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【关键词】 human
Institute of Physiology, University of Zurich, Zurich, Switzerland
ABSTRACT
We have characterized the kinetics of substrate transport in the renal type IIa human sodium-phosphate cotransporter (NaPi-IIa). The transporter was expressed in Xenopus laevis oocytes, and steady-state and pre-steady-state currents and substrate uptakes were characterized by voltage-clamp and isotope flux. First, by measuring simultaneous uptake of a substrate (32Pi, 22Na) and charge in voltage-clamped oocytes, we established that the human NaPi-IIa isoform operates with a Na:Pi:charge stoichiometry of 3:1:1 and that the preferred transported Pi species is HPO42. We then probed the complex interrelationship of substrates, pH, and voltage in the NaPi-IIa transport cycle by analyzing both steady-state and pre-steady-state currents. Steady-state current measurements show that the apparent HPO42 affinity is voltage dependent and that this voltage dependency is abrogated by lowering the pH or the Na+ concentration. In contrast, the voltage dependency of the apparent Na+ affinity increased when pH was lowered. Pre-steady-state current analysis shows that Na+ ions bind first and influence the preferred orientation of the transporter in the absence of Pi. Pre-steady-state charge movement was partially suppressed by complete removal of Na+ from the bath, by reducing extracellular pH (both in the presence and absence of Na+), or by adding Pi (in the presence of 100 mM Na). None of these conditions suppressed charge movement completely. The results allowed us to modify previous models for the transport cycle of NaPi-II transporters by including voltage dependency of HPO42 binding and proton modulation of the first Na+ binding step.
Xenopus laevis oocyte; electrophysiology; two-electrode voltage clamp; stoichiometry; structure-function
INORGANIC PHOSPHATE (Pi) is vital to all cells and organisms for both their structural and metabolic needs. As most cells maintain an inside-negative membrane potential, the anionic Pi cannot accumulate in the cytosol by passive diffusion. In epithelial cells, regulated Pi uptake is principally mediated by type II sodium-phosphate cotransporters (NaPi-II), which drive uphill transport of Pi by coupling it to the Na+ gradient. In mammals, NaPi-II transporters mediate Pi absorption in the intestine and reabsorption of Pi in the kidney and are thus in position to control both uptake and loss of Pi from the organism (for a recent review, see Ref. 28).
Three distinct forms of the type II Na-Pi cotransporters have been identified. The first one cloned was the type IIa isoform, which was obtained from rat and human kidney cortex by expression cloning (22). The transporter is expressed in the brush-border membrane in the proximal tubule of the kidney and is the principal target for regulators of Pi reabsorption (for reviews, see Refs. 27 and 28). The related type IIb isoform is expressed in the apical membrane of intestinal enterocytes and in a variety of other tissues (6, 16). Both of these isoforms are electrogenic and transport one Pi with three Na+ ions, carrying one net positive charge. The electroneutral type IIc isoform is most highly expressed in kidneys of weaning rats and thus plays a role in Pi reabsorption mainly during development (32).
The cloning of cDNAs for NaPi-II cotransporters has enabled the study of their transport mechanism in more detail by exogenous expression, notably in Xenopus laevis oocytes. Work focusing mainly on the renal type IIa isoform has established that Pi transport is electrogenic and that transport involves the ordered binding and translocation of three Na+, one Pi, and one net positive charge per transport cycle (for reviews, see Refs. 8 and 10). The most detailed kinetic analysis has been carried out on rat and flounder NaPi-II isoforms, where pre-steady-state current analysis was employed to show that Na+ binds first, followed by Pi and two more Na+ ions (7, 12). Translocation of the fully loaded carrier is electroneutral, with electrogenicity arising from the return of the empty carrier to its initial conformation in an electrogenic step. In addition to cotransport, in the absence of Pi, NaPi-IIa mediates a Na+-dependent leak current that is blocked by phosphonoformic acid (PFA), a competitive inhibitor of Pi transport. Pi transport by NaPi-IIa decreases with decreasing pH, in part because of titration of the preferred transported substrate (HPO42) (11) and in part because protons are able to interact with H+-dependent partial reactions in the transport cycle (9).
Understanding the kinetics of Pi transport by NaPi-IIa is complicated because of the number of interdependent parameters involved. The rate of transport is influenced by the availability of Na+ and Pi, by pH and by membrane voltage. Furthermore, alterations in any one of these parameters affect the interactions of the others with the transport cycle. In this work, we have characterized the complex interrelationship of substrate concentrations, pH, and membrane voltage in human NaPi-IIa by analyzing the dependency of steady-state and pre-steady-state currents on these parameters. This has allowed us to identify additional partial reactions in the transport cycle that are influenced by pH and/or voltage.
MATERIALS AND METHODS
Molecular Biology and Oocyte Expression
The cDNA encoding for human NaPi-IIa was subcloned into a KSM expression vector to improve its expression in X. laevis oocytes, as described previously (36). The plasmid was linearized using Xba1 (Promega) and used as a template for the synthesis of capped cRNA using a Message Machine T3 kit (Ambion).
Stage V-VI defolliculated oocytes from X. laevis were isolated and maintained as described previously (40). Oocytes were injected with 50 nl of cRNA (0.2 μg/μl) encoding NaPi-IIa. Control oocytes were injected with 50 nl of water. Oocytes were incubated at 18°C in modified Barth's solution containing (in mM) 88 NaCl, 1 KCl, 0.41 CaCl2, 0.82 MgSO4, 2.5 NaHCO3, 2 Ca(NO3)2, and 7.5 HEPES, pH 7.5, adjusted with TRIS. The solution was supplemented with 5 mg/l doxycyclin. Electrophysiology and radiotracer flux experiments were performed 25 days after injection. Each data set was obtained from at least two batches of oocytes from two different donor frogs.
