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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Characterization of transport mechanisms and determinants critical for Na⁺-dependent P_i symport of the PiT family paralogs human PiT1 and PiT2

¹Department of Molecular Biology and ²Institute of Clinical Medicine, University of Aarhus, Aarhus; ³August Krogh Institute, University of Copenhagen, Copenhagen; ⁴Department of Medical Physiology, The Panum Institute, University of Copenhagen, Copenhagen; ⁵NeuroSearch A/S, Ballerup; ⁶Department of Chemistry, University of Aarhus, Aarhus; and ⁷Department of Basic Animal and Veterinary Sciences, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark

Submitted 14 January 2006 ; accepted in final form 24 May 2006

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The general phosphate need in mammalian cells is accommodated by members of the P_i transport (PiT) family (SLC20), which use either Na⁺ or H⁺ to mediate inorganic phosphate (P_i) symport. The mammalian PiT paralogs PiT1 and PiT2 are Na⁺-dependent P_i (NaP_i) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with ³²P_i as a traceable P_i source. For PiT1, the Michaelis-Menten constant for P_i was determined as 322.5 ± 124.5 µM. PiT2 was analyzed for the first time and showed positive cooperativity in P_i uptake with a half-maximal activity constant for P_i of 163.5 ± 39.8 µM. PiT1- and PiT2-mediated Na⁺-dependent P_i uptake functions were not significantly affected by acidic and alkaline pH and displayed similar Na⁺ dependency patterns. However, only PiT2 was capable of Na⁺-independent P_i transport at acidic pH. Study of the impact of divalent cations Ca²⁺ and Mg²⁺ revealed that Ca²⁺ was important, but not critical, for NaP_i transport function of PiT proteins. To gain insight into the NaP_i cotransport function, we analyzed PiT2 and a PiT2 P_i transport knockout mutant using ²²Na⁺ as a traceable Na⁺ source. Na⁺ was transported by PiT2 even without P_i in the uptake medium and also when P_i transport function was knocked out. This is the first time decoupling of P_i from Na⁺ transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids E⁵⁵ and E⁵⁷⁵ are responsible for linking P_i import to Na⁺ transport in PiT2.

inorganic phosphate transport; retroviral receptor; SLC20

The mammalian type III transporters are part of the P_i transport (PiT) family [SLC20 (43); TC #2.A.20 (29)], but several members were originally identified as receptors for retroviruses belonging to the gammaretrovirus genus (18, 19, 21, 37, 42, 45); thus PiT1 and PiT2 are proteins that exhibit dual functions.

The PiT paralogs are highly related, and human PiT1 and PiT2 share overall 62% amino acid sequence identity with a higher degree of homology at the NH₂- and COOH-terminal ends (21, 42). Hydropathy profiles show that the PiT paralogs are polytopic proteins and predict 10–12 transmembrane domains (4, 9, 14, 32) and a large hydrophilic domain near the center of each protein that has been experimentally assigned to the cytoplasmic space in PiT2 (6).

PiT1 and PiT2 show a broad tissue distribution, both being expressed in all investigated human tissues, albeit at different levels (40), and low extracellular P_i levels can result in upregulated PiT1 and PiT2 expression in mammalian cells (7, 17), which, together with the current knowledge, albeit limited, on their transport characteristics, suggests that they constitute the housekeeping P_i uptake system between cells and the extracellular fluid (7, 16, 17, 40). This entails that PiT1 and PiT2 have to possess the abilities to adjust to a wide range of extracellular surroundings and organ-specific milieus in the body. Moreover, the PiT proteins also were recently found to be implicated in chondro- and osteoblastic mineralization (3, 20, 24) as well as the phenomenon of transdifferentiation of vascular smooth muscle cells to osteoblast-like cells in the process of vascular calcification under hyperphosphatemic conditions (reviewed in Ref. 11).

Despite the essential role of P_i in life processes and the specific roles of PiT1 and PiT2 in supplying mammalian cells with their general P_i need, as well as the increasing evidence for the role of type III transporters in normal and pathological calcification, little is known about the basic transport functions of the individual proteins. Using the Xenopus laevis oocyte as a "host" in the present study allowed us to investigate the basal capacities of either of the human PiT proteins in surroundings without the influence of uncontrollable variables coupled to cell line-specific characteristics dependent on, for example, organ of origin. No previous study has addressed the basic NaP_i transport characteristics of human PiT2, but some of these characteristics already have been analyzed for human PiT1 (16, 17) and rodent PiT proteins (rat PiT1 and mouse and rat PiT2) (1, 17, 40).

The functional characterization of human PiT1 and PiT2 presented in this study shows that although they differ in their transport kinetics with PiT2, showing positive cooperativity in P_i uptake, they also behave remarkably alike. For example, they both support Na⁺-dependent P_i uptake at acidic and alkaline pH values, and their Na⁺ requirements for P_i transport also are similar. However, only human PiT2 supports Na⁺-independent P_i uptake, albeit at low levels, at acidic pH, underscoring another difference in the transport capacities among these two quite similar proteins. Moreover, Ca²⁺ or Ca²⁺ and Mg²⁺ were found to increase PiT1- and PiT2-mediated NaP_i import, but neither cation was critical for the transport functions in that, compared with transport in the presence of both divalent cations, omitting Ca²⁺ and Mg²⁺ only resulted in an 50% reduction in P_i uptake for both proteins. Interestingly, in the absence of Ca²⁺, Mg²⁺ could partially substitute Ca²⁺ for PiT1 but not for PiT2, implying another difference in their P_i transport mechanisms. We also addressed the Na⁺ transport function versus the P_i import function of PiT2 and included a mutant PiT2 protein lacking the ability to sustain P_i uptake (5). These studies showed that wild-type PiT2 transported Na⁺ regardless of whether P_i was available for import or not. Moreover, the mutant PiT2 protein with knocked out P_i uptake function supported wild-type levels of Na⁺ transport, allowing us to suggest two putative transmembrane glutamate residues as being responsible for the coupling of P_i import to Na⁺ transport in wild-type PiT2.

