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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Institute of Physiology and Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland
Submitted 16 February 2007 ; accepted in final form 28 April 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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0.05 mM and increased to 0.2 mM at pH 5.0 and pH 8.0. Xenopus PiT-1 stoichiometry was determined by dual 22Na-32Pi uptake and suggested a 2:1 Na+:Pi stoichiometry. A correlation of 32Pi uptake and net charge movement indicated one charge translocation per Pi. Changes in oocyte surface pH were consistent with transport of monovalent Pi. On the basis of the kinetics of substrate interdependence, we propose an ordered binding scheme of Na+:H2PO4–:Na+. Significantly, in contrast to type II Na+-Pi cotransporters, the transport inhibitor phosphonoformic acid did not inhibit PiT-1 or PiT-2 activity. Na+-Pi cotransport; two-electrode voltage clamp; surface pH electrode; SLC20; retroviral receptor
The two known mammalian PiT members, PiT-1 and PiT-2, have a broad tissue distribution (3, 30, 33, 50). This suggests that they may play a housekeeping role for Pi homeostasis in cells, but other roles for PiT proteins are also emerging. For example, PiT-mediated Pi transport appears to play an important role in providing Pi for the formation of mineralized bone (8, 46, 62). Furthermore, PiT proteins have been strongly implicated in pathological calcification of vascular tissue in response to hyperphosphatemia (31, 34, 39). Recently it was shown (43) that the malaria parasite Plasmodium falciparum expresses a PiT protein that is essential for providing the parasite with Pi for growth.
Although Pi plays a central role in cell metabolism as well as in normal and pathological calcification, the PiT transporters that provide cells with Pi have not been well characterized. Most studies have relied on radionuclide uptake measurements, where lack of membrane-potential control makes interpretation of kinetic studies of electrogenic transport processes difficult. Data analysis is further complicated by the low transport rates attained when expressing mammalian PiT-1 and PiT-2 heterologously. This may explain why, apart from the initial reports by Kavanaugh et al. (32, 33), no further electrophysiological characterization has been done.
We reasoned that Xenopus oocytes might express a Xenopus protein better than mammalian ones and decided to express a Xenopus homolog (XlPiT-1) as well as human PiT (hPiT)-1 and hPiT-2 in Xenopus oocytes. The Pi transport attained with XlPiT-1 far exceeded that of either mammalian isoform, and therefore most of our kinetic characterization was done using XlPiT-1. Using electrophysiology, radiotracer flux, and surface pH measurements, we show that PiT transports two Na+ ions for each Pi and that the preferred Pi species is monovalent H2PO4–. PiT transport is affected by pH because of the dependence of the H2PO4–:HPO42– ratio on pH, and between pH 6.2 and 8.0 there is no effect of H+ per se on PiT function. On the basis of the kinetics of substrate interdependence, we propose an ordered binding scheme of Na+:H2PO4–:Na+; however, the lack of significant pre-steady-state current precludes the assignment of voltage dependence to a particular partial reaction in the transport cycle.
During the preparation of this manuscript, a report on PiT-mediated Pi transport in vascular smooth muscle cells appeared (53). This study, based on 32Pi-uptake assays, provides confirmation of our findings concerning phosphonoformic acid (PFA), Li+, and pH dependence. However, because PiT-mediated Pi transport is electrogenic, controlling the membrane potential (Vm) is essential for determining transport kinetics, and thus the electrophysiological findings that we report herein both complement and extend those of Ref. 53 and previous tracer-flux studies.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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cDNAs encoding hPiT-1, XlPiT-1, and hPiT-2 from the German Resource Center for Genome Research (RZPD) were subcloned into a KSM expression vector (56) to improve expression in Xenopus laevis oocytes. In some experiments, we used a type II Na+-Pi transporter cloned from flounder (flounder NaPi-IIb) for comparison. The plasmids were linearized and were used as a template for the synthesis of capped cRNA by using the mMESSAGE mMACHINE T3 kit (Ambion).
Stage V–VI defolliculated oocytes from X. laevis were isolated and maintained as described previously (57). Oocytes were injected with 50 nl of cRNA (50 ng). Control oocytes were either injected with 50 nl of water or not injected. Oocytes were incubated at 16°C in modified Barth's solution, containing (in mM) 88 NaCl, 1 KC1, 0.41 CaCl2, 0.82 MgSO4, 2.5 NaHCO3, 2 Ca(NO3)2, and 7.5 HEPES, pH 7.4, adjusted with Tris. The solution was supplemented with 5 mg/l doxycycline.
Electrophysiology and radiotracer flux experiments were performed 2–5 days after injection. Each data set was obtained from at least two batches of oocytes from two different donor frogs.
Reagents and Solutions
The solution compositions were as follows. Control superfusate (ND100) contained (in mM) 100 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 10 HEPES adjusted to pH 7.4 using Tris, unless otherwise stated (different pHs, different concentration of Ca2+ or Mg2+). For pH 
6.2, MES was substituted for HEPES. Na+-free superfusate composition was as for ND100 with isosmotic substitution of choline chloride for NaCl (ND0) or LiCl (LD100). Solutions with intermediate Na+ concentrations were prepared by mixing ND0 and ND100 in appropriate proportions. For substrate test solutions, Pi was added from 1 M K2HPO4 and KH2PO4 stocks premixed to give the required pH. PFA was added from 100 mM stock; arsenate and sulfate were added from 1 M stocks.