Reagents
All standard chemicals and reagents were obtained from either Sigma or Fluka Chemie. [32P]orthophosphate and 22Na were purchased from New England Nuclear and Amersham, respectively.
Two-Electrode Voltage Clamp and Radiotracer Uptake
Simultaneous voltage clamp and 32Pi/22Na uptake. For simultaneous voltage-clamp and radiotracer uptake experiments, we used an OC-725C oocyte clamp (Warner Instruments) connected to data-acquisition hardware (Digidata 1322A, Axon Instruments). A computer running pClamp8 (Axon Instruments) was used to control the clamp and record currents. The oocyte was placed in a superfusion chamber (based on a design by D. D. F. Loo, UCLA) with an effective volume of 24 μl and superfused at a rate of 100 μl/min using a peristaltic pump, with excess solution removed by a suction pump. The oocyte was initially voltage clamped to 50 mV and superfused with ND-100 solution (in mM: 100 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 10 HEPES, titrated to pH 7.4 using TRIS) until a stable baseline was reached. The superfusate was then switched to a solution containing 40 mM Na+ (ND-40, Na+ replaced by choline) at a pH of either 7.5 or 6.8. Amiloride (0.8 mM final concentration) was included to suppress endogenous Na+ transport activity. After the holding current had again reached a stable baseline, the oocyte was exposed for 1015 min to ND-40 solution containing 2 mM cold Pi (added as a mixture of K2HPO4/KH2PO4 of the appropriate pH) and [32P]orthophosphate (specific activity 3,0004,000 dpm/nmol Pi) and 22Na (specific activity 1,000 dpm/nmol Na). After a 10- to 15-min time period, the perfusate was switched back to ND-40. Both application and washout of Pi were monitored from the current tracing. After the holding current returned to the baseline level, the oocyte was removed from the chamber and rinsed with ice-cold ND-0 solution. The oocyte was placed in a scintillation vial and lysed in 250 μl 10% SDS. 32P and 22Na activities of individual oocytes were counted using a Packard Tri-Carb 2900TR scintillation counter. The counter was programmed with a Dual DPM assay with quench curves to separate the counts of the two isotopes. To ensure that the counting assay was reliable, samples were recounted over a time period of 2 mo, and counts due to 32Pi (T 14.3 days) were separated from those of 22Na (T1/2, 2.6 days) based on their differing rates of decay. Total Na+ and Pi uptakes (in pmol/oocyte) were calculated from the specific activities of 22Na and 32Pi.
The net charge Q translocated by NaPi-IIa was calculated from current tracings by first subtracting the baseline holding current and then integrating the area under Pi-induced current curves using the Clampfit module in pClamp. The Na+ and Pi uptakes for each oocyte were then correlated to Q using linear regression analysis.
Two-electrode voltage clamp: kinetic characterization. We used a custom-built voltage clamp optimized for fast clamping speed (7) to make recordings from control oocytes and oocytes expressing NaPi-IIa. The voltage clamp was controlled and data were acquired using a computer running pClamp 8 software (Axon Instruments), which also controlled the valves for solution switching. Solutions were cooled to 2022°C before introduction to the oocyte-recording chamber at a rate of 5 ml/min. The oocyte was initially superfused with ND-100 solution, pH 7.4. Different concentrations of Pi were obtained by adding K2HPO4/KH2PO4, the proportions of which were adjusted to give the desired pH. PFA was first dissolved in water before addition to the desired solution. In some experiments, it was necessary to substitute CaCl2 with BaCl2 to suppress the activation of endogenous Cl channels at hyperpolarizing voltages. In solutions where the Na+ concentration was varied (ND-0 to ND-100), NaCl was substituted equimolarly with choline Cl.
Measurement of apparent Pi and Na+ affinities. The oocyte was initially perfused with ND-100 solution, and the membrane potential was clamped to 50 mV. The holding current was recorded continuously. To measure Pi-induced currents, the superfusate was switched to one containing Pi, and deflection in the holding current was monitored. When the current had reached its maximum, the perfusate was switched back and washout of Pi was monitored by observing the return of the holding current to baseline. When Pi-induced currents were to be recorded for another Na+ concentration or pH, the holding current was first allowed to stabilize in the new baseline solution before the switch to one including Pi,
For determining apparent Km for Pi (KmPi), Pi-induced current deflections were measured using different Pi concentrations while the Na+ concentration was kept constant. For determining the apparent Km for Na (KmNa), the oocyte was first perfused with a specific concentration of Na+ before the switch to a solution containing Pi and the same Na+ concentration. To enable comparison between different oocytes and to monitor current rundown, Pi-induced currents were recorded using a standard solution (ND-100, 1 mM Pi, pH 7.4) at the beginning and at the very end of each experiment. Pi-induced currents recorded in other conditions were then normalized to the current obtained with the standard solution.
The apparent KmPi and KmNa were determined by fitting data with the modified Hill equation
(1)
where IPi is the Pi-induced current, [S] is the concentration of the substrate (Na+ or Pi), KmSis the concentration of the substrate that gives a half-maximum response, and n is the Hill coefficient. For determination of KmPi, n was constrained to 1.
