 amino acids (last three TDs plus the C-terminal tail) from IIb. Finally, to make IIa4N-IIb we fused the first 380 amino acids (N-terminal tail plus the first four TDs) of IIa to the last 300 residues (last four TDs plus the C-terminal tail) from IIb. As shown in Fig. 2, all the chimeras were able to mediate Na+-dependent Pi uptake when expressed in oocytes. Based on Western blot the reduced activities of the chimeric constructs as compared with both WT IIa and WT IIb was mostly due to a smaller amount of chimeric transporter expressed in the oocyte's plasma membrane (data not shown). Figure 2Open in figure viewerPowerPoint Na+-dependent 32Pi uptake mediated by WT and chimeric constructs Oocytes were injected either with water or cRNA corresponding to the WT (IIa, IIb) and different chimeric cotransporters (IIa-IIb, IIb-IIa, IIaN-IIbC, IIbN-IIaC, IIaN5-IIb, IIaN4-IIb). Na+-dependent 32Pi uptake was assayed 4 days after injection at pH 7.4, 0.5 mm Pi and 100 mm Na+. The bars represent the means ±s.e.m. obtained from 8-10 oocytes per group of a representative experiment. At least 3 independent experiments were performed. The Na+ activation characteristics of the WT IIa and WT IIb as well as the chimeric cotransporters were analysed in oocytes by measuring the Na+-dependent 32Pi uptake at pH 7.4 and several external Na+ concentrations. Figure 3A shows the Na+ dependence of both WT cotransporters. WT IIa showed only 6 % of its maximal activity at 10 mm external Na+, 18 % at 25 mm Na+, 46 % at 50 mm Na+, 94 % at 75 mm Na+ and was saturated at 100 mm Na+. In contrast, IIb already exhibited 30 % of its maximal activity at 10 mm external Na+, 62 % at 25 mm Na+, 84 % at 50 mm Na+, 98 % at 75 mm Na+ and was saturated at 100 mm Na+. These patterns of Na+ dependence are in agreement with our previous reports (Magagnin et al. 1993; Busch et al. 1994; Hartman et al. 1995; Hilfiker et al. 1998; Forster et al. 1998). The percentage of activity at 25 mm relative to 100 mm Na+ was used as a criterion to distinguish between the characteristic kinetic of the two cotransporters: at 25 mm external Na+ type IIa exhibits less than 20 % of its maximal activity, whereas under the same conditions type IIb already shows 60 % of its maximal activity. As shown in Fig. 3B, the behaviour of the IIb-IIa chimera was similar to WT IIa, showing only a small activity at low external Na+ concentration; in contrast, the IIa-IIb chimera behaved similarly to WT IIb, already exhibiting 70 % of its maximal activity at the 25 mm Na+. Based on these data, the segment containing the last five TDs and the C-terminal tail appeared to confer the different Na+ activation properties of both type II Na+-Pi cotransporters. Within this segment most of the differences between the IIa and IIb cotransporters are located in their C-terminal tails whereas the remainder is highly conserved. Therefore we investigated the potential role of these tails by studying the behaviour of the chimeras in which only the C-terminal tails were interchanged. As shown in Fig. 3C, the IIaN-IIbC chimera had a Na+ dependence pattern that was similar to WT IIa, whereas the IIbN-IIaC chimera behaved like the WT IIb. These results indicated that the cytoplasmic tails are not involved in the different kinetic properties displayed by both types of cotransporters. Therefore we concluded that the region responsible for these differences must be located within the last five TDs and the associated intracellular loops of the proteins. Figure 3Open in figure viewerPowerPoint Na+-dependent Pi uptake mediated by WT and chimeric constructs 32P uptakes was measured at pH 7.4 in the presence of 10, 25, 50, 75 and 100 mm external Na+ (Nao). Then, the percentage of activity was calculated as the ratio of the activity at a given external Na+ concentration versus the activity at 100 mm Na+. A, WT IIa and WT IIb. B, IIa-IIb and IIb-IIa chimeras. C, IIaN-IIbC and IIbN-IIaC chimeras. D, IIa5N-IIb and IIa4N-IIb chimeras. Each bar represents the mean ±s.e.m. of 8-10 oocytes per group of a representative experiment. At least 3 independent experiments were performed. To narrow down this region we analysed two additional chimeras, IIa5N-IIb and IIa4N-IIb, that differ from each other in only one TD. As shown in Fig. 3D, IIa5-IIb behaved like the WT IIa, with only 20 % of its maximal activity at 25 mm Na+. In contrast, the IIa4N-IIb chimera had about 60 % of activity at the same Na+ concentration, like the WT IIb. These data strongly implicate the region encompassing the last half (16 residues) of the second intracellular loop (IL2), the fifth transmembrane domain (TD5) and the first part (10 residues) of the third extracellular loop (EL3) in the different Na+ activation properties displayed by both types of cotransporters. Site directed mutagenesis in the TD5 Figure 4 A shows a sequence comparison of the mouse IIa