Radiotracer Uptake
A group of oocytes (7–10 oocytes/group) was first allowed to equilibrate in uptake solution without tracer. After aspiration of this solution, we added 100 µl uptake solution containing radiotracer (32Pi alone or both 32Pi and 22Na). The uptake was allowed to proceed for 15–20 min before it was stopped by washing the oocytes four times with 4 ml ice-cold ND0 solution containing 0.5 mM cold Pi. Uptake of 32Pi alone was carried out by using ND100 solution and 1 mM cold Pi, to which 32Pi (specific activity 10–20 mCi/mmol Pi) was added. Simultaneous uptake of 32Pi and 22Na was done in ND50 solution (50 mM NaCl; pH 6.2 or pH 7.4) with 2 mM cold Pi. These concentrations of cold Pi and Na+ were used to balance the specific activities of the two radionuclides. 32Pi was added to obtain 7 mCi/mmol Pi, and 22Na was added to obtain 1.4 mCi/mmol Na. After being washed, oocytes were placed individually in a scintillation vial and lysed in 250 µl 10% SDS. 32Pi activities of individual oocytes were counted by using a Packard Tri-Carb 2900TR scintillation counter. To separate the counts of 32Pi and 22Na, we programmed the counter for dual DPM assay with quench curves. Net Pi and Na+ uptakes for each individual oocyte were plotted, and linear regression analysis was performed to obtain the Na:Pi transport stoichiometry.
Electrophysiology
Standard two-electrode voltage-clamp hardware was used [GeneClamp, Model 500 (Molecular Devices) or a laboratory-built clamp (17)]. Clamp hardware was controlled and data were acquired by using pClamp 8 software (Molecular Devices), which also controlled valves for solution switching. At the onset of each experiment, the oocyte was clamped to a holding potential (Vh) = –50 mV and was superfused with ND100 solution. To measure Pi-induced currents (IPi), the superfusate was switched to one containing Pi and the change in the holding current was monitored. When the current had reached a steady state, the perfusate was switched back and washout of Pi was monitored by observing the return of holding current to baseline.
Rundown of Electrogenic Activity and Estimation of Membrane Capacitance
Repeated application of Pi to voltage-clamped oocytes often resulted in a progressive loss of activity, which in severe cases amounted to >50% loss by the end of an experiment (see 
Fig. 2A). To take account of rundown in those experiments where repeated Pi applications induced a reduction in IPi, successive test applications were bracketed with control substrate applications (1 mM Pi in ND100). These were normalized to the response at time 0 and were fitted with a single exponential that satisfactorily described the time course of rundown. The test values were then rescaled by a factor derived from the fit. To obtain a mechanistic insight into the rundown of IPi, we correlated changes in the oocyte membrane capacitance (Cm, indicative of changes in membrane area), with the decrease in IPi documented with repeated exposure of Pi. Cm was determined by measuring the current transient in response to voltage steps from –50 mV to –60 mV and to –40 mV, integrating the capacitive transients and calculating the mean from the magnitude of the two charge estimates (23).
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The oocyte was voltage clamped to Vh = –50 mV, and the holding current was continuously recorded. To measure IPi, the superfusate was switched to one containing Pi and change in the holding current was monitored. When the current had 'reached a steady state, the superfusate was switched back and washout of Pi was monitored by observing the return of holding current to baseline. When IPi was recorded for another Na+ concentration or pH, the holding current was first allowed to stabilize at the new reference solution before being switched to one containing Pi. To control for current rundown, each Pi test pulse was bracketed by a control (1 mM Pi) application, which was used to correct the measurements. To determine the apparent affinity constant for Pi (K
), IPi was measured by using different Pi concentrations while keeping the Na+ concentration constant. For determining the apparent affinity constant for Na+ (K0.5Na), the oocyte was first perfused with a specific concentration of Na+ before being switched to one containing Pi (i.e., the Pi concentration was kept constant throughout the experiment). Estimates of K0.5Pi and K0.5Na were obtained by fitting data with the modified Hill equation given by
 | (1) |
The voltage dependence of IPi was measured by applying potentials from –160 or –140 mV to +40 mV and subtracting the current records for the same potential in the presence and absence of Pi, as described previously (17, 55). To account for the differences in expression levels between individual oocytes, data obtained from each oocyte were normalized to IPi recorded at –100 mV with 100 mM Na+ and 1 mM Pi in the bath at pH 7.4 before the data was fitted with Eq. 1. The offset was included to account for the leak current that we have documented in NaPi-IIa-expressing oocytes in the absence of Pi but that is blocked by Pi with an unknown affinity (12, 55).
Pre-Steady-State Current Measurements
Pre-steady-state currents were recorded by applying voltage steps from Vh = –60 mV to test potentials. To improve the signal resolution without distorting the current during the membrane-charging phase when exogenous charge movements might occur, the endogenous capacitive transient was partially suppressed by using a capacitive transient simulator.
Simultaneous Voltage Clamp and 32Pi Uptake
These experiments were carried out as previously described (2, 55). Briefly, an oocyte was placed in a superfusion chamber and the membrane voltage was clamped to –80 mV. After the holding current had stabilized, the superfusate was switched from ND100 (pH 7.4 or pH 6.2) to ND100 solution (with the same pH) containing 1 mM cold Pi and 32Pi at a specific activity of 5 mCi/mmol Pi. After 5 min, the perfusate was switched back to ND100 solution, and washout of Pi was monitored by following the return of the current to baseline. The oocyte was then removed from the chamber and lysed in 10% SDS, and oocyte radioactivity was counted by using a scintillation counter.
The net charge (Q) translocated by the transporter was calculated by first subtracting the baseline holding current and then by integrating the IPi. Net Pi uptake and Q were plotted for each individual oocyte, and linear regression analysis was used to obtain the Pi:Q translocation ratio.
Surface pH Measurements
pH-sensitive microelectrodes of the liquid-membrane type (1) were manufactured as previously described (19). For surface pH measurements, we used electrodes with a large tip diameter (20 µM) that were fire polished to obtain a smooth surface. The pH electrode potential was measured by using a laboratory-built unity-gain electrometer amplifier. The signal from the pH electrode was electronically subtracted from that of the Vm electrode of the two-electrode voltage clamp. Because the Vm electrode was intracellular and the pH electrode extracellular, the Vh (intracellular – bath) was then subtracted from the signal to obtain extracellular pH. The pH electrodes were calibrated by using a two-point calibration (pH 8.0 and 6.0).