The voltage dependencies of KmPi and KmNa were measured by applying holding potentials from 160 to +40 mV and recording currents in the presence and absence of Pi, as described previously (7). Currents recorded in the absence of Pi were subtracted from those recorded in the presence of Pi to obtain IPi. The amount of leak current mediated by NaPi-IIa in ND-100 was estimated by substituting Pi with 1 mM PFA, a competitive blocker of NaPi-IIa (17). To account for the differences in expression levels between individual oocytes, the 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. KmPi and KmNawere determined by fitting the data at each voltage with the modified Hill equation plus a variable offset.1
Pre-steady-state charge movement. Pre-steady-state current relaxations were acquired using voltage steps from a holding potential of 60 mV to test voltages in the range 160 to +80 mV. Signals were sampled at 50-μs intervals, low-pass filtered at 500 Hz, and four current tracings were averaged for the final recording. To improve signal-to-noise ratios, capacitive current compensation was used to partially suppress the capacitive transient and thereby allow recordings to be made at a high gain without amplifier clipping distorting the acquired signal. Relaxations were quantified by fitting a decaying double exponential function using a fitting algorithm based on the Chebychev transform supplied with pClamp. The fast component ( 0.7 ms) was assumed to represent endogenous charging of the oocyte membrane, as it showed little voltage dependence. The exogenous charge movement Q at each voltage was obtained by numerically integrating the slower component, extrapolated to the step onset. For some oocytes, it was necessary to apply a linear baseline correction to eliminate contamination from endogenous currents activated at extreme depolarizing or hyperpolarizing potentials.
The total charge (Q) moved per transport cycle was estimated by fitting the charge-voltage (Q-V) data with a two-state Boltzmann equation of the form
(2)
where Qmax is the total charge available for translocation, Qhyp is the charge translocated at the hyperpolarizing limit, V0.5 is the voltage at which the charge is distributed equally between two states, z is the apparent valence (a product of the valency of the charge and the fraction of the transmembrane field sensed by that charge), e is the elementary charge, k is Boltzmann's constant, and T is the absolute temperature.
Statistical Analysis and Curve Fitting
Statistical analysis of the data was carried out using a two-tailed t-test or one-tailed ANOVA with Tukey's posttest, using GraphPad Prism 3.0 software (GraphPad Software). Data were fit with the fitting algorithms supplied with Prism 3.0 software using the appropriate equation as indicated in the text.
RESULTS
Voltage Clamp and Dual Uptake of Na+ and Pi
To determine the stoichiometry of human NaPi-IIa, we performed simultaneous measurements of Na+, Pi, and charge transfer in oocytes expressing human NaPi-IIa. In addition, we identified the preferred transported Pi species by performing the assay at two different pHs (the pKa for H2PO4/HPO42 is 6.8). Figure 1A shows a representative current trace for a NaPi-IIa-expressing oocyte voltage clamped to 50 mV in ND-40 solution at pH 7.5. At the indicated time, 2 mM Pi was added, together with radioactive 22Na and 32Pi. Application of Pi induces an inward current, and by integrating the area under the curve, we obtained the total amount of charge moved during the application of Pi. The amount of Pi and Na+ taken up by the oocyte during this time period was calculated from accumulated radioactivity in each individual cell. The Na+ and Pi uptake data were plotted as a function of the charge in Fig. 1, B (pH 7.5) and C (pH 6.8). Linear regression analyses showed that for both pH values tested, the ratio of Na+ to charge (3:1) and Pi to charge (1:1) remained constant. Also, when we plotted Na+ as a function of Pi, the ratio to Na to Pi was constant (3:1, not shown).
The purpose of performing the experiment at two different pH values was to determine the contribution of monovalent (H2PO4) and divalent (HPO42) Pi to the movement of charge. At pH 7.5, the ratio of divalent to monovalent Pi is 5:1, whereas at pH 6.8 monovalent and divalent forms are present in equal amounts. If both species were transported with similar efficiencies, we would expect that the ratio of Pi to charge would decrease as the pH is lowered from 7.5 to 6.8, assuming a constant Na+:charge ratio of 3:1. Neither the Pi:charge, Na+:charge, nor Pi:Na+ ratio was affected by a change in pH, in agreement with data obtained previously on flounder and rat isoforms (11). We therefore conclude that the preferred substrate for human NaPi-IIa is divalent Pi.
Effect of pH on Na+ Transport Kinetics in NaPi-IIa
The effect of pH on apparent KmNa was first investigated using a constant holding potential of 50 mV. At this potential, contamination of the leak pathway to the Pi-dependent current is very small; i.e., the Pi-dependent current equals cotransport current. The protocol is also less challenging for the oocyte and allows a more reliable estimate of IPi, max to be made, compared with the voltage-jump protocol used below.
We measured Pi-activated currents by continuously recording the holding current at 50 mV and monitoring Pi-induced downward deflection (Fig. 2A). The Pi-induced current (IPi) was plotted as a function of Na+ concentration and fitted with Eq. 1 to obtain apparent KmNa, Hill coefficient n, and IPi, max (Fig. 2B). Experiments were carried out at pH 7.4 and 6.6. Pi was applied at 1 or 0.1 mM concentration for pH 7.4, and at 1 or 2 mM concentration for pH 6.6 (at pH 6.6, the HPO42 concentration at 2 mM Pi is equal to the HPO42 concentration at 1 mM Pi at pH 7.4). For each oocyte, IPi was also measured at pH 7.4, 1 mM Pi, and all data were normalized to this value for each individual oocyte.
Reducing pH from 7.4 to 6.6 at either a fixed total Pi or total HPO42 concentration resulted in a large increase in KmNa, as did reducing the total Pi from 1 to 0.1 (see Table 1). The Hill coefficient and IPi, max were less sensitive to pH; however, lowering the pH at a fixed total Pi of 1 mM caused a statistically significant increase in the Hill coefficient as well as a decrease in IPi, max. Thus KmNa is highly sensitive to both pH and the concentration of the substrate HPO42, whereas the Hill coefficient and IPi, max are less so.