	MATERIALS AND METHODS

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Expression plasmids. The pcDNA1- and pcDNA1A^RtkpA-derived expression plasmids pOJ9 and pOJ75, respectively (Wyeth-Ayerst Research, Pearl River, NY), encode human PiT1 and have been described elsewhere (15, 25). The pcDNA1A^RtkpA-derived expression plasmid pOJ74 (Wyeth-Ayerst Research) encoding human PiT2 and the PiT2-derived NaP_i transport knockout mutant PiT2 E⁵⁵Q E⁹¹Q E⁵⁷⁵Q also have been described previously (5, 25).

X. laevis oocytes. Female X. laevis frogs were obtained from Nasco (Modesto, CA) and were kept and handled according to guidelines approved by the Danish National Committee for Animal Studies. Oocytes were isolated from frogs anesthetized in a 0.1–0.2% MS.222 (3-aminobenzoic acid ethyl ester; Sigma, St. Louis, MO) solution for 10–30 min. A 1- to 1.5-cm incision was made in the abdomen, and several ovaries were removed surgically by authorized personnel.

The oocytes were manually dissected and subsequently treated with collagenase (Sigma) and maintained in modified Barth's solution [88 mM NaCl, 1 mM KCl, 0.82 mM MgSO₄, 0.4 mM CaCl₂, 0.33 mM Ca(NO₃)₂, 2.4 mM NaHCO₃, 10 mM HEPES-KOH, pH 7.5, 100 IU/ml penicillin, and 100 µg/ml streptomycin] at 18°C as described previously (5). The following day, the oocytes were ready for cRNA or H₂O injection.

³²P_i transport assays in X. laevis oocytes. The ³²P_i transport assays in X. laevis oocytes were essentially done as described previously (5). Briefly, restriction enzyme Bln1 or Not1 was used to linearize the pOJ74 plasmid, Bln1 or Apa1 was used to linearize the pOJ75 plasmid, and Sfi1 was used to linearize the pOJ9 plasmid. Notice that Apa1, Bln1, and Sfi1 cut in the vector sequence downstream of the polylinker and Not1 cuts in the polylinker immediately after the 3'-untranslated region (UTR) of PiT2. Moreover, except for a few bases, Bln1 and Sfi1 cut pOJ75 and pOJ9, respectively, in corresponding positions. Thereafter, we applied the mMESSAGE mMACHINE kit (Ambion, Austin, TX) to produce cRNAs according to the manufacturer's instructions. The quality of the cRNA preparations was verified by agarose gel electrophoresis before injection as well as on remaining samples of the diluted cRNA preparations used for injection.

Stage V and VI oocytes were microinjected with 12.5 ng of cRNA (or H₂O as negative control) 1 day after surgery and incubated at 18°C. After 2 or 3 days, we applied a standardized Na³²P_i transport protocol in which the oocytes were washed in P_i-free uptake solution (100 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, and 10 mM HEPES-KOH, pH 7.5), and uptake was allowed for 1 h at 18°C in 0.1 mM KH₂³²PO₄ (2 mCi/ml; New England Nuclear, Boston, MA) diluted in P_i-free uptake solution. The oocytes were subsequently washed in ice-cold P_i-free uptake solution containing 5 mM KH₂PO₄, and ³²P_i uptake of each oocyte was measured in a liquid scintillation counter as described previously (5). This protocol was modified in individual experiments as indicated.

For determination of the affinity constants K_m and K_0.5 for P_i, oocytes were incubated for 5 min in uptake solution containing KH₂³²PO₄ in concentrations varying from 1,000 to 7.8 µM. For determination of the pH-dependent Na³²P_i uptake pattern, oocytes were incubated for 1 h in uptake solutions modified to reach pH values ranging from 4.5 to 5.5 and 6.5 with HCl and to 8.5 with KOH. More acidic conditions could not be analyzed because the oocytes did not tolerate pH 3.5.

Na⁺-independent ³²P_i transport was analyzed at pH values ranging from 5.5 to 7.5 with Tris·HCl substituting for NaCl; pH was adjusted to 6.5 and 7.5 with KOH and to 5.5 with HCl, otherwise the uptake solutions were comparable to the ones used for pH-dependent Na³²P_i uptake studies. The dependency of Na³²P_i transport on Mg²⁺ and/or Ca²⁺ was analyzed by incubating oocytes in 0.1 mM KH₂³²PO₄ in P_i-free uptake solution without Mg²⁺ or Ca²⁺ or without both Mg²⁺ and Ca²⁺.

For analysis of Na⁺ dependency of ³²P_i transport function, ³²P_i uptake was analyzed using Na⁺ concentrations ranging from 0.5 to 100 mM. The uptake solution was supplemented with Tris·HCl at pH 7.5 to reach a final concentration of 100 mM Tris/Na⁺.