Continuous current and pH recordings were obtained from voltage-clamped (Vh = –10 mV, –50 mV, or –100 mV) control oocytes or oocytes expressing XlPiT-1 or flounder NaPi-IIb. In these experiments, the buffering power of the ND100 solution was decreased by reducing the amount of HEPES from 10 to 2 mM. The response of the pH electrode to application of 1 mM Pi was recorded with the pH electrode either in the bath or pressed firmly against the oocyte surface. Because applying Pi with the pH electrode in the bath caused a deflection in the pH signal of 5–8 mV, possibly due to an unspecific interaction of the Pi ion with the liquid membrane, we subtracted the Pi-induced deflection obtained with the electrode in the bath from the one obtained with the electrode pressed against the oocyte surface.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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We first investigated the Pi transport capabilities of the different clones by performing 32Pi uptake in 100 mM Na, 100 mM choline, and 100 mM lithium. Figure 1A shows Pi uptake measured in oocytes expressing hPiT-1, XlPiT-1, and hPiT-2 and in uninjected oocytes. Pi uptake was much higher in oocytes expressing XlPiT-1 than in either human isoform. Pi uptake was highest in the presence of Na+ for all isoforms, but we also observed Pi uptake in choline and Li+ that was significantly larger than in control oocytes (except for hPiT-1 in choline solution).
Next, we investigated the electrogenic response of hPiT-1, XlPiT-1, and hPiT-2 to 1 mM Pi in ND100 solution. First we recorded a continuous current response to 1 mM Pi by using Vh = –50 mV (Fig. 1B). Application of 1 mM Pi induced an inward current, which indicated transport of positive charge into the cell. The magnitude of the IPi was highest in oocytes expressing XlPiT-1, intermediate in hPiT-2, and lowest in hPiT-1, which paralleled the Pi uptake results documented in Fig. 1A. It is conceivable that the difference in 32Pi transport and IPi results from different expression levels of the three isoforms; however, because the turnover rate and the number of active transporters in the membrane are not known for any of the isoforms, this remains speculative. Uninjected oocytes from the same batch did not show Pi-induced inward current (data not shown).
The voltage dependence of IPi in XlPiT-1 and hPiT-2 is shown in Fig. 1C. Again, XlPiT-1-expressing oocytes showed a higher IPi than the human isoform, but the voltage dependence of IPi was similar for the two clones. We were unable to determine with confidence the voltage dependence of IPi in oocytes expressing hPiT-1 because of the small currents.
Rundown of IPi.
On repeated and continuous Pi application, we observed that IPi in XlPiT-1-expressing oocytes decreased. This phenomenon was particularly severe in oocytes giving large IPi (–100 nA at Vh = –50 mV). Figure 2A shows a current recording from a representative oocyte where we applied 1 mM Pi for 20 s at 1-min intervals. Such a loss of activity could result from accumulation of substrate of the trans side of the membrane, resulting in inhibition of the forward transport rate. Alternatively, it could result from a reduction in the number of active cotransporters in the membrane due to endocytosis (23). To investigate the latter hypothesis, we investigated whether changes in the oocyte whole cell capacitance accompanied the decrease in IPi. The capacitance was taken as a measure of the oocyte membrane area, and a decrease in capacitance (Cm) would indicate that endocytosis had occurred. Figure 2B shows IPi plotted against the change in Cm (
Cm) induced by repeated Pi applications. The decrease in IPi was accompanied by a decrease in Cm that strongly suggested that endocytosis of transporters was induced by repeated Pi exposure. However, the correlation between the decrease in IPi and Cm was not linear, and therefore other mechanisms must also play a role.1
Pi Dependence of XlPiT-1 at pH 7.4
To investigate the electrogenic response of XlPiT-1 to Pi in more detail, we determined the Pi dependence at different Vm. Pooled current-voltage (I-V) data are shown in Fig. 3A for Pi varying from 0.01 mM to 1 mM with 100 mM Na+, pH 7.4. At the lowest Pi concentration tested (0.01 mM), the slope of the I-V data was positive and reversed direction in the range –30 mV to –50 mV. The response to 0.01 mM Pi varied between batches of oocytes and suggested that a Pi-dependent leak component was also present, as previously reported for the type II Na+-Pi cotransporter (17). For all other Pi concentrations, the currents did not reverse, even up to +60 mV, for those oocytes where endogenous activating currents were judged to be negligible (data not shown). By transposing the I-V data, we obtained the Pi dependence at each test potential (Fig. 3B). The data were well described analytically by fitting them with a form of the Michaelis-Menten function (Eq. 1, with nH = 1) for test potentials 0 mV. We accounted for the putative Pi-inhibitable leak component by including a variable offset in the fit function (see Measurement of Apparent Pi and Na+Affinities) as previously described (12, 55). For potentials >0 mV, the Pi dependence was prone to contamination by endogenous activating currents, which precluded analysis in this region. The Michaelian behavior suggested that there was one Pi interaction site for each XlPiT-1 transporter. These fits yielded an estimate for the apparent K that was independent of the test potential (Fig. 3F), and the predicted maximum electrogenic activity (IPimax) showed a curvilinear voltage dependence with no evidence of rate-limiting behavior at the hyperpolarizing limit (Fig. 3E). The lack of voltage dependence of K0.5Pi with 100 mM Na+ superfusion suggested that Pi did not interact with the transmembrane electric field.
