1 Department of Physiology, University of California at Los Angeles, School of Medicine, Los Angeles, California 90095-1751; and 2 Physiologisches Institut, Universität Zürich, CH-8057 Zurich, Switzerland
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The stoichiometry of the rat and flounder isoforms of the renal type II sodium-phosphate (Na+-Pi) cotransporter was determined directly by simultaneous measurements of phosphate (Pi)-induced inward current and uptake of radiolabeled Pi and Na+ in Xenopus laevis oocytes expressing the cotransporters. There was a direct correlation between the Pi-induced inward charge and Pi uptake into the oocytes; the slope indicated that one net inward charge was transported per Pi. There was also a direct correlation between the Pi-induced inward charge and Na+ influx; the slope indicated that the influx of three Na+ ions resulted in one net inward charge. This behavior was similar for both isoforms. We conclude that for both Na+-Pi cotransporter isoforms the Na+:Pi stoichiometry is 3:1 and that divalent Pi is the transported substrate. Steady-state activation of the currents showed that the Hill coefficients for Pi were unity for both isoforms, whereas for Na+, they were 1.8 (flounder) and 2.5 (rat). Therefore, despite significant differences in the apparent Na+ binding cooperativity, the estimated Na+:Pi stoichiometry was the same for both isoforms.
electrogenic phosphate transport; Xenopus oocyte; voltage clamp; substrate coupling ratio
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE REABSORPTION of inorganic phosphate (Pi) in the kidney is mediated by a secondary active Na+-coupled transport mechanism localized at the proximal tubular apical brush-border membrane (18, 19). The downhill Na+ concentration gradient, together with the membrane potential, leads to an influx of Pi under normal physiological conditions. The steady-state kinetics for this type II Na+-Pi cotransport system were first characterized in vitro using brush-border membrane vesicles (BBMV) isolated from proximal tubular epithelia (4, 14) and more recently by expressing cloned transporters in Xenopus laevis oocytes (5, 6, 11-13, 17).
An important unresolved issue is the Na+:Pi stoichiometry. Early studies on BBMVs assumed that cotransport was electroneutral at pH 7.4 and postulated a Na+:Pi stoichiometry of 2:1, given that divalent Pi would be the preferred species transported (4, 14). Nevertheless, using the BBMV preparation, it has been difficult to obtain a definitive measure for the stoichiometry because of uncertainties regarding the electrogenicity of the transport mechanism and the species of Pi transported (i.e., monovalent vs. divalent Pi) (1-3, 24). Indeed, electrophysiological studies on cloned renal Na+-Pi cotransporters expressed in Xenopus oocytes have shown unequivocally that type II Na+-Pi cotransport is electrogenic and involves the inward movement of positive charge (5, 6, 11-13). This finding necessitates the inward transport of at least one net positive charge with the cotransport of the phosphate species. Electrical measurements on the rat and flounder isoforms of type II Na+-Pi cotransporter expressed in Xenopus oocytes show Michaelis-Menten kinetics for steady-state Pi activation. In contrast, the Hill coefficients for Na+ vary from less than 2 in the case of the flounder (NaPi-5) (11) and up to 3 for the rat (NaPi-2) cotransporter (5, 12). The reported differences in the Hill coefficients indicate a difference in cooperativity with respect to the binding of Na+ ions to the transporters; however, it is not known whether this difference in cooperativity is also reflected in the Na+:Pi stoichiometry of cotransport.
To investigate this possibility and to obtain a direct measure of cation/substrate coupling ratio for type II Na+-Pi cotransport, we have correlated the radiolabeled flux of each substrate (Na+ and Pi) with the net charge transported across the plasma membrane in the same oocyte (16), for both the rat and flounder isoforms. Our findings indicate that in the pH range 6.8-7.4, the inward current evoked by phosphate is carried by both Na+ and the divalent form of Pi and that the Na+:Pi stoichiometry is 3:1 for both isoforms.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Oocytes. Stage VI Xenopus laevis oocytes (Nasco, Fort Atkinson, WI) were defolliculated and injected with cRNA coding for the rat (NaPi-2) (17) or flounder (NaPi-5) (27) renal type II Na+-Pi cotransporter. Cells were maintained at 18°C in modified Barth's medium (20) with 10 mg/l gentamicin sulfate. Electrophysiological and uptake assays were performed 3-5 days after injection. All experiments were performed at 22 ± 1°C.
Solutions. Control superfusate (Na+ buffer) contained (in mM) 100 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES-Tris, adjusted to pH 7.4. For experiments at pH 6.8, the control solution was buffered with 10 mM PIPES (pH 6.8) and adjusted using NaOH. Pi was added at the required concentration as K2HPO4/KH2PO4, the proportion of which was adjusted to give the desired pH. For Na+ dose-response determinations, Na+ was replaced by equimolar choline.