²²Na⁺ transport assay in X. laevis oocytes. Plasmids encoding PiT2 (pOJ74) and PiT2 E⁵⁵Q E⁹¹Q E⁵⁷⁵Q were linearized with Not1, and cRNAs were made as described above. H₂O or 25 ng of cRNA was injected into each oocyte, and after 3 days, ²²Na⁺ uptake studies were performed in P_i-free uptake solution (100 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, and 10 mM HEPES-KOH, pH 7.5) containing 5.8 µM ²²NaCl (11.6 mCi/ml: Amersham Biosciences, Uppsala, Sweden). Moreover, ²²Na⁺ uptake in the presence of P_i was analyzed in the same way by supplementing the uptake solution with 5 mM KH₂PO₄. Uptake was allowed for 1 h, and the oocytes were washed in ice-cold uptake solution. Fifteen oocytes were used per setup, and ²²Na⁺ uptake was measured as gamma decay on pools of five oocytes in a Packard Cobra II Auto gamma counter (Packard Instruments, Canberra, Australia).

Kinetics and statistical analyses. Substrate kinetics calculations for PiT1 were determined by nonlinear regression to the Michaelis-Menten equation: , where represents the initial rate of uptake at a given substrate concentration, V_max is the maximal rate of uptake at saturating substrate concentrations, [S] is the concentration of substrate, and K_mPi is the Michaelis-Menten constant representing the substrate concentration that produces one-half V_max. Substrate kinetics calculations for PiT2 were determined by nonlinear regression to a sigmoidal dose-response (variable slope) equation: , where the slope h denoting the Hill coefficient reveals positive cooperativity if >1, and K_0.5 represents the substrate concentration that produces one-half V_max. Both analyses were performed using GraphPad Prism version 4.01 for Windows (GraphPad Software, San Diego, CA; www.graphpad.com).

For ordinary transport function comparison of two sets of data, the hypothesis that two mean values were identical was tested using a two-tailed Student's t-test. Values were considered different at a 95% confidence level.

Calculation of chemical speciation. Calculation of the chemical speciation in the uptake solutions was performed using MINEQL+ version 3.01b Chemical Equilibrium Management System, distributed by Environmental Research Software (33, 34). For most uptake solutions used at a pH >5.5, the program predicts the presence of precipitates. However, no precipitates were evident in any of the uptake solutions used except at pH 8.5, where slight precipitation was formed after 3 days. Therefore, in all calculations precipitates were neglected. HEPES was not included in the calculations because it does not form complexes with the cations present in the uptake solutions (12); however, its contribution to the ionic strength was taken into consideration.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Kinetic analysis of human PiT1 and PiT2 expressed in X. laevis oocytes. To determine the fundamental kinetic parameters of human PiT1 and PiT2 for P_i, we injected cRNAs encoding PiT1 and PiT2 or H₂O into oocytes and measured Na³²P_i uptake as a function of the extracellular P_i concentration. ³²P_i concentrations in the range of 62.5–1,000 µM at pH 7.5 resulted in a dose-dependent approximately linear uptake of ³²P_i by PiT1-expressing oocytes when the background uptake was not subtracted (Fig. 1A). After correction for background, the Michaelis-Menten constant for PiT1, K_mPi, was determined to be 322.5 ± 124.5 µM (Fig. 1B). For PiT2, the data in Fig. 1A suggested cooperative substrate binding and were instead analyzed using the variable slope curve fitting (Fig. 1C), which gave a PiT2 K_0.5 value for P_i (K_0.5Pi) of 163.5 ± 39.8 µM and a Hill coefficient, h, of 2.0 ± 0.7. This is the first time K_0.5Pi for human PiT2 expressed in oocytes has been determined. V_max for PiT1 and PiT2 were determined to be 452.8 ± 71.7 and 449.5 ± 57.3 pmol·oocyte^–1·h^–1, respectively (Fig. 1).

View larger version (14K): [in this window] [in a new window]   Fig. 1. Uptake behavior and kinetics of PiT1- and PiT2-mediated Na32Pi uptake at different 32Pi concentrations analyzed in Xenopus laevis oocytes. Oocytes were injected with cRNAs (transcribed from Bln1-linearized pOJ74 and pOJ75 plasmids) encoding PiT2 and PiT1 or with H2O. Two days later, 32Pi uptake at pH 7.5 was allowed for 5 min in KH232PO4 varying from 7.8 to 1,000 µM and in the presence of 100 mM NaCl. A: Na32Pi uptake behavior of oocytes expressing PiT2 or PiT1 and of H2O-injected oocytes at different 32Pi concentrations. Data are means ± SE; 15–20 oocytes were used per setup. B and C: mean 32Pi uptake levels obtained for PiT2- and PiT1-expressing oocytes from A are deducted the mean background 32Pi transport observed in H2O-injected oocytes. Each point represents means ± SE. B shows nonlinear regression of PiT1 uptake data to the Michaelis-Menten equation; KmPi and approximated Vmax values are shown. C shows nonlinear regression of PiT2 uptake data to the sigmoidal dose-response equation; K0.5Pi and approximated Vmax values are shown.

Expression of PiT1 protein in X. laevis oocytes is influenced by enzyme choice in the cRNA preparation process. An important aspect of working with protein expression in oocytes is the step of making cRNAs from plasmid preparations. At first, we used the enzymes Apa1 and Not1 for linearization of PiT1- and PiT2-encoding plasmids, respectively. Apa1 linearizes 549 base pairs (bp) downstream of the 126-bp-long PiT1-specific 3'-UTR fragment in pOJ75, whereas Not1 linearizes 13 bp downstream of the 541-bp-long PiT2-specific 3'-UTR fragment in pOJ74. However, to make relative comparisons of PiT2 and PiT1 more feasible, we switched to an enzyme (Bln1) applicable for both pOJ74 and pOJ75; Bln1 linearizes 845 bp and 306 bp downstream of the Not1 site in pOJ74 and the Apa1 site in pOJ75, respectively.