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at 100 mM Na+ slightly and deviated at depolarizing potentials. This trend was even more obvious for 25 mM Na+; at –100 mV, K0.5Pi doubled and the voltage dependence was clearly evident in the depolarizing direction (Fig. 3F). The behavior suggested that voltage-dependent transitions in the transport cycle were rate determining and dependent on Na+ ion availability. The maximum Pi-induced current (IPimax) obtained from the fits was also dependent on external Na+ (Fig. 3E), which implied that the interaction of Na+ and Pi with XlPiT-1 was ordered (45). Moreover, if we assume that IPi is a measure of the number of carrier proteins with fully bound substrate the dependence of IPimax on Na+ would be consistent with Na+ ions being the last substrate to bind before translocation (45). Na+ Dependence of XlPiT-1 at pH 7.4
To investigate the dependence of IPi on external Na+, we determined the I-V relationship for IPi by varying the external Na+ from 0 to 100 mM, with Pi = 1 mM (Fig. 4A). As expected, at all test potentials, IPi decreased in a concentration-dependent manner with decreasing external Na+. I-V data were pooled and normalized to IPi induced by 1 mM Pi at –100 mV, to take account of different expression levels. As for the Pi-dependence assays, for Vm > 0 mV, the data were less reliable due to contamination from endogenous activating currents, and analysis was only performed for V 0 mV. The data were transposed into a Na+-dependence relationship that showed obvious saturation at all test potentials. Together with our finding of a saturable Pi dependence of the electrogenic response (Fig. 3B), the Na+ dependence provided complementary evidence for carrier-type behavior for XlPiT-1 with respect to Na+ as the variable substrate. The Na+-dependence data were well described by fitting with the modified Hill equation (Eq. 1; Fig. 4B). With all four fit parameters unconstrained, the fits predicted a nH close to unity. An F-test that compared the fits for the unconstrained case (nH as a free parameter) with the Michaelian model (nH = 1) indicated that there was no statistical difference (P > 0.3) between the two models for all test potentials. All subsequent fitting for determination of the apparent affinity constant for Na+ (K
) and maximum Pi-inducible current (IPimax) was therefore constrained with nH = 1 to reduce the uncertainty in estimating these parameters. If we assume that more than one Na+ ion is translocated per transport cycle, which would be the most straightforward explanation for the observed electrogenicity, the Michaelian behavior of the Na+ dependence suggests that there was little cooperativity for the interaction of Na+ ions with the transporter.
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. For 0.3 mM total Pi (data not shown) and 0.1 mM total Pi (Fig. 4C), we also observed a monotonic dependence of IPi on Na+. To compare the different Pi conditions, we normalized each data point to IPi induced by 1 mM Pi at –100 mV and superfusion in 100 mM Na+. After transposition of these data, saturation with respect to Na+ was also evident, and the data were similarly well described by a Michaelian relationship (Fig. 4D) for test potentials –40 mV. The normalized IPimax was obviously dependent on Pi (Fig. 4E). The apparent affinity for Na+ was also voltage dependent, and K0.5Na increased with membrane hyperpolarization (Fig. 4F). There was no statistical difference for K0.5Na with Pi = 1 and 0.3 mM; however, for Pi = 0.1 mM, K0.5Na increased markedly in the depolarizing direction. At hyperpolarizing potentials, the K0.5Na data suggested that there was an asymptotic limit for this parameter that was independent of Pi and Vm. Transport Stoichiometry of XlPiT-1
Simultaneous voltage clamp and uptake of 32Pi. We measured the Q transferred for each Pi molecule transported by performing simultaneous voltage clamp and 32Pi uptake measurements in oocytes expressing XlPiT-1. Uninjected oocytes served as controls. This assay was performed at two pH values (6.2 and 7.4) to determine whether the transporter has a preference for mono- or divalent Pi. For a pKa of 6.8, the ratio H2PO4–:HPO42– at pH 6.2 is 4:1, whereas at pH 7.4 it is 1:4. If both species were transported with similar efficiencies, we would expect the Q:Pi ratio to change with different pH (assuming that the number of Na+ ions transported per Pi remain unchanged). Figure 5A shows an original current trace of and oocyte expressing XlPiT-1 and held at Vh = –80 mV. The oocyte was initially superfused with ND100 solution; then 1 mM Pi with 32Pi as a tracer was applied as indicated. After washout of Pi, the holding current was allowed to return to baseline before the oocyte was removed and lysed for scintillation counting to measure intracellular 32Pi. The Q moved was calculated by integrating the area under the IPi trace.
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Dual uptake of 32Pi and 22Na. Next we performed simultaneous uptake of 32Pi and 22Na at pH 6.2 and 7.4 to measure the Na:Pi transport stoichiometry. These experiments were performed without voltage clamping the oocytes. We reduced the Na+ concentration in the solution to 50 mM and increased the Pi concentration to 2 mM to ensure that the energy spectra of the radioactive isotopes were well separated by the scintillation counter. The amount of Na+ and Pi taken up by each oocyte was calculated from the amount of radioactivity accumulated in each cell. Figure 5C shows a plot of Na+ uptake as a function of Pi uptake for each individual cell. Using linear regression analysis, we obtained a Na:Pi ratio of 1.6 ± 0.1 for pH 7.4 and 1.7 ± 0.1 for pH 6.2, suggesting that the Na:Pi stoichiometry is 2:1. However, because the measured Na:Pi ratio was not exactly an integer, it is possible that another substrate, for example H+, may substitute for Na+. However, because there was no difference between the slopes of the regression lines measured at pH 6.2 and 7.4, representing a 16-fold difference in H+ concentration, it seems unlikely that H+ would play a significant role as transported substrate.
Surface pH measurements.