and IIb cotransporters focused on the segments suggested by the chimera studies to be involved in the different Na+ activation properties. The first 16 residues that constitute the last half of the IL2 show a low degree of conservation; however, these positions are also poorly conserved among the IIa cotransporters cloned from different species (Magagnin et al. 1993; Sorribas et al. 1994; Hartman et al. 1995; Verri et al. 1995). On the other hand, the first 10 residues of the EL3 are identical in both cotransporters, with the exception of a conserved substitution (M-I). Therefore, we considered that none of these loops was likely to confer different Na+ activation characteristics. In contrast, the 19 residues that are suggested to constitute the TD5 show a high degree of homology. Out of six different positions, four represent conserved substitutions (represented by '.' in Fig. 4A) and only two are non-conserved changes (represented by '-' in Fig. 4A): F402 and S409 from IIa are substituted by L418 and G425 in IIb. These residues are conserved throughout evolution in all the members that belong to the same isoform (Magagnin et al. 1993; Sorribas et al. 1994; Verri et al. 1995; Hartman et al. 1995; Hilfiker et al. 1998). Therefore, we investigated if these non-conserved residues play a role in conferring the different Na+ activation characteristics of IIa and IIb, by using a site-directed mutagenesis approach (Fig. 4B). The point mutations did not induce large changes in the activity of the cotransporters compared with their respective WT (Fig. 5). The effect of these single amino acid substitutions on the Na+ activation of IIa and IIb cotransporters is shown in Fig. 6. Substitution of the specific F402 of IIa by the corresponding L residue of IIb yielded a cotransporter (IIaL) that behaved like the WT IIb, by displaying about 60 % of its maximal activity at 25 mm Na+ (Fig. 6a). In contrast, substitution of the S409 from the IIa by the corresponding G residue from IIb did not modify the behaviour of the cotransporter (IIaG), whose activity pattern remained similar to that of the WT IIa (Fig. 6a). The apparent Km values (see Table 2) showed a similar tendency: IIaL had a Km for Na+ very close to that of WT IIb (26 mm) whereas the value for IIaG remained very similar to that of WT IIa (67 mm). These data suggested a role of F402/L418 in the different Na+ interaction of both cotransporters. This hypothesis was further supported by complementary point mutations, in which the specific residues of IIb were replaced by the corresponding residues of IIa. As shown in Fig. 6B substitution of L418 of IIb by the equivalent F from IIa (IIbF) led the phenotype of the mutant to revert to that of the WT IIa, while substitution of G425 by the equivalent S from IIa (IIbS) was without effect. The apparent Km values for Na+ (Table 2) provided similar information (105 mm for IIbF and 24 mm for IIbS). Therefore, the mutagenesis data suggested that residues F402/L418 located in the TD5 are determinants for the different Na+ interaction exhibited by type IIa and type IIb cotransporters. Figure 4Open in figure viewerPowerPoint A, sequence comparison of the predicted second intracellular loop and fifth transmembrane domain (TD5) of IIa and IIb. Conserved changes are shown by '.'; non conserved changes are shown by '-'; residues interchanged by site-directed mutagenesis are shown by '*'. The position of the putative TD5 is indicated. B, schematic representation of the point mutated cotransporters. Figure 6Open in figure viewerPowerPoint Na+-dependent Pi uptake mediated by the point mutated cotransporters 32Pi uptake was measured at pH 7.4 in the presence of 10, 25, 50, 75 and 100 mm external Na+ (Nao). Then, the percentage of activity was calculated as described in Fig. 3. A, WT IIa, IIaL and IIaG mutants. B, WT IIb, IIbF and IIbS mutants. C and D, the pH dependency was studied by measuring the Na+-dependent Pi uptakes at pH 6.2, 7.4 and 8 (100 mm Na+, 0.5 mm Pi). C, WT IIa and IIaL, IIaG mutants. D, WT IIb and IIbF, IIbS. At least 3 independent experiments were performed. We have recently reported that the predicted third extracellular loop (EL3), which connects TD5 and TD6, contains the domain responsible for the specific pH dependency of the type IIa cotransporter: three charged amino acids (REK) present in type IIa are substituted by three uncharged residues (GNT) in type IIb (de la Horra et al. 2000). Therefore we investigated whether or not the F402/L418 residues also influenced the pH response. We found that the characteristic pH dependence of each type of cotransporter was not affected by these point mutations: IIaL and IIaG behaved like WT IIa, showing higher activity at higher pH values, whereas the activities of the IIbF and IIbS were independent of the external pH, similar to WT IIb (Fig. 7C and D). Conversely, substitutio