Comparison of PiT2 cRNAs transcribed from Bln1- and Not1-linearized pOJ74 with our standardized Na³²P_i transport protocol resulted in PiT2-expressing oocytes supporting equal (P = 0.171) levels of Na³²P_i uptake (see Table 1, experiment 1), thus both Not1 and Bln1 are suitable enzymes when preparing PiT2 cRNAs from pOJ74. Moreover, we compared PiT1 cRNA transcribed from Apa1-linearized pOJ75 with PiT2 cRNA from Not1-linearized pOJ74 (see Table 1, experiment 2) as well as PiT1 cRNA transcribed from Bln1-linearized pOJ75 with PiT2 cRNA made from Bln1-linearized pOJ74 (see Table 1, experiments 1 and 3). PiT1 cRNA from Apa1-linearized pOJ75 gave rise to oocytes transporting significantly lower levels of ³²P_i than oocytes injected with PiT2 cRNA from Not1-linearized pOJ74 (P < 0.001) (experiment 2). However, cRNAs made from Bln1-linearized pOJ74 and pOJ75 plasmids resulted in ³²P_i uptake levels that were not statistically significantly different (P = 0.921 and P = 0.662, experiments 1 and 3, respectively). Thus, for PiT1 cRNA production from pOJ75, we suggest that Bln1 is used for linearization of the plasmid.

Human PiT2 displays linear NaP_i uptake from 1 to 4 h when expressed in X. laevis oocytes. The time course of P_i uptake in the presence of Na⁺ showed that linear transport of ³²P_i supported by PiT2-expressing and H₂O-injected oocytes occurred in the range from 1 to 4 h (Fig. 2). We chose 1-h uptake for future experiments, because this time span resulted in data with lesser variation than longer transport periods did (see SE on Fig. 2). Allowing oocytes to transport for 1 h resulted in a PiT2 uptake of 114.4 ± 20.6 pmol ³²P_i/oocyte (Fig. 2), in agreement with our former results obtained with PiT2-expressing oocytes (Table 1, experiments 1 and 2) (4, 5).

View larger version (8K): [in this window] [in a new window]   Fig. 2. PiT2 Na+-dependent 32Pi uptake for different time periods analyzed in X. laevis oocytes. Oocytes were injected with cRNA encoding human PiT2 (transcribed from Not1-linearized plasmid) or with H2O. Two days later, 32Pi uptake was measured at pH 7.5 in the presence of 100 mM Na+ and 0.1 mM KH232PO4. Uptake was allowed for 1–4 h, after which the 32Pi uptake in individual oocytes was measured. Data are means ± SE; 9–11 oocytes were used per setup. Linear regression curves are included to illustrate the linear relationship of 32Pi uptake over time (PiT2, R2 = 0.98; H2O, R2 = 0.96). The average 32Pi uptake levels for 1 h for PiT2-expressing and H2O-injected oocytes were 114.4 ± 20.6 and 30.4 ± 6.7 pmol/oocyte, respectively. For PiT2 relative to H2O: P = 0.003 (1 h), P = 0.001 (2 h), P = 0.008 (3 h), and P = 0.003 (4 h).