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Effect of pH on Pi Transport Kinetics in NaPi-IIa
Next, we characterized how pH affects the apparent affinity for Pi by studying the kinetics of IPi over a range of pH values. Experiments were carried out as above using a constant holding potential of 50 mV and by applying Pi at various concentrations (Fig. 3). In addition, we acquired a data set for pH 7.4 with 50 mM Na+. For each oocyte, IPi obtained for each condition was normalized to that measured at ND-100, 1 mM Pi, pH 7.4.
IPi was plotted as a function of the concentration of divalent HPO42 (Fig. 3B). Data were fitted with Eq. 1, and the calculated apparent affinity for HPO42 (KmHPO42) and IPi, max was plotted as a function of pH in Fig. 3C. IPi, max was reduced, and KmHPO42 increased, by reduced pH. Thus reducing pH affects both apparent KmHPO42 and IPi, max. Note that if the affinity for phosphate were calculated as a function of the total Pi concentration (as KmPi) the increase in Km would be much larger for a given reduction in pH, because the concentration of the transported substrate (HPO42) is pH dependent (pKa 6.8). However, the calculated IPi, max would be the same in both cases. Fitting IPi, max data in Fig. 3C with Eq. 1 gave an apparent inhibition constant (Ki) for H+ of 400 ± 40 nM (corresponding to a pH of 6.4) and a Hill coefficient of 2.1 ± 0.6.
When we compare the effect of pH on the two cosubstrates Na+ and HPO42, we see that, for a reduction in pH from 7.4 to 6.6, KmHPO42 was increased by 50% and IPi, max was reduced by 25%. For Na+ as the variable substrate, we find in Fig. 2 and Table 1 that KmNa was increased by 57% and IPi, max was reduced by 15% for the same decrease in pH. Thus pH affects the kinetic parameters of steady-state transport of both substrates in a similar manner. Note that reducing Na+ from 100 to 50 mM causes a fourfold increase in KmHPO42, a significantly larger effect than the twofold increase inKmHPO42 seen with the maximal reduction of pH to 6.4. The opposite effect is seen for IPi, max, where lowering Na from 100 to 50 mM has a much smaller effect than lowering pH.
Voltage Dependency of KmPi
Figure 4A shows IPi (filled symbols) and current blocked by PFA () as a function of the holding voltage in oocytes expressing NaPi-IIa. The PFA-blocked current represents current mediated by the leak pathway that operates in the absence of Pi. The true Na-Pi cotransport current (not shown) mediated by NaPi-IIa is therefore the sum of Pi-induced and leak currents, because the latter current is subtracted from current recordings made in the presence of Pi (note that for nonsaturating Pi concentrations, some leak current will remain in the recordings made in the presence of Pi, which will reduce the error associated with the subtraction procedure). It can be seen (Fig. 4, AC) that as the Pi (HPO42) concentration is reduced, the slope of the current-voltage (I-V) relationship shallows and is ultimately inverted for the lowest HPO42 concentration (0.008 mM). This is a consequence of the leak current and the subtraction procedure used to obtain the Pi-dependent current. Since the affinity of Pi for blocking the leak pathway is not known, estimating how much leak is left in the recordings made with a lower Pi concentration is not feasible. We have therefore chosen not to correct the data for leak.
The voltage dependency of the Pi-dependent current was also measured at pH 6.6 (Fig. 4B) and 50 mM Na, pH 7.4 (Fig. 4C). All data were normalized for each cell to the Pi-dependent current obtained at 100 mV with 100 mM Na, 1 mM Pi, and pH 7.4 ( in B and C). The data in Fig. 4, B and C, show that Pi-induced currents were reduced at all Pi concentrations, compared with the data in Fig. 4A. The flattening of the I-V curves at hyperpolarizing potentials, seen, in particular, for 50 mM Na+ (Fig. 4C), indicates that the transport rate is determined by voltage-independent, rate-limiting steps under these conditions.
KmHPO42 was calculated from data in Fig. 4, AC, using Eq. 1 (we assume that only divalent HPO42 is transported; Fig. 1), and plotted as a function of the holding potential in Fig. 4D. At 100 mM Na+ and pH 7.4, KmHPO42 is voltage dependent and varies inversely with voltage. This voltage dependency is reduced when the pH is lowered to 6.6. The voltage dependency of KmHPO42 in ND-50, pH 7.4, also appears to be reduced; however, the error in estimating the parameter was exacerbated by the reduced signal-to-noise ratio at 50 mM Na+. Reducing Na+ significantly increased KmHPO42 throughout the whole voltage range measured.
The voltage dependency of IPi, max was extracted using Eq. 2 and plotted in Fig. 4E. Reducing pH from 7.4 to 6.6, or reducing the Na+ concentration from 100 to 50 mM, caused a reduction in IPi, max that became more pronounced at hyperpolarizing voltages. Interestingly, this decrease in IPi, max was very similar in both conditions, although reducing the Na+ concentration had a much stronger effect on KmHPO42 than lowering pH.
Voltage Dependency of KmNa
Figure 5A shows the voltage dependency of the current induced by 1 mM Pi (0.8 mM HPO42) at variable Na+ concentrations and pH 7.4 for oocytes expressing NaPi-IIa. Reducing the Na+ concentration also reduced the voltage dependency of IPi, so that at 10 mM Na+ the I-V curve was essentially flat. When pH was reduced to 6.6 while total Pi concentration was maintained at 1 mM (Fig. 5B), IPi decreased for all Na+ concentrations tested. At low Na+, the I-V curves acquired a negative slope due to uncompensated leak current (see above).