Finally, we made surface pH measurements to determine if XlPiT-1 preferentially transported monovalent or divalent Pi. If the transporter were to remove monovalent H2PO4– from the extracellular solution, we would expect an alkalinization to occur, because excess HPO42– left behind would combine with H+ to form H2PO4–, thus causing an increase in pH. On the other hand, if divalent HPO42– were the preferred species, we would expect to observe an acidification. Inside the oocyte, the pH changes would be reversed. However, we did not see any Pi-induced changes in intracellular pH in oocytes expressing NaPi-IIb (I. C. Forster, unpublished observations) or in XlPiT-1-expressing oocytes (data not shown). This is most likely due to the low transport rate accomplished when expressing these transporters in oocytes and the high buffering capacity of the oocyte [20 mM/pH (e.g., Ref. 10)].
To observe pH changes in the bath solutions due to XlPiT-1 transport activity, we fabricated pH-sensitive microelectrodes with a large tip area and fire-polished them to be very smooth, so that they did not damage the oocyte surface. Pressing the tip of such an electrode onto the top of an oocyte creates a microenvironment to which diffusion of substrate from the bulk medium is slowed down (but not blocked). This enables pH changes caused by Pi transport to be recorded, provided that the Pi removal rate by the transporter exceeds the H+ diffusion rate to or from the bulk medium. To magnify any pH change occurring, we reduced the buffering power of the ND100 solution from 7.2 to 1.4 mM/pH by reducing the HEPES concentration from 10 to 2 mM.
Figure 6A shows surface pH recording from a control oocyte, an oocyte expressing XlPiT-1, and an oocyte expressing flounder NaPi-IIb, along with the corresponding current recordings. Application of 1 mM Pi caused a small deflection in the voltage signal from the pH electrode, which translated into an apparent decrease in pH of 0.02–0.05 pH units. We believe, however, that this may be an artifact caused by interaction of Pi ions with the liquid membrane electrode, because we observed no difference in pH between the two solutions with a glass electrode. When we repeated the Pi application with the pH electrode pressed against the surface of a control oocyte ("ON") we observed an identical deflection in the signal from the pH electrode. However, when the pH electrode was pressed against the surface of an XlPiT-1-expressing oocyte, the direction of the pH change was reversed, showing that an alkalinization occurred on the surface of the oocyte. This would be consistent with transport of monovalent H2PO4– by XlPiT-1. In contrast, when we pressed the pH electrode onto the surface of an oocyte expressing flounder NaPi-IIb, the magnitude of the pH deflection was increased. This acidification of the oocyte surface indicated that NaPi-IIb transports divalent HPO42–, which is in excellent agreement with previous studies (2, 22, 55) on the stoichiometry and substrate specificity of the type II Na+-Pi cotransporter family.
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Role of metal-ion complexes.
Bottger et al. (4) recently reported that 32Pi uptake into oocytes expressing hPiT-1 and hPiT-2 was reduced by 46 and 42%, respectively, when Ca2+ and Mg2+ were omitted from the incubation medium. They suggested that divalent cations modulate the Pi-transport capacities of the proteins rather than Pi being transported as a metal complex, as has been reported for some bacterial Pi transporters (51, 52). We addressed the role of divalents by measuring the current induced by 1 mM Pi in oocytes expressing XlPiT-1. We found that IPi was reduced by 29 ± 2% when both Ca2+ and Mg2+ were removed and by 15 ± 2 and 12 ± 1% when either Ca2+ or Mg2+ was removed, respectively (n = 6). These values were significantly smaller than reported by Bottger et al. (4).
pH Dependence of Pi Transport in XlPiT-1
To determine the pH dependence of Pi transport by PiT-1, we measured the apparent affinity constant for Pi (K0.5Pi) in XlPiT-1-expressing oocytes at seven pH values in the range 5.0–8.0. The Vm was held at –50 mV, and deflections in IPi induced by application of Pi at different concentrations were monitored. The oocyte responses were normalized to IPi obtained with 1 mM Pi, pH 7.4, to allow pooling of data from oocytes with different expression levels. We plotted IPi as a function of Pi concentration (Fig. 7A) and fitted the data with Eq. 1 (nH was constrained to 1). For clarity, only four pH conditions are shown. Figure 7B shows the fitted parameters K0.5Pi (left axis) and IPimax (right axis) as a function of pH. The data show that IPimax is unaffected by a change in pH between 5 and 7.4. IPimax slightly decreased only when pH increased to 8.0. In contrast, large changes were documented for the apparent Pi affinity. K0.5Pi increased both when pH was lowered below pH 6.2 or increased above pH 6.8 (Fig. 7B). However, given a pKa for H2PO4–:HPO42– of 6.8, and because the preferred substrate for XlPiT-1 is H2PO4– (Fig. 6), it would appear that the increase in K0.5Pi seen when pH was increased above pH 6.8 resulted from a reduced availability of monovalent H2PO4–. Indeed, if we calculate the apparent affinity constant for H2PO4– (K0.5H2PO4–) instead of for Pi (Fig. 7B), we see essentially no change in K0.5H2PO4– for pH values >6.8.
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and IPi. As shown in Fig. 7C, the normalized maximum attainable current showed the same voltage dependence for all pH values. Furthermore, for pH 7.4 and 8.0, we documented no significant alteration in either the magnitude or voltage dependence of K, whereas at pH 6.2, K showed little variation with voltage and the apparent affinity for Na+ increased. This behavior reflects the interdependence of substrates in determining their apparent affinities because at pH 6.2, there is an increased availability of the preferred species, H2PO4–. We also observed that at pH 8.0, fits to the Na+-dependence data with nH as a free parameter were improved compared with those by fixing nH = 1. This behavior suggested that at the higher pH the cooperativity of Na+ interaction increased. Substrate Specificity
Arsenate is structurally similar to Pi and is a known substrate for members of the SLC34 family of Na+-Pi cotransporters (7, 27). To investigate whether arsenate is also a substrate for the type III family, we performed 32Pi uptake and electrophysiology assays. First, we performed 32Pi uptake on uninjected oocytes and oocytes expressing hPiT-1, XlPiT-1, and hPiT-2 by using 0.3 mM cold Pi with or without 1 mM arsenate. Arsenate induced a significant reduction in Pi transport in the two PiT-1 isoforms. However, arsenate did not affect Pi transport by hPiT-2 (Fig. 8).