View larger version (19K): [in this window] [in a new window]   Fig. 3. PiT1 and PiT2 Na+-dependent 32Pi uptake under varying pH conditions analyzed in X. laevis oocytes. Oocytes were injected with H2O or cRNAs encoding PiT2 (from Bln1-linearized pOJ74) or PiT1 (from Bln1-linearized pOJ75). Two days later, 32Pi uptake from pH 4.5 to 8.5 in the presence of 100 mM Na+ and 0.1 mM KH232PO4 was allowed for 1 h. A: 32Pi uptake in PiT2-expressing and H2O-injected oocytes. Data are means ± SE; 15–20 oocytes were used per setup. For comparison of PiT2 32Pi transport at different pH values with that of PiT2 at pH 7.5: P = 0.208 (pH 4.5), P = 0.202 (pH 5.5), P = 0.242 (pH 6.5), and P = 0.262 (pH 8.5). For comparison of PiT2 32Pi transport at different pH values with that of H2O-injected oocytes at the same pH: P = 0.005 (pH 4.5), P = 0.002 (pH 5.5), P < 0.001 (pH 6.5), P < 0.001 (pH 7.5), and P < 0.001 (pH 8.5). The average 32Pi uptake levels for PiT2-expressing and H2O-injected oocytes at pH 7.5 were 117.6 ± 16.8 (16 oocytes) and 7.9 ± 0.9 (20 oocytes) pmol·oocyte–1·h–1, respectively. B: 32Pi transport in PiT1-expressing and H2O-injected oocytes. Data are means ± SE; 17–20 oocytes were used per setup. For comparison of PiT1 32Pi transport at different pH values with that of PiT1 at pH 7.5: P = 0.247 (pH 4.5), P = 0.148 (pH 5.5), P = 0.948 (pH 6.5), and P = 0.128 (pH 8.5). At all pH values, P < 0.001 for 32Pi transport of PiT1-expressing oocytes compared with that of H2O-injected oocytes at the same pH. The average 32Pi uptake levels for PiT1-expressing and H2O-injected oocytes at pH 7.5 were 114.5 ± 24.7 (19 oocytes) and 7.9 ± 0.9 (20 oocytes) pmol·oocyte–1·h–1, respectively.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Impact of Ca2+ and/or Mg2+ on Na+-dependent 32Pi uptake supported by PiT1- and PiT2-expressing X. laevis oocytes. Experimental conditions were as described for pH 7.5 in the legend to Fig. 3, except that Ca2+ and/or Mg2+ was omitted from the uptake solution as indicated; all uptake solutions contained 100 mM Na+. Data are means ± SE; 17–24 oocytes were used per setup. For comparison of PiT2 and PiT1 32Pi transport in the presence of Mg2+ and Ca2+ with 32Pi uptake performed in the absence of Ca2+ and/or Mg2+: PiT2, P = 0.896 (Ca2+), P = 0.031 (Mg2+), and P < 0.001 (no divalent cations); PiT1, P = 0.455 (Ca2+), P = 0.050 (Mg2+), and P < 0.001 (no divalent cations). Moreover, in all cases, the PiT2- and PiT1-expressing oocytes transported Pi significantly different from H2O-injected oocytes (P < 0.001). Uptake conditions in the presence of Ca2+ and Mg2+ were equivalent to those applied in the standardized Na32Pi transport protocol and, in agreement with PiT1- and PiT2-expressing and H2O-injected oocytes, supported 32Pi uptake levels of 94.5 ± 9.0 (24 oocytes), 111.4 ± 9.1 (24 oocytes), and 17.7 ± 1.0 (21 oocytes) pmol·oocyte–1·h–1, respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 5. Na+-independent 32Pi uptake supported by PiT2-expressing X. laevis oocytes analyzed at different pH conditions. Oocytes were injected with either H2O or cRNA encoding PiT2 (transcribed from Bln1-linearized plasmid). Two days later, 32Pi uptake was allowed for 1 h at different pH values in the presence of 100 mM Tris. A: 32Pi transport of PiT2-expressing and H2O-injected oocytes at pH conditions ranging from 5.5 to 7.5. Data are means ± SE; 14–17 oocytes were used per setup. For comparison of PiT2-sustained 32Pi transport relative to that of H2O-injected oocytes at the same pH: P = 0.015 (pH 5.5), P = 0.024 (pH 6.5), and P = 0.477 (pH 7.5). As control, Na+-dependent 32Pi uptake levels for PiT2-expressing and H2O-injected oocytes at pH 7.5 were investigated in parallel, and they supported 32Pi uptake levels of 112.1 ± 26.6 (11 oocytes) and 10.5 ± 0.4 (12 oocytes) pmol·oocyte–1·h–1, respectively (not shown). B: 32Pi transport of PiT2-expressing and H2O-injected oocytes at pH 6.0. Data are means ± SE; 13 oocytes were used per setup. For comparison of PiT2-sustained 32Pi transport relative to that of H2O-injected oocytes at pH 6.0, P = 0.009. As control, Na+-dependent 32Pi uptake levels for PiT2-expressing and H2O-injected oocytes at pH 7.5 were investigated in parallel, and they supported 32Pi uptake levels of 128.6 ± 22.6 (13 oocytes) and 10.4 ± 0.9 (13 oocytes) pmol·oocyte–1·h–1, respectively (not shown). The osmolarities (in mosM) of the uptake solutions were 225–227 (pH 5.5–6.5) and 242 (pH 7.5), respectively.

PiT1 lacks Na⁺-independent P_i transport function. We also investigated whether human PiT1 could sustain Na⁺-independent ³²P_i transport function. Oocytes were injected with cRNAs from pOJ75 (Fig. 6A) or, in an independent experiment, with cRNAs transcribed from pOJ9 (Fig. 6B). Surprisingly, PiT1-expressing oocytes were not able to import ³²P_i in a Na⁺-independent manner significantly better than H₂O-injected oocytes at pH 5.5 (Fig. 6). However, oocytes injected with PiT1 cRNA made from pOJ75 plasmid did import ³²P_i at a level significantly different from that of H₂O-injected oocytes at pH 6.5 (P = 0.010) (Fig. 6A), but the difference in transport was less than twofold and was not observed in the experiment shown in Fig. 6B; thus PiT1 is unable to import P_i at acidic pH in a Na⁺-independent manner. Our study confirms another study analyzing human PiT1 in MDTF cells at pH 6.5 (22).

View larger version (14K): [in this window] [in a new window]   Fig. 6. Na+-independent 32Pi uptake supported by PiT1-expressing and H2O-injected X. laevis oocytes analyzed at different pH conditions. Experimental conditions were as described in the legend to Fig. 5. A: oocytes were injected with cRNA transcribed from Bln1-linearized pOJ75 plasmid. Data are means ± SE; 14–19 oocytes were used per setup. For comparison of PiT1 32Pi transport relative to that of H2O-injected oocytes at the same pH: P = 0.062 (pH 5.5), P = 0.010 (pH 6.5), and P = 0.056 (pH 7.5). As control, Na+-dependent 32Pi uptake levels for PiT1-expressing and H2O-injected oocytes at pH 7.5 were investigated in parallel, and they supported 32Pi uptake levels of 97.9 ± 18.6 (12 oocytes) and 10.5 ± 0.4 (12 oocytes) pmol·oocyte–1·h–1, respectively (not shown). B: oocytes were injected with cRNA encoding PiT1 made from Sfi1-linearized pOJ9 plasmid. Data are means ± SE; 10–17 oocytes were used per setup. For comparison of PiT1 32Pi transport relative to that of H2O-injected oocytes at the same pH: P = 0.493 (pH 5.5), P = 0.983 (pH 6.5), and P < 0.001 (pH 7.5). As control, Na+-dependent 32Pi uptake levels for PiT1-expressing and H2O-injected oocytes at pH 7.5 were investigated in parallel, and they supported 32Pi uptake levels of 36.4 ± 7.3 (12 oocytes) and 10.5 ± 0.4 (12 oocytes) pmol·oocyte–1·h–1, respectively (not shown). The osmolarities (in mosM) of the uptake solutions are given in the legend to Fig. 5.