Data for each voltage were plotted as a function of Na+ concentration, and KmNa was calculated using Eq. 1 with a variable offset. Figure 5C shows KmNa measured at pH 7.4 and 6.6 as a function of the holding potential. No voltage dependency is seen for voltages more positive than 80 mV. However, below 80 mV, KmNa increased with hyperpolarization. At pH 6.6, KmNa also became voltage dependent at more positive voltages and was voltage independent only for potentials above 0 mV.
Figure 5D shows IPi, max at pH 7.4 and 6.6 plotted as a function of voltage. There is an apparent reduction in IPi, max at pH 6.6 compared with 7.4 throughout the voltage range tested. This result should be interpreted with caution, however, as the lack of saturation in Fig. 5B precludes a sufficiently reliable fit of the data to Eq. 1. We were unable to obtain reliable data using the voltage-jump protocol on oocytes using solutions with Na+ concentrations above 125 mM.
Pre-Steady-State Kinetics: Na+ Dependence of Charge Movement
To identify partial reactions in the NaPi-IIa transport cycle that are influenced by substrate and pH, we performed pre-steady-state current analysis. First, we examined the Na+ dependence of pre-steady-state currents by exposing an oocyte to solutions with different Na+ concentrations. Figure 6A shows a representative recording of currents acquired using the voltage-jump protocol in an oocyte expressing NaPi-IIa bathed in ND-100 solution at pH 7.4. After baseline correction and rescaling (Fig. 6B, left), pre-steady-state relaxations were clearly observed, superimposed on the oocyte capacitive charging. These relaxations were partially suppressed in the presence of 1 mM Pi (Fig. 6B, middle) and were absent in control oocytes (Fig. 6B, right).
The charge Q moved from 60 mV to each target potential was extracted (see MATERIALS AND METHODS), plotted as a function of target potential and fitted with Eq. 2. Figure 6C shows a Q-V plot for oocytes superfused with Na+ concentrations from 0 to 125 mM, and 100 mM Na+ and 1 mM Pi. The parameters obtained from the fits are shown in Table 2. The maximum amount of charge available (Qmax) was suppressed when all Na+ was removed from the superfusate. Maximum suppression of charge movement was observed when 1 mM Pi was added to the superfusate containing 100 mM Na; however, none of the conditions used in Table 2 were able to fully suppress pre-steady-state currents. Quantitatively similar results were obtained when we extracted the charge moved from each test potential back to 60 mV (not shown), which confirmed charge balance.
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The midpoint voltage V0.5 was shifted in the negative direction when the Na+ concentration was reduced, from 8 ± 12 mV at 125 mM Na+ to 77 ± 7 mV at 10 mM Na+ (Fig. 6D, Table 2). Removing all Na+ in the bath caused no further change in V0.5. Plotting V0.5 as a function of log[Na+] revealed a near-linear relationship for Na+ concentrations 25 mM, with a slope of 94 ± 6 mV/log[Na+] (Fig. 6D, inset). The apparent valency (z) reported by the fit was between 0.40 and 0.52 for all the conditions tested, but the differences were not statistically significant.
Pre-Steady-State Kinetics: Effect of pH
To identify pH-sensitive steps in the transport cycle of NaPi-IIa, we examined the effect of lowering pH on pre-steady-state charge movement. Q-V data were acquired in the presence and absence of Na+, with and without 1 mM Pi. The Q-V data shown in Fig. 7 were fitted with Eq. 2, and the parameters obtained from the fit are shown in Table 3. Lowering pH from 7.4 to 6.6 in ND-100 suppressed Qmax and shifted V0.5 from 30 ± 2 to 23 ± 9 mV. After the addition of 1 mM Pi at either pH, Qmax was suppressed to the same level in both cases (40%). In the absence of external Na+, a condition that already suppresses Qmax, reducing pH from 7.4 to 6.6 resulted in an additional but weak suppression of Qmax (10%) and a shift in V0.5 from 62 ± 9 to 52 ± 6 mV. Including 1 mM Pi in the bath in ND-0 solution did not cause any changes in the measured parameters, indicating that Pi is only able to alter charge movement in the presence of external Na+.
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We also attempted to resolve pre-steady-state charge movements using the voltage protocol at pH 5.0 (not shown). At this pH, the holding current of the oocyte was unstable for voltage steps below 140 mV and above +40 mV, precluding further analysis. However, inspection of the current transients (acquired in either the presence or absence of Na+) revealed that no pre-steady-state charge movement was visible and the transients looked like those obtained from water-injected (control) oocytes.
DISCUSSION
Stoichiometry of hNaPi-IIa
Determining the stoichiometry of electrogenic transporters is often accomplished by simultaneously measuring the transfer of charge and substrate (e.g., Refs. 3, 4, 21, and 33). Substrate transport is measured either as the flux of a radioactive isotope (unidirectional transport) or from changes in substrate concentration (net transport). Another approach, based on thermodynamics, is to measure changes in the current reversal potential (Erev) in response to changes in substrate concentration (2, 37); however, this approach requires that Erev can be measured with the voltage window usable for the preparation, and for many Na+-coupled transporters this is not often possible. Also, this approach is compromised if the substrate induces significant nonstoichiometric current, as for neurotransmitter transporters (5, 13, 24, 34). Using a kinetic approach, the Hill coefficient is often used to infer stoichiometry. However, since the Hill coefficient is a measure of cooperativity of substrate binding, it can only provide a lower estimate of the number of binding sites (see Ref. 39, for example).
As we were unable to record current reversal potentials in oocytes expressing NaPi-IIa (for a 3:1 Na:Pi stoichiometry, the expected Erev exceeds +150 mV), we measured simultaneous uptake of Na+, Pi, and charge. The results show a Na+:charge ratio of 3:1, Pi:charge ratio of 1:1, and a Na+:Pi ratio of 3:1 for both pH 7.5 and 6.8. From these data, we conclude that the transport cycle of human NaPi-IIa involves the translocation of 1 HPO42, 3 Na+, and 1 positive net charge. This result is in full agreement with previous studies of the rat and flounder isoforms (11).