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40% of current induced by 1 mM Pi (Fig. 8B). This suggested that arsenate is indeed transported by XlPiT-1. In contrast, no significant change in the holding current was observed when applying sulfate (1 mM; Fig. 8B). We then measured the apparent arsenate affinity constant (K
) in XlPiT-1-expressing oocytes and normalized the current response to that obtained with 1 mM Pi for each oocyte. Fitting the data in Fig. 8C with Eq. 1 gave a K of 0.83 ± 0.10 mM, which indicated that the arsenate affinity of XlPiT-1 was significantly smaller than its affinity for Pi (0.19 ± 0.02 mM at pH 7.4 in Fig. 7A). In addition, the maximum attainable arsenate current (I
), as reported by the fit of Eq. 1 to the arsenate data, was smaller compared with Pi (0.74 compared with 1.2 for similarly normalized data), which indicated that the transporter transports arsenate at a slower rate than Pi.
Lithium is known to support transport in some Na+-coupled transporters such as the Na+-dicarboxylate transporter (42) and Na+-driven Cl–/HCO3– exchanger (54). Our results from the 32Pi uptake experiment (Fig. 1A) and others (6, 53) indicated that Pi transport through PiT isoforms could be driven by Li+. We revisited this question by using electrophysiology in oocytes expressing XlPiT-1. We recorded currents induced by 1 mM Pi in 100 mM Na+, 100 mM Li+, and 100 mM choline+ solutions at Vh = –50 mV. Under these conditions, Pi elicited a current response in Li+ that was 15 ± 1% of the response seen in Na+, whereas in 100 mM choline, Pi did not elicit inward currents (data not shown). Thus it appears that Li+ also can act as a substrate for PiT, whereas choline is excluded.
Inhibitors of PiT
PFA is a well-documented competitive inhibitor of the type II Na+-Pi cotransporter (7, 47) and has also been reported to inhibit PiT-mediated Pi uptake (3, 49). Recently, Villa-Bellosta et al. (53) showed by using 32Pi uptake assays that PFA is a very poor inhibitor of Pi uptake mediated by rat PiT-1 and PiT-2 both in oocytes and in native rat vascular smooth muscle cells. We investigated the effect of PFA at a concentration of 1 mM on oocytes expressing PiT proteins by means of 32Pi uptake and electrophysiology. First, we measured 32Pi uptake in uninjected oocytes and oocytes expressing hPiT-1, XlPiT-1, or hPiT-2 with 0.3 mM cold Pi with or without 1 mM PFA. Figure 9 A shows that PFA did not cause any statistically significant differences in Pi uptake in any of the isoforms studied.
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Technetium-99m dimercaptosuccinic acid [99mTc-(V)-DMSA] is a radiopharmaceutical agent with potential in the medical imaging of tumors. Recently, Denoyer et al. (11) reported that entry of 99mTc-(V)-DMSA in tumor cell lines is mediated by PiT transporters. We investigated whether DMSA could affect IPi or whether we could observe DMSA-induced currents in XlPiT-1-expressing oocytes. We observed no effect of 1 mM DMSA alone nor any effect of 1 mM DMSA on currents induced by 0.1 mM Pi in XlPiT-1-expressing oocytes at either pH 6.2 or 7.4 (data not shown), indicating that DMSA is neither transported by nor able to block Pi transport mediated by XlPiT-1. It is, however, possible that an interaction of DMSA with PiT transporters requires chelation with technetium [Denoyer et al. (11) only used 99mTc-(V)-DMSA, not DMSA alone, in their studies]. This question will remain open for further studies.
Pre-Steady-State Kinetics
Pre-steady-state current relaxations induced by voltage steps are a common property of electrogenic members of the SLC34 Na+-Pi cotransporter family (NaPi-IIa/b; reviewed in Ref. 21), and relaxations in the millisecond range are readily observed superimposed on the oocyte capacitive-charging transient. We applied the same experimental protocols to XlPiT-1-expressing oocytes (e.g., Refs. 17 and 55); however, from the raw data alone for oocytes exhibiting comparable IPi (–200 nA) as observed for the type II cotransporters, we were unable to distinguish pre-steady-state relaxations from the endogenous response either in ND100 or ND0 (not shown).
At saturating concentrations, Pi is known to suppress pre-steady-state charge movements (17), and therefore, in a further attempt to detect charge movements, we subtracted records in ND100 from the corresponding traces in ND100 + 1 mM Pi and then examined the time course of currents at the voltage-step onset at high time resolution (Fig. 10A). The current did not change immediately at the onset of the voltage step, and we observed a finite time dependence as the current settled to the new steady-state value. This was qualitatively similar to the time course documented for the type II Na+-Pi cotransporter (e.g., Ref. 24) under similar experimental conditions. Moreover, the relaxations extended beyond the range of the membrane-charging time (1–2 ms) and were therefore unlikely to have originated from a voltage-clamp artifact. For uninjected oocytes from the same donor frog, no relaxations were observed under the same experimental conditions (data not shown). Interestingly, the apparent charge movements were not balanced, i.e., the area under the relaxation curve was not the same for the ON and OFF steps as illustrated in Fig. 10B for two traces at extreme hyper- and hypopolarizing potentials. Under the assumption that we had detected all transient charges for both steps, the lack of charge balance suggested that the relaxations do not arise from a conserved charge movement within the transmembrane field. Nevertheless, the apparent charge associated with the ON transition correlated with IPi over a wide range of expression levels (n = 12), which strongly suggested that the relaxations were associated with the presence of XlPiT-1 in the membrane (Fig. 10C).