View larger version (16K): [in this window] [in a new window]   Fig. 7. PiT1 and PiT2 mediated 32Pi uptake in X. laevis oocytes at different Na+ concentrations ([Na+]). Oocytes were injected with cRNAs (from Bln1-linearized pOJ74 and pOJ75 plasmids) encoding PiT2 or PiT1 or with H2O. Two days later, 32Pi uptake was measured for 1 h in the presence of [Na+] varying from 0.5 to 100 mM and 0.1 mM KH232PO4. A and B: 32Pi uptake in PiT2- or PiT1-expressing oocytes and in H2O-injected oocytes. Data are means ± SE; 16–20 oocytes were used per setup. The conditions for uptake performed in the presence of 100 mM Na+ were equivalent to those applied in the standardized Na32Pi transport protocol and, in agreement with that in the presence of 100 mM Na+, the average 32Pi uptake was 117.6 ± 16.8 (16 oocytes) and 114.5 ± 24.7 (19 oocytes) pmol·oocyte–1·h–1 for PiT2- and PiT1-expressing oocytes, respectively, and 7.9 ± 0.9 (20 oocytes) pmol·oocyte–1·h–1 for H2O-injected oocytes. For comparison of PiT1 and PiT2 32Pi transport at different [Na+] with that of H2O-injected oocytes at the same [Na+]: PiT1, P < 0.001 (all [Na+]); PiT2, P = 0.012 (0.5 mM), P = 0.003 (1 mM), P < 0.001 (5 mM), P < 0.001 (10 mM), and P < 0.001 (100 mM). The osmolarities (in mosM) of the uptake solutions were 241 at 0.5–5 mM NaCl, 240 at 10 mM NaCl, and 225 at 100 mM NaCl, respectively.

Na⁺ and P_i symport functions can be uncoupled in PiT2. Na⁺ transport and P_i import are the two active events conducted by the PiT proteins, so to gain insight into the NaP_i cotransport function, we analyzed PiT2 and a PiT2-derived P_i transport knockout mutant protein (PiT2 E⁵⁵Q E⁹¹Q E⁵⁷⁵Q; mutant QQQ in Ref. 5) in oocytes by using ²²Na⁺ as a traceable Na⁺ source. The mutant protein PiT2 E⁵⁵Q E⁹¹Q E⁵⁷⁵Q lacks P_i transport function (Fig. 8B) (5), but the retroviral receptor function for two PiT2 cognate viruses is intact, implying that the mutant protein is correctly processed and that overall topology is preserved (5).

View larger version (18K): [in this window] [in a new window]   Fig. 8. 22Na+ uptake in PiT2- and PiT2 E55Q E91Q E575Q-expressing and H2O-injected X. laevis oocytes in the presence or absence of Pi. A: oocytes were injected with either H2O or cRNA encoding PiT2 or PiT2 E55Q E91Q E575Q. Three days later, 22Na+ uptake was allowed for 1 h in the presence or absence of Pi by using 100 mM "cold" NaCl and 5.8 µM 22NaCl as traceable Na+ source. Data are means ± SE expressed as pmol Na+·oocyte–1·h–1 calculated on the basis of 15 oocytes per setup randomly distributed in 3 vials with 5 oocytes per vial. For comparison of PiT2- and PiT2 E55Q E91Q E575Q-mediated 22Na+ transport in the presence of Pi with that in the absence of Pi, P = 0.363 and P = 0.143, respectively. In the presence of Pi, comparison of 22Na+ transport by PiT2 and PiT2 E55Q E91Q E575Q resulted in P = 0.928, and comparison of 22Na+ uptake by H2O-injected oocytes with that mediated by PiT2 and PiT2 E55Q E91Q E575Q resulted in P = 0.002 and P = 0.045, respectively. In the absence of Pi, comparison of 22Na+ transport of PiT2 with that of PiT2 E55Q E91Q E575Q resulted in P = 0.732, and comparison of 22Na+ uptake by H2O-injected oocytes with that mediated by PiT2 and PiT2 E55Q E91Q E575Q resulted in P = 0.112 and P = 0.007, respectively. In the presence of Pi, PiT2- and PiT2 E55Q E91Q E575Q-expressing oocytes supported 22Na+ uptake levels of 9,132 ± 906 and 9,307 ± 1,579 pmol·oocyte–1·h–1, respectively. Moreover, at the same conditions, the H2O-injected oocytes supported a 22Na+ uptake of 2,325 ± 208 pmol·oocyte–1·h–1. B: oocytes from the same batch of frogs used in A were injected with the same amounts of cRNAs of the same preparations used in A, and 3 days later they were allowed to take up 32Pi for 1 h at pH 7.5. The average 32Pi uptake levels for PiT2- and PiT2 E55Q E91Q E575Q-expressing oocytes and H2O-injected oocytes were 505.9 ± 29.0 (7 oocytes), 41.9 ± 6.1 (9 oocytes), and 24.1 ± 5.6 (7 oocytes) pmol·oocyte–1·h–1, respectively. For PiT2 and PiT2 E55Q E91Q E575Q relative to H2O: P < 0.001 and P = 0.053, respectively. Notice that the oocytes used in experiments shown in A and B are from a batch of frogs that in general tolerated a higher cRNA amount and thus sustained a higher Pi uptake level than those in previous experiments.