This finding contrasts with other studies, which concluded that both monovalent and divalent Pi is transported by human NaPi-IIa heterologuosly expressed in oocytes (1, 26). Busch and co-workers (1) reported that decreasing pH from 7.8 to 6.8 at 150 mM Na+ enhanced the Pi-induced current, which would result from transport of more monovalent Pi, and hence more charge, at acidic pH. We repeated these experiments, but in our hands reducing the pH from 7.8 to 6.8 at 150 mM Na+ produced an 10% reduction in the Pi-induced current (Virkki LV, unpublished observations). More recently, Moschèn and co-workers (26) used oocytes expressing the human NaPi-IIa isoform to compare changes in intracellular pH (pHi) induced by Pi at pH 6.0 and 8.0, concluding that both monovalent and divalent Pi are transported. However, their data show that applying Pi at pH 6.0 (monovalent/divalent ratio 6.3:1) induces significant alkalinization of the oocyte cytosol (pHi 7.3, monovalent/divalent ratio 1:1.6), which shows that the affinity of the transporter for divalent Pi exceeds at least 20-fold its affinity for monovalent Pi. Their data thus support the conclusion that divalent Pi is the preferred transported species, whereas transport of H2PO4 is negligible.
Substrate Interactions
The experiments in Figs. 3 and 4 show that with Pi (HPO42) as the variable substrate, KmHPO42 is increased and IPi, max is decreased when the Na+ concentration is reduced. A similar dependency of KmPi (IPi, max was not determined) on Na+ was observed for the mouse NaPi-IIa isoform (14), the flounder isoform (12), and the rat isoform (7). However, when Na+ is the variable substrate, an increase in KmNa is observed with reduced Pi concentrations, but in this case IPi, max is minimally affected (Table 1). A similar effect was observed for the rat isoform (7). In contrast, Hartmann et al. (14) saw no effect of reducing Pi on KmNa of the mouse isoform (IPi, max was not determined). Given an apparent KmPi of 0.07 mM at pH 7.4, this discrepancy might be explained by the smaller reduction in Pi used by Hartmann et al. (from 1 to 0.3 mM Pi), compared with this study (from 1 to 0.1 mM Pi).
Hartmann and co-workers (14) conclude from their data on the mouse NaPi-IIa isoform that binding of a substrate is ordered and that Na+ binds before Pi. However, according to classic enzyme kinetics theory of ordered substrate binding, the concentration of either substrate affects the apparent Km of the other (35). On the other hand, the maximum reaction velocity depends on the concentration of the substrate that binds last but is independent of the concentration of the substrate that binds first. Our result that the Na+ concentration affects both KmHPO42 and IPi, max, but that the Pi concentration only affects KmNa but not IPi, max, suggests that Pi is the first substrate to bind, followed by Na+, in agreement with results obtained on the flounder (12) and rat (7) isoforms.
The fact that NaPi-IIa cotransport involves three Na+ ions per transport cycle complicates the interpretation of the results based on analysis of steady-state current only. Analysis of pre-steady-state charge movement on the flounder (12) and rat (7) NaPi-IIa isoforms indicates that at least one of the three Na+ ions binds before Pi, as shown in the eight-state kinetic scheme presented in Fig. 8. The model is based on the results of this and previous studies (7, 9, 12). In this model, one Na+ binds first, followed by one HPO42 and then two more Na+ ions. The substrate-bound carrier then transitions to the internal side and returns empty after releasing the substrate.
The dependencies of Km and IPi, max on the cotransported substrate described above can be reconciled with the model presented in Fig. 8 if the first Na+ binding site (transitions 12) has a higher affinity for Na+ than the latter ones (transitions 34). In that case, the first Na+ site is fully occupied at Na+ concentrations where only a fraction of the latter Na+ binding sites are occupied, and the system behaves (at saturating Na+ concentrations) as if Pi was the first substrate to bind.
Voltage Dependency of Steady-State Transport
Voltage dependencies of KmPi and KmNa were previously examined in the rat NaPi-IIa isoform (7). At maximal Na+ (100 mM), the apparent Pi affinity of rat NaPi-IIa was weakly voltage dependent, with a decrease in KmPi seen at hyperpolarizing potentials. The effect was more pronounced at a lower Na+ concentration (50 mM). This contrasts with the behavior seen for the human NaPi-IIa isoform, where KmPi (KmHPO42) increased markedly with hyperpolarization (Fig. 4). Lowering the Na+ concentration and decreasing pH from 7.4 to 6.6 abrogated the voltage dependency. In contrast, the voltage dependency of KmNa of human NaPi-IIa was increased when pH was lowered to 6.6 in the voltage range examined (Fig. 5), with an increase in KmNa seen at hyperpolarizing potentials. Thus the voltage dependencies of KmPi and KmNa of human NaPi-IIa are modulated in opposite ways by pH.
The finding of a voltage-dependent KmPi is novel for type II Na-Pi cotransporters. Previous models based on the rat NaPi-IIa isoform assume that Pi binding is voltage independent (7, 12). The finding that at increasingly negative potentials the apparent affinity for negatively charged HPO42 is decreased suggests that at least in the human isoform, the Pi binding site lies within the membrane electrical field.