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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reported in these studies lay between 50 and 500 µM for measurements in cell lines (14, 33, 41, 48, 49, 60) and between 40 and 300 µM in oocytes (3, 4, 48). Whereas cell-specific environmental issues might influence K measured in different environments, it is also most likely that the lack of membrane-potential control as well as contamination from endogenous Pi-transport systems may have contributed to the spread in the measurements. The problem is further exacerbated by the low Pi-transport levels attained in these systems, possibly explaining why only limited electrophysiological characterization has previously been carried out (32, 33).
Reasoning that Xenopus oocytes might express a Xenopus protein better than a mammalian one, we compared the Pi transport levels of XlPiT-1, hPiT-1, and hPiT-2 by heterologous expression in Xenopus oocytes. Indeed, our results show that XlPiT-1 expressed far better than either of the mammalian isoforms, and therefore we performed most of our kinetic characterization on XlPiT-1. At the amino acid level, XlPiT-1 is 78% identical to hPiT-1 and 60% identical to hPiT-2, and it is possible that the isoforms differ to some extent in their kinetic profiles. However, the main transport-function characteristics, such as substrate specificity, binding order, transport stoichiometry, pH sensitivity, and voltage dependence are probably very similar in the different isoforms, given the high degree of amino acid identity, thus validating the use of XlPiT-1 as a model for vertebrate PiT-1 characteristics in general.
Transport Stoichiometry Determination
Our results from the simultaneous 22Na vs. 32Pi and charge vs. 32Pi uptake experiments strongly suggest that the transport cycle of XlPiT-1 involves the transport of 2 Na+ ions, one Pi in the form of the monovalent H2PO4– ion, and one net positive charge. Because the transport stoichiometries were the same, whether measured at pH 6.2 (H2PO4–:HPO42– ratio 4:1) or at pH 7.4 (H2PO4–:HPO42– ratio 1:4), it appears that the transporter has an absolute preference for monovalent Pi. Furthermore, pH changes recorded at the oocyte surface are consistent with transport of H2PO4–. This contrasts with the behavior of the flounder NaPi-IIb, for which surface pH changes were consistent with transport of HPO42–. Thus the results from the surface pH measurements are in excellent agreement with the stoichiometry measurements from simultaneous radionuclide uptake and voltage-clamp experiments both for type II (22, 55) and type III (this study) Na+-Pi cotransporters.
A 2:1 Na:Pi transport stoichiometry for PiT was initially proposed by Kavanaugh and Kabat (32), but the experimental data were never published. Using 32Pi uptake experiments in oocytes expressing mouse PiT-2, Bai et al. (3) observed that the Na+ dependence of Pi transport showed a very weak positive cooperativity with a nH of 1.1. This low cooperativity agrees with our electrophysiological findings for XlPiT-1, in which the cooperativity of Na+ binding became apparent only at pH 8.0, where the H2PO4– concentration is 0.059 mM. At higher H2PO4– concentrations, we observed no cooperativity for the Na+ dependence (see Fig. 3), and fitting the data with a Michaelian function was statistically indistinguishable from a free fit to the Hill equation. Recently, Saliba et al. (43) showed elegantly that the intraerythrocytic malaria parasite Plasmodium falciparum uses a type III Na+-Pi cotransporter (PfPiT) to mediate Na+-driven Pi uptake. By using isolated parasites, they estimated a nH of 2.1 for the Na+ dependence of 32Pi influx, and by using a "static head" experiment, they confirmed the 2:1 Na+:Pi stoichiometry.
Thus the 2:1 Na+:H2PO4– stoichiometry of PiT has been unequivocally demonstrated in these studies. Because PiT proteins transport H2PO4–, we would expect that this would cause a concomitant acid loading of the cell. In contrast, type II Na+-Pi cotransporters, which mediate HPO42– transport, would alkalinize the cell. Whether the transport rates of type II or type III Na+-Pi cotransporters in their natural environments are high enough to affect cell pH is not known and has not, to our knowledge, been studied in any cell system. Our own experience with expressing type II and type III Na+-Pi cotransporters in Xenopus oocytes would suggest that the pH changes would be minimal at most. However, with respect to acid-base regulation in general, the question as to which Pi species is preferential might be of some consequence. For example, in the average human body, 70% of filtered Pi is reabsorbed in the form of divalent HPO42– in the kidney proximal tubule via type II Na+-Pi cotransporters. Because H2PO4– remains, this effectively constitutes a "secretion" of H+ into the primary urine. For a glomerular filtration rate of 180 l/day, a serum Pi concentration of 1 mM, and a pH of 7.4 in the lumen of the proximal tubule, we estimate that 40 mmol of H+ a day is "excreted" into the proximal tubular lumen this way.
Other Substrates
Anions.
In addition to Na+ and H2PO4–, it appears that PiT-1 and PiT-2 transport few other substrates, with the exception of arsenate. Arsenate is a phosphate mimetic and is highly toxic to cells because it can substitute for Pi in glycolytic and cellular respiration pathways. Accordingly, it is transported both by type II Na+-Pi cotransporters (7, 27) and PiT (this study). In both transporter families, K is three to four times higher than K and the transport rate of arsenate is slower than that for Pi, showing that it is not a perfect Pi substitute. In contrast to arsenate, SO4 did not induce any currents in XlPiT-1-expressing oocytes.
Cations.
In PiT proteins, Li+ ions can act as cosubstrates, as evidenced by both tracer flux (6, 53) and electrophysiology, but the transport rate is much lower than for Na+ (the magnitude of the IPi in XlPiT-1 using 1 mM Pi in 100 mM Li+ solution was only 15% of that in 100 mM Na+). We do not know if this is because the affinity for Li+ is much lower than that for Na+ or because Li+ is transported less efficiently than Na+, because the currents were too small to carry out a dose-dependence measurement. Other cations, such as choline or K+ (53), do not support Pi transport.