Moreover, comparison of the ²²Na⁺ uptake levels obtained for wild-type PiT2 and PiT2 E⁵⁵Q E⁹¹Q E⁵⁷⁵Q in the presence of P_i (P = 0.928) and in the absence of P_i (P = 0.732) revealed that both proteins supported comparable levels of ²²Na⁺ uptake whether P_i were absent or present. Thus, in a P_i-rich environment, the P_i transport knockout PiT2 E⁵⁵Q E⁹¹Q E⁵⁷⁵Q supported the same amount of ²²Na⁺ transport as the wild-type PiT2 in an environment devoid of P_i (Fig. 8), showing that Na⁺ transport of human PiT2 can be uncoupled from P_i transport.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
We have investigated a variety of physiological features of the human Na⁺-dependent P_i transporters PiT1 and PiT2 and used the X. laevis oocyte as expression system. Using the oocyte as "host" allowed us to investigate some basal capacities of the PiT proteins in surroundings without the influence of uncontrollable variables coupled to cell line-specific characteristics.

Whereas rat and mouse PiT2 have been analyzed previously in oocytes, this is the first report on human PiT2. Interestingly, our data reveal positive cooperativity (Hill coefficient of 2) in human PiT2 P_i transport function. This is in agreement with the observations that human PiT2 can be at dimeric form (5) and that P_i starvation promotes human PiT2 dimerization, suggesting that the transporting form is a dimer (30). We determined the K_0.5Pi value of human PiT2 to be 163.5 ± 39.8 µM; however, rodent PiT2 were reported to obey Michaelis-Menten kinetics, and their K_mPi values were determined to be 25.3 ± 6.0 µM (rat) (17) and 38 ± 12 µM (mouse) (1), respectively. The disparities regarding human and rodent PiT2 might be due to amino acid differences, because the overall homology between human and mouse PiT2 is 91% and that between mouse and rat PiT2 is 95% (1).

We determined the PiT1 K_m value for P_i to be 322.5 ± 124.5 µM, and others have determined K_mPi values for human (17) and rat PiT1 (38) in oocytes to be 24.1 ± 5.5 and 89 ± 13 µM, respectively. It cannot be excluded that the difference in K_mPi values between human (present work) and rat PiT1 (38) reflects amino acid differences (human and rat PiT1 exhibit 90% overall sequence homology). Moreover, the K_mPi value for human PiT1 determined in the present study and that determined by Kavanaugh et al. (17) differ by >13-fold. Kavanaugh et al. performed NaP_i uptake in the presence of 1.8 mM CaCl₂, whereas we applied 1.0 mM CaCl₂, and because we found that human PiT1 can sustain P_i uptake without the presence of Ca²⁺ (or Mg²⁺) but that the presence of either at 1 mM increased human PiT1 P_i uptake (Fig. 4), we cannot exclude the possibility that the difference in Ca²⁺ levels contributes to the differences in K_mPi values obtained for human PiT1.

The PiT1 K_mPi and PiT2 K_0.5Pi values we obtained in oocytes are in good agreement with former studies on endogenously expressed human PiT proteins, which revealed K_mPi values of 454 ± 61 µM in human osteoblastic SaOS2 cells (23), 280 ± 30 µM in human primary erythrocytes (35), 360 µM in human erythroid precursor-like K562 cells (39), 110 ± 20 µM in human cervical epithelial-like HeLa cells (27), and 147 ± 14 µM in human embryonic kidney HEK-293 cells (10). Although Fernandes et al. (10) assigned their K_mPi values in HEK-293 cells to PiT1, their cells most likely also expressed PiT2 mRNA, because another study (40) showed its presence in HEK-293 cells and kidney. These results are in agreement with the previous assignment of PiT1 as the major NaP_i transporter in osteoblastic cells (20, 23) and type III transporters as the housekeeping NaP_i transporters in human cells (16, 17, 40).

We found that human PiT1 and PiT2 sustained substantial P_i uptake in oocytes at pH values from 4.5 to 8.5 with a tendency to pH optima at 6.5 and 7.5 for both proteins. Human PiT1 NaP_i uptake was previously investigated in oocytes at pH 7.5 and 8.5, and a higher reduction in uptake at pH 8.5 (55%) compared with pH 7.5 was observed (17). As found in the present study for human PiT2, mouse PiT2 was reported to sustain comparable levels of P_i transport in oocytes at pH 5.5–7.5. Moreover, mouse PiT2 also supported substantial levels of P_i uptake at pH 8.0 and 8.5, albeit significantly impaired compared with transport at pH 7.5 (1). The same tendency was observed for rat PiT2 at pH 7.5 and 8.5 (17); however, the P_i uptake levels in rat PiT2-expressing oocytes were in general surprisingly low (17) compared with those supported by human PiT2 (4, 5) (present work) and mouse PiT2 (1), hampering the full interpretation of these data. In mammalian cell lines originating from kidney, ovary, and connective tissue, the pH optima of the PiT proteins did not differ substantially from those found in oocytes; however, severely impaired uptake at pH 8.0 and 8.5 was observed (10, 22, 44). This underscores the difficulties in addressing general characteristics of proteins in mammalian cells, where a cell type-specific behavior might determine the outcome of the protein characterization.