Electrophysiology. A two-electrode
voltage clamp system was used for electrophysiological measurements.
This was interfaced to an integrated
analog-to-digital/digital-to-analog data acquisition system (Labmaster,
TL-1; Axon Instruments, Foster City, CA) and driven by
pCLAMP software (Axon Instruments). Data were recorded using AXOTAPE software (sampling at 1 s1) (Axon
Instruments). All data were filtered at 1 Hz. The experiments were
performed as previously described (16). Oocytes were placed in a small
perfusion chamber (functional volume
20 µl) and continuously perfused at the rate of
90 µl/min using a peristaltic pump. The superfusate was removed by suction. For all experiments, the membrane voltage was held at a fixed value of
50 mV. Each cell was
superfused with the standard Na+
buffer solution (see above) until a stable baseline was achieved (±5 nA). Only cells that showed a holding (baseline) current
magnitude smaller than 50 nA were used for subsequent
electrophysiological and isotope uptake assays. The
Pi-induced current was obtained as
the difference between the baseline current and the current in the
presence of Pi. Net charge
transfer into the oocyte was obtained by numerically integrating the
Pi-induced inward current record
until the current returned to the baseline after washout of
Pi. Charge was converted to a
molar equivalent using Faraday's constant, assuming monovalency.
Isotope uptake assay. Radiolabeled superfusate was made up with 32P (final specific activity, 5-50 mCi/mmol) or 22Na (final specific activity, 135 µCi/mmol) (Amersham) together with the appropriate substrates. Immediately after washout of the isotope, the oocyte was removed from the chamber, washed in choline buffer at 4°C, transferred to a scintillation vial, solubilized with 10% SDS, and assayed for tracer (22Na+ or 32Pi) content. The Na+ or Pi influx obtained in this manner, together with the net inward charge obtained from time integral of the Pi-induced inward current (see above) from the same oocyte, allowed for a determination of net inward charge/substrate flux ratio in individual oocytes. For each experiment, the current was recorded and uptake of radiolabeled substrate (Na+ or Pi) was assayed in at least three control (noninjected) oocytes from the same batch, the mean count of which was used to correct for endogenous Na+ or Pi uptake.
Curve fitting and data analysis. The Hill equation of the form
![]() |
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Charge transfer and substrate uptake are linearly
correlated. Figure 1 shows
a typical current record of an oocyte expressing the flounder
Na+-Pi
cotransporter (NaPi-5), voltage clamped to a holding potential of
50 mV. When 0.1 mM Pi + 32P (25 mCi/mmol) were added to
the external solution, there was a peak
Pi-induced inward current of
approximately
200 nA. Upon removal of
Pi from the bathing medium, the
current returned to baseline. The same experiment performed on control
noninjected oocytes indicated that endogenous electrogenic phosphate
transport was negligible in these cells. The net inward charge
(Q), which represents the net
positive charge transported across the plasma membrane, was obtained by
integration of the Pi-induced
current with time, and the corresponding
Pi uptake was obtained from the determination of 32P content in
this oocyte.
|
Figure 2 shows data for NaPi-2 and NaPi-5
obtained for a number of oocytes having different levels of expression
and assayed over time intervals varying from 5-20 min. A linear
correlation was found between the inward charge
(Q) and
Pi uptake, indicating that the
coupling between Q and
Pi uptake was independent of the length of the assay period and expression level. Data for both isoforms
gave a slope
(Q/Pi)
of unity (NaPi-2, 0.95 ± 0.03; NaPi-5, 1.04 ± 0.02). Thus one
net positive charge was transferred per Pi transported for both isoforms.
|
For the complementary experiment using 22Na, the low specific activity at the normal extracellular Na+ concentration of 100 mM necessitated that we reduce the external Na+ concentration to 30 mM to obtain a sufficiently large specific activity for accurate counting (135 µCi/mmol). Since it is known that the Na+ concentration influences both the maximum rate of Na+-Pi transport and the apparent affinity for Pi (11, 12), it was first necessary to establish whether the Q/Pi ratio was a function of the Na+ concentration. We therefore repeated the above protocol to determine the net inward charge per Pi uptake at 30 mM Na+ and 1 mM Pi (and 32P; 5 mCi/mmol). Similar to the result at 100 mM Na+, the slope (Q/Pi) at 30 mM Na+ was unity for both isoforms (data not shown), indicating that the net inward charge coupled to Pi influx was unaffected by the reduction in external Na+ concentration.