Protons.
Protons serve as the driving ion in PiT family members from plants and bacteria (5), whereas in vertebrates the driving force of PiT is provided by Na+. Bottger et al. (4) first reported Na+-independent Pi transport via hPiT-2. Their finding was recently confirmed by Villa-Bellosta et al. (53), who reported that in oocytes expressing rat PiT-2, lowering pH from 7.5 to 6.0 in choline solution resulted in small (7% of that observed in Na+) but statistically significant Pi uptake. These findings indicate that in PiT-2 protons may, to some extent, substitute for Na+. No such effect was seen for rat PiT-1 in their study, which indicated that this effect is specific for PiT-2. For XlPiT-1, we observed identical Na:Pi and charge:Pi ratios at pH 6.2 and 7.4 despite a 16-fold difference in the H+ gradient, indicating that in XlPiT-1 H+ does not substitute for Na+.
Previous studies have shown that PiT-mediated Pi transport is reduced at alkaline pH when measured at a constant total Pi concentration (3, 4, 53). However, protons do not appear to have a strong effect on PiT per se (see Fig. 7), and thus the mechanism by which changes in H+ concentration affect the transport rate of PiT-1 is mainly through alteration of the H2PO4–:HPO42– ratio. In XlPiT-1, we observed a decrease in the apparent H2PO4– affinity at pH < 6.2 (Fig. 7B); however, the maximum attainable transport rate was unaffected.
This contrasts with the type II Na+-Pi cotransporters, where increasing H+ reduces the Pi transport rate via several mechanisms, including modulation of the state occupancy of the empty carrier and competitive interaction with Na+ binding (19, 24, 55). Thus the two families of Pi transporters respond differently to changes in pH, and these differences in the pH dependence may serve as a useful tool to identify the dominant Pi transport system in a system where the molecular identity of the transporter is unknown.
Divalent cations.
Divalent cations are essential for some Pi-transport systems in bacteria (51, 52), where Pi is transported as a neutral metal-ion (MeHPO4) complex driven by the proton-motive force. Recently, Bottger et al. (4) reported that Pi transport rates for hPiT-1 and hPiT-2 were decreased by 46 and 42%, respectively, when both Ca2+ and Mg2+ ions were removed from the medium, and they suggested that this is because divalent cations modulate PiT function. We replicated their experiment by using electrophysiology and found that removing all divalent cations from the solution decreased Pi-dependent currents in XlPiT-1-expressing oocytes by 30%. This result indicated that both Pi transport and net charge transfer were reduced when divalent cations were omitted, which suggested that the cotransport, charge-transferring transport cycle was affected and not a bacteria-like MeHPO4 leak pathway. The Ca2+/Mg2+ dependence of Pi transport could be explained by, for example, modulation of surface charge of a membrane by divalent cations, and they have been shown to allosterically modulate the transport activity of the vitamin C transporter SVCT2 (26).
PFA does not inhibit PiT
PFA is a well-known competitive inhibitor of Na+-dependent Pi transport (35, 47). Inhibition studies using heterologously expressed type II Na+-Pi cotransporters have unequivocally shown that PFA inhibits NaPi-II-mediated Pi transport (7, 61). In contrast, only limited data is available on the effect of PFA on PiT proteins. Bai et al. (3) expressed mouse PiT-2 in Xenopus oocytes and documented a 40% decrease in 32Pi uptake by using a 50-fold excess of PFA (5 mM). Recently, Villa-Bellosta et al. (53) reported that high concentrations of PFA (
2.5 mM, 50-fold excess of PFA) reduced Pi uptake in vascular smooth muscle cells and in oocytes expressing PiT-1 or PiT-2.
We found no effect of PFA on 32Pi uptake in any of the PiT isoforms studied and no effect of PFA on IPi mediated by XlPiT-1 (Fig. 9C), which confirms the findings of Villa-Bellosta et al. (53) with 1 mM PFA found by uptake assays alone. To avoid nonspecific effects, we did not use concentrations of PFA > 1 mM, because these usually increase the oocyte endogenous leak (I. C. Forster, unpublished experiments). Recently, it was reported that PFA inhibits Pi-induced calcification in smooth muscle cells (31, 34) and that that it inhibits matrix calcification in osteoblast-like cells (46). Although other experiments carried out by Li et al. (34) and Suzuki et al. (46) point to an important role of PiT in the calcification process, the effect of PFA in inhibiting calcification is probably more related to the ability of phosphonates and bisphosphonates to inhibit the formation of calcium phosphate crystals (15, 16, 59) than to inhibition of Pi transport through PiT. Alternatively, perhaps PFA-inhibitable type II Na+-Pi cotransporters play a more important role in mineral formation than previously thought (37).
PiT-1 Substrate-Binding Scheme
Our substrate-dependence data strongly suggest that XlPiT-1 binds substrates in an ordered manner, as indicated by the dependence of K on Na+ concentration (Fig. 3F) and K0.5Na on Pi (Fig. 4F) (45). For Pi as the variable substrate, the dependence of I
on Na+ is consistent with Na+ being the last substrate to bind. Consistent with the 2:1 Na+:Pi stoichiometry, a possible binding order, based on analogy with the electrogenic SLC34 transporters, might therefore be Na+:H2PO4–:Na+. The increase in apparent cooperativity for Na+ interaction observed at pH 8.0 would also support this scheme, whereby the decreased availability of H2PO4– at pH 8.0 would increase the apparent dissociation constant associated with the proposed first Na+-binding step, thus conferring a greater cooperativity to the overall Na+ interaction. On the other hand,
) and XlPiT-1 (
). Data points are joined for visualization only. Asterisks indicate statistically significant uptake compared with NI oocytes under same external bath conditions (Student's t-test, P < 0.05).
. Left axis, K0.5Pi and K