Human PiT1 and PiT2 supported comparable levels of P_i uptake at Na⁺ concentrations varying from 0.5 to 100 mM when analyzed in oocytes. This P_i uptake was Na⁺ dose dependent and in agreement with results from similar studies of mouse PiT2 (1). Voltage-clamp studies of oocytes expressing rat PiT2 and human PiT1 proposed transport of 2 Na⁺:1 H₂PO₄^– or 3 Na⁺:1HPO₄^2– (16); our analyses of pH-dependent uptake indeed support the suggestion that both H₂PO₄^– and HPO₄^2– can be transported. Moreover, our ²²Na⁺ uptake studies also are in agreement with uptake of a surplus Na⁺ to P_i. We also found that the PiT proteins are capable of sustaining striking levels of P_i uptake at low levels of Na⁺; however, in the human body, the physiological Na⁺ levels is in the range of 130–145 mM, and thus it is unlikely that this ability is of physiological relevance.

The cotransport of Na⁺ and P_i by the PiT proteins are the two actions responsible for P_i import into cells. Human PiT2 residues D²⁸, D⁵⁰⁶, E⁵⁵, and E⁵⁷⁵ (4, 5) as well as S¹¹³ and S⁵⁹³ (31) were found to be critical for P_i transport function of PiT2 (4, 5, 31). The present study is the first to address the Na⁺ transport function of a PiT protein. We found that wild-type PiT2 was capable of transporting Na⁺ without the presence of P_i, emphasizing that P_i and Na⁺ transport occur as two distinct functions. However, a full understanding of the implication and relevance of this finding, i.e., whether PiT2 exhibits substrate-independent Na⁺ uptake, a phenomenon referred to as slippage, in its physiological environment in the mammalian cell, demands more investigations. Nevertheless, the analyses of a P_i transport knockout PiT2 mutant protein, PiT2 E⁵⁵Q E⁹¹Q E⁵⁷⁵Q, did provide knowledge on the mechanism of the Na⁺ transport, showing that it can occur even though the P_i uptake function is abolished. The glutamates in human PiT2 positions 55, 91, and 575 are believed to locate to transmembrane domains (4, 9, 14, 32). A former study (5) revealed that E⁹¹ is dispensable, whereas E⁵⁵ and E⁵⁷⁵ are critical for P_i transport of PiT2. The putative location of E⁵⁵ and E⁵⁷⁵ make them unlikely to fulfill structural roles in the PiT proteins, and instead we have previously suggested that they were involved in function-dependent conformational changes or being parts of a ligand site for the coupling cation (5). However, the data presented in the present study clearly show that they are not participants in the Na⁺ translocation pathway. Rather, we suggest that E⁵⁵ and E⁵⁷⁵ are involved in the coupling of Na⁺ transport to P_i uptake. Noticeably, the conserved nature of the glutamates in PiT2 positions 55 and 575 among members of the PiT family, including in those dependent on H⁺ for P_i uptake, suggest that the residues have similar roles in other PiT family members.

The modulation of PiT1- and PiT2-mediated P_i uptake by Ca²⁺ within 1 h of exposure is interesting. The Ca²⁺ concentration used in the present experiments is slightly lower than the free serum Ca²⁺ level (1.1–1.3 mM) in adult healthy individuals (26). We found an increase in P_i uptake by adding 1 mM Ca²⁺ to the uptake solution; however, it is tempting to speculate that Ca²⁺ plays a general regulating function in P_i uptake via PiT1 and PiT2, because serum Ca²⁺ levels are tightly controlled and serum P_i levels are not. Whether the effects of Ca²⁺ and Mg²⁺ occur directly on the PiT proteins is presently not known.

In summary, we have in the present study characterized human PiT1 and PiT2 transport functions at different levels. We have determined kinetic constants of both proteins for P_i and found that they correspond well to K_mPi values determined for endogenously expressed human PiT proteins. We also have found that both proteins supported NaP_i uptake at pH 4.5–8.5, but only PiT2 was capable of HP_i uptake at acidic pH. Moreover, Ca²⁺ increased both PiT1- and PiT2-mediated P_i uptake in a manner suggesting a regulatory function of Ca²⁺. Dissection of the transport function of human PiT2 revealed glutamate residues critical for the coupling of P_i import to Na⁺ transport.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by Lundbeck Foundation Grants 14/02 and 68/04, the Novo Nordisk Foundation, Danish Medical Research Foundation Grants 9802349 and 22-03-0254, the Karen Elise Jensen Foundation, and Danish Cancer Society Grant DP00092.

	ACKNOWLEDGMENTS

 
We thank Bryan O'Hara for pOJ74, pOJ75, and pOJ9, and Jan Egebjerg Jensen for use of the X. laevis oocyte facilities.

	FOOTNOTES

 

Address for reprint requests and other correspondence: L. Pedersen, Dept. of Molecular Biology, Univ. of Aarhus, C. F. Møllers Allé, Bldg. 1130, DK-8000 Aarhus C, Denmark (e-mail: LP{at}mb.au.dk. http://www.mb.au.dk and http://www.ki.au.dk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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