Pre-Steady-State Charge Movement: Na+ Dependency
Pre-steady-state currents have been analyzed for several different Na+-coupled transporters, such as Na+-dependent glucose (20, 29, 30), GABA (25), glutamate (38), iodine (4), and nucleoside (33) transporters, and are thought to arise from voltage-dependent conformational changes in the carrier and/or movement of ion(s) to binding site(s) within the membrane electric field. The pre-steady-state charge distribution is often approximated by a two-state Boltzmann function (15, 20, 23), yielding estimates of Qmax, V0.5, and z. In the absence of a substrate, all Na+-coupled transporters mentioned above show a negative shift in V0.5 with reducing Na+. In contrast, large differences between transporters are observed with respect to the Na+ dependency of Qmax. For example, Qmax was fully suppressed by removal of Na+ in the GABA transporter mGAT3 (31), whereas Qmax appears virtually independent of Na+ in the iodine transporter NIS (4).
Analysis of Na+ dependence of the pre-steady-state charge movement for human NaPi-IIa shows that Qmax is relatively independent of Na+ concentration and that significant charge movement was still observed in the complete absence of Na+ (Table 2). This indicates that both the empty carrier (transitions 81 in Fig. 8) and the first Na+ binding step (transitions 12 in Fig. 8) contribute to charge movement. Furthermore, a marked negative shift in V0.5 is seen with reduced external Na+ (Fig. 6D). For Na+ concentrations of 25125 mM, V0.5 was a near-linear function of log[Na+], with a slope of 100 mV per 10-fold change in the Na+ concentration. This is similar to that seen for other Na+-dependent transporters, such as SGLT1 (15) and GAT1 (25), and is reconcilable with a Nernstian dependency of V0.5 on a single Na+ that moves through a fraction of the transmembrane field to its binding site. According to the slope of the V0.5 vs. log[Na] data (Fig. 6D, inset), the hypothetical Na+ binding site would be located at an electrical distance corresponding to 60% of the transmembrane electrical field, relative to the external membrane face.2 2 When the Na+ concentration was reduced to below 25 mM, the linearity was lost. This is most likely a consequence of fitting a single Boltzmann to the Q-V data, although at least two voltage-dependent steps (binding of Na+ and transition of the empty carrier) are expected to contribute charge movement. As the Na+ concentration is changed, the relative contribution of these steps is altered, but the relative contribution of each remains unquantified. Unfortunately, it was not possible to fit the data to more than one Boltzmann function, a procedure that could have enabled us to separate the two components (18). Nevertheless, we can conclude that Na+ ions affect the preferred orientation of the Na+-bound carrier. As the external Na+ concentration is raised, the transporter is increasingly, assuming the outward-facing configuration, presumably ready for a Pi (HPO42) ion to bind.
Applying Pi (at 100 mM Na) significantly suppressed pre-steady-state charge movements (Table 2). However, in contrast to previous studies on rat (7) and flounder (12) isoforms, significant charge movement could still be observed in the human isoform in the presence of Pi. That also the Pi-bound carrier contributes to pre-steady-state charge movement is paralleled by the observation that the apparent affinity for Pi binding in steady-state current measurement is voltage dependent, further supporting the idea that the Pi (HPO42) binding site lies within the transmembrane electrical field.
Pre-Steady-State Charge Movement: Effect of pH
Examining the effect of reducing extracellular pH on pre-steady-state charge movement reveals that decreasing external pH from 7.4 to 6.6 markedly suppresses the maximum charge detected in the presence of 100 mM Na+ (see Fig. 7 and Table 3). Also, in the absence of external Na+ we observed suppression of charge movement by low pH. The results indicate that protons can interact with both the first Na+ binding step and the transition of the empty carrier. Analysis of the pH dependency of the Pi-induced steady-state current supports the notion that protons are able to interact with at least two sites in the protein, as a Hill coefficient of 2 was found from fitting Eq. 1 to the data in Fig. 3C. A similar indication of cooperativity binding of H+ was found for the flounder and rat isoforms (9).
The steady-state charge distribution (V0.5 in Table 3) was shifted in the positive direction by low pH, which is opposite of what we observed when the Na+ concentration was lowered. This indicates that protons are not merely hindering Na+ from reaching the first binding site, since this would be expected to cause a shift of V0.5 in the negative direction, as with lowering the Na+ concentration. One possible explanation is that protons interact with the first Na+ binding site, but in contrast to Na+, they are not able to support charge movement or steady-state transport. In this work, we limited the range of pH values examined to pH 7.4 and 6.6, since lowering the pH further caused marked instabilities in the holding currents for extreme potentials. Examining current records acquired at pH 5.0 using a narrower voltage range revealed that at this pH, pre-steady-state charge movements were completely suppressed and the transients looked like those obtained in water-injected control oocytes (not shown), as previously observed (9).
Earlier work on rat and flounder isoforms (9) examined the pH dependency of pre-steady-state charge movement over a significantly broader range. Over the pH range 8.05.6, V0.5 was shifted in the positive direction in both the presence and absence of Na. However, in contrast to the human isoform, Qmax was only minimally affected by reduced pH for both flounder and rat isoforms whether in the presence or absence of Na+. Furthermore, no pre-steady-state charge movement was observed at pH 5.0, presumably because at this low pH the steady-state charge distribution was shifted so far toward positive potentials that no charge movement could be detected in the voltage range normally possible with oocytes. From these studies, it was postulated that protons mainly interact with the transition of the empty carrier and with the binding of the last two Na+ ions. It appears that the human isoform differs from the rat and flounder isoforms in that the first Na+ binding step shows more proton sensitivity.