In the absence of Pi, type II
Na+-Pi
cotransporters exhibit uncoupled
Na+ leak or slippage (12), which
could lead to errors in estimating the coupled
Na+ uptake. Therefore, we first
measured Na+ uptake in the absence
of Pi. For both isoforms at 30 mM
Na+, this amounted to 250 pmol
Na+ influx during the recording
period (Fig. 3) which was significantly higher than background Na+ influx
(
50 pmol). We then measured
Pi-induced
Na+ influx (coupled
Na+ transport) in NaPi-2- and
NaPi-5-expressing oocytes from the same batch. As shown in Fig. 3,
there was a linear correlation between the inward charge and
Na+ uptake for different oocytes,
indicating that similar to Pi, cotransport of Na+ was also
strictly coupled to net charge transfer. The ratio
Q/Na+
was determined by constraining the linear regression line through the
uncoupled Na+ influx, and the
slope
(Q/Na+)
was close to 0.3 (NaPi-2, 0.29 ± 0.02; NaPi-5, 0.33 ± 0.02) for
both isoforms. This finding is consistent with three
Na+ ions being transported per net
inward charge. Therefore, based on the
Q/Pi
ratio of 1 and
Q/Na+
of 0.3, a 3:1
Na+-to-Pi
coupling ratio was predicted which, moreover, would be consistent with the transport of divalent
Pi
(HPO2
4).
|
Inward charge transfer and external
pH. Between pH 7.4 and 6.8, the
Pi divalent-to-monovalent ratio
varies from 4:1 to 1:1. If there were a concomitant change in the
preferred Pi species transported,
then we would predict a change in the
Q/Pi
ratio at the lower pH. To test this hypothesis, we determined the
Q/Pi ratio at pH 6.8 for both isoforms. At the lower pH, the
Pi concentration was 1 mM and the
Na+ concentration was maintained
at 100 mM, to ensure sufficient accuracy for both the charge and uptake
measurements. Similar to the behavior at pH 7.4 (Fig. 2), we obtained a
linear correlation between Pi
influx and total inward charge movement for NaPi-2 and NaPi-5 at pH 6.8 as illustrated in Fig. 4, where the data were normalized to aid comparison between the two data sets. For each
isoform, the slope
(Q/Pi)
was not significantly different from the corresponding slope at pH 7.4 (P 0.05; Student's
t-test for comparison of regression
coefficients), which indicated that the preferred
Pi species was
HPO2
4 over this pH range.
|
Steady-state
Na+ and
Pi activation kinetics.
A common method used to estimate substrate coupling ratios of
transporters is to fit the Hill equation to the steady-state dose-response activation curve for each cosubstrate. To compare this
with the direct method used in this study, we determined the Hill
coefficients for Pi and
Na+ under similar experimental
conditions. As summarized in Table 1, the
Hill coefficient for Pi activation
was unity for both isoforms, whereas for
Na+ activation, a significant
difference in the Hill coefficient was obtained for the two isoforms
(2.5 ± 0.2 for NaPi-2 vs. 1.8 ± 0.1 for NaPi-5;
P < 0.05, unpaired Student's
t-test). The values for the affinity
constants for each substrate
(KPim and
KNam) agreed well with
previously published values (11, 12).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The substrate stoichiometry is an important thermodynamic parameter for cotransporters, because it determines the concentrating capacity for coupled solute transport and the energetic expenditure required to maintain such transport. A common, indirect method by which to estimate ion:substrate stoichiometry has been to use the Hill coefficient (e.g., 5, 11, 12, 20, 25); however, this parameter is no more than a qualitative indicator of apparent binding cooperativity (10) and at best can provide a lower estimate of the number of binding sites (21, 26). To complicate matters further for cotransporters, the sigmoidicity observed in the Hill plot may merely indicate the cooperativity of transport activation, by binding at a modifier site, and not transport of the substrate per se (23). Thermodynamic approaches have yielded more reliable estimations of stoichiometric ratios (7, 8), but it is difficult to accurately control the imposed potential difference across the membrane and simultaneously measure solute fluxes.
The activation of electrogenic cotransporters leads to the depolarization of the membrane potential, which in turn affects cotransporter activity (5, 11, 20). Therefore, to determine the stoichiometry, isotope flux assays must be performed under conditions in which the membrane potential is controlled, thereby allowing the simultaneous measurements of the isotope flux and the substrate-coupled current in the same cell. For the Na+-Pi cotransporter, there is an additional complication in that both Na+ and Pi contribute to the net charge transported across the membrane, and the relative contribution of each cannot be determined based on current measurements alone. In addition to establishing the Na+:Pi stoichiometry, the main advantage of the direct method used in this study is that by simultaneous measurement of the translocated charge and the unidirectional flux of the substrate, we could identify the preferred species of Pi transported.
A characteristic feature of type II Na+-Pi
cotransport is that transport activity is decreased by low external
pH (1-4). Since pH also determines the ratio,
[HPO24]/[H2PO
4], it has been difficult to draw definite conclusions, based on
radiolabeled uptake (1, 3, 24) or electrophysiological studies (5, 6,
13) alone, as to which Pi species
is transported. Our finding of a constant
Q/Pi
ratio for pH 7.4 and 6.8 strongly suggests that, despite a fourfold
variation in the divalent/monovalent Pi ratio, divalent
Pi remains the preferred phosphate
species transported over this pH range.
As previously demonstrated for the Na+-glucose (16) and Na+-iodide (9) cotransporters, simultaneous electrophysiological and unidirectional isotope flux assays on the same cell provide a direct, model-independent means of estimating substrate coupling ratios. In the present case, our findings that at pH 7.4 1) one net inward charge is transported for every three Na+ ions cotransported and 2) one net inward charge is transported for every Pi cotransported are consistent with a 3:1 Na+:Pi stoichiometry. This indicates that the divalent form of Pi is preferentially transported. Significantly, this finding was consistent for both the rat and flounder Na+-Pi cotransporters, suggesting that both isoforms use the same substrate coupling mechanism.
The Na+ and Pi fluxes were directly proportional to the Pi-induced inward charge. For some neurotransmitter transporters (e.g., glutamate, dopamine, and serotonin transporters), the substrate-evoked currents have been shown to exceed what could be accounted for by substrate fluxes, and thus the activity of these transporters has been likened to that of ligand-gated ion channels (for review, see Ref. 22). This behavior does not appear to be a general rule among Na+-coupled transporters. For the Na+-glucose (16), Na+-iodide (9), and Na+-Pi (this study) cotransporters, fluxes of Na+ and the substrate are directly proportional to transporter-mediated currents. Since currents in excess of fluxes are not observed, permeation through these transporters appears to be strictly coupled to transporter conformational changes (15).
Both the rat and flounder
Na+-Pi
cotransporter isoforms showed Michaelis-Menten kinetics for
Pi activation
(n 1). However, Na+ activation curves for the two
isoforms gave significantly different estimates for the Hill
coefficient (Table 1) in agreement with previous studies (11, 12).
Agreement between the Hill coefficient and the stoichiometry for
Na+ indicates high cooperativity
between the Na+ binding sites (21,
26). Therefore, since the
Na+:Pi
stoichiometry was identical for the two isoforms, we conclude that
there is greater cooperativity between the
Na+ binding sites in the rat than
the flounder isoform. Despite this difference in
Na+ binding cooperativity between
the two isoforms, their substrate concentrating capacity remains the same.
In summary, using simultaneous radiolabeled influx and inward charge determinations in the same oocyte, we now provide direct evidence that the Na+:Pi coupling ratio for both the rat and the flounder type II Na+-Pi cotransporter isoforms is 3:1 and that divalent Pi is the species transported. Our data further indicate that reliance on the Hill coefficient alone can be misleading in the determination of cation:substrate stoichiometry of cotransporters.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Elsa Gallardo and Daisy Leung for preparation of cRNA and oocytes, respectively.
![]() |
FOOTNOTES |
---|
We gratefully acknowledge the support and encouragement of Professors E. M. Wright (UCLA) and H. Murer (Zurich). This project was made possible through financial support from National Institutes of Health Grants DK-19567 and NS-25554 (to E. M. Wright) and Swiss National Science Foundation Grant 31-46523 (to H. Murer).
I. C. Forster and S. Eskandari contributed equally to this work.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: I. C. Forster, Physiologisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland (E-mail: forster{at}physiol.unizh.ch).
Received 21 October 1998; accepted in final form 7 January 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Amstutz, M.,
M. Mohrmann,
P. Gmaj,
and
H. Murer.
Effect of pH on phosphate transport in rat renal brush border membrane vesicles.
Am. J. Physiol.
248 (Renal Fluid Electrolyte Physiol. 17):
F705-F710,
1985[Medline].
2.
Béliveau, R.,
and
H. Ibnoul-Khatib.
Electrogenicity of phosphate transport by renal brush-border membranes.
Biochem. J.
22:
801-806,
1988.
3.
Bindels, R. J. M.,
L. A. M. Van den Broek,
and
C. H. Von Os.
Effect of pH on the kinetics of Na+-dependent phosphate transport in rat renal brush-border membranes.
Biochim. Biophys. Acta
897:
83-92,
1987[Medline].
4.
Burckhardt, G.,
H. Stern,
and
M. Murer.
The influence of pH on phosphate transport into rat renal brush border vesicles.
Pflügers Arch.