Revised Kinetic Model for the NaPi-IIa Transport Cycle
The results of the steady-state and pre-steady-state analysis in this study were used to modify the kinetic model originally proposed for NaPi-IIa (7, 9, 12). We simulated steady-state and pre-steady-state currents by assigning rate constants to the transitions between the states shown in Fig. 8A, as described earlier (7). Voltage-dependent rates were formulated according to Eyring transition rate theory, assuming sharp energy barriers. Similar to flounder and rat NaPi-II, voltage dependency was assigned to the transitions of the empty carrier (transitions 81), the first Na+ binding step (transitions 12), and the last Na+ debinding step (transitions 78). However, to simulate the voltage dependency of KmHPO42 seen in Fig. 4, it was necessary to introduce voltage dependency for HPO42 binding (transitions 23). In addition, the voltage-dependent rates associated with the Na+ binding steps (transitions 12 and 78) were simulated with assymmetrical energy barriers. Using the rate constants as defined (see legend for Fig. 8), we could simulate the voltage dependencies of the Pi-induced currents at variable Pi (100 mM Na) and variable Na+ (1 mM Pi) and reproduce the experimentally observed voltage dependency of KmHPO42 and KmNa. Thus a major difference from previous models (developed for the rat and flounder isoforms) is that HPO42 binding is voltage dependent, indicating that HPO42 ions bind in an area within the membrane electrical field.
In general, our model presented in Fig. 8 incorporates more skewness into rate constants and the factors that influence voltage-dependent transitions than previous models developed for the rat and flounder NaPi-II isoforms. Instead of assuming equal and slow forward and backward rate constants for the transition of the fully loaded carrier (transitions 45), the new model requires that, upon binding of all substrates on the external side, the fully loaded carrier (state 4) favors transitions 45 over the reverse reaction. Furthermore, we incorporated a larger voltage dependency for the last Na+ debinding step (transitions 78) than for the first Na+ binding step (transitions 12). Finally, whereas in previous models, mobile charges were assumed to move over symmetrically located barriers, here we found it necessary to introduce strong asymmetry into the first Na binding step (12) and the transition of the empty carrier (18) to obtain satisfactory agreement between the measured and simulated data.
The model presented in Fig. 8 is limited. We were not able to satisfactorily reproduce suppression of pre-steady-state charge movement (Qmax) caused by reducing extracellular pH from 7.4 to 6.6. Since we obtained the data in Fig. 7 by integrating the full oocyte pre-steady-state current response, it is possible that a transient with a faster time constant could have been masked by the oocyte capacitative transient and thus account for the "missing" charge in the data at pH 6.6. Nevertheless, we introduced H+ modulation of the first Na+ binding, since the pH-dependent decrease in Qmax is much larger in the presence of external Na+ than in its absence. Furthermore, from the slope V0.5/log[Na] in Fig. 6D, we estimated that the first Na+ binding (transitions 12) would sense 60% of the transmembrane electrical field. In the model in Fig. 8, this value is set to 1 = 0.2, and if we estimate this fraction from the slope of the simulated V0.5/log[Na] data in Fig. 8F, the value is close to 1. The discrepancy most likely results from fitting one single Boltzmann function to a charge distribution containing contributions from three charge-carrying transitions (12, 78, and 81). Finally, little information is available about factors influencing the intracellular reaction steps (transitions 58), such as the order of substrate release and their affinities. These issues would be more easily resolved using methods, such as the cut-open oocyte technique, that allow a faster voltage clamp of the membrane as well as manipulation of intracellular ion concentrations
Conclusions
We have examined the interactions of Na, Pi, and pH on the transport cycle of the human NaPi-IIa isoform. The interdependencies of the two substrates are complex, with the Na+ concentration affecting the Pi binding affinity and vice versa. The whole process is modulated by pH, which acts via titration of HPO42, by reducing the Na+ affinity of one or more Na+ binding sites, and by modulating the transition of the empty carrier. The data have enabled us to modify previous models of NaPi-II transport by including proton modulation of the first Na+ binding step and voltage dependency of HPO42 binding.
GRANTS
This work was supported by grants to H. Murer from the Swiss National Science Foundation, the Gebert Rüf Stiftung (www.grstiftung.ch), the Hartmann-Müller-Stiftung (Zürich), the Olga Mayenfish-Stiftung (Zürich), and the Union Bank of Switzerland.
FOOTNOTES
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.
1 It is necessary to include an offset in the equation, because an error is introduced in the estimate of IPi when current recordings obtained in the presence and absence of Pi are subtracted. This occurs because in the absence of Pi, NaPi-IIa operates in a Na+-dependent leak mode. Because the leak current is suppressed by Pi (17, 19), recordings made in the absence of Pi contain more leak current than recordings made in the presence of Pi. The amount of leak current present in each case is hard to define, since it is a function of both Na+ and Pi concentrations. Furthermore, although the current is fully suppressed at 1 mM Pi and 100 mM Na (7, 19), the Ki for the suppression of the leak current is not known. PFA is a blocker of NaPi-II and fully suppresses the leak current at 1 mM (and 100 mM Na+) as well as provides an estimate for the total amount of leak. However, its Ki for blocking the leak current is not known. In the human NaPi-IIa isoform, the leak current, measured as the PFA-inhibitable component, has a reversal potential around 25 mV and contributes little to the total current measured at 50 mV (see Fig. 4A). However, at more extreme potentials the error introduced by the subtraction procedure becomes larger. Hence, we decided to apply the offset in calculating KmPi and KmNa in experiments where a broad range of voltages was applied.
2 We assume a simple two-state model for the first Na+ binding (states 1 and 2 in Fig. 8), from which the fraction of the membrane electrical field sensed by the ion can be estimated. V0.5 is the voltage at which the two states are equally occupied at each extracellular Na concentration ([Na]o), at which k12 = k21, and the slope of the relationship V0.5/ln[Na]o = kT/e. However, because the empty carrier also contributes to charge movement, and its relative contribution is higher at low [Na]o, the method underestimates the slope, and hence overestimates . For a definition of symbols, see the legend to Fig. 8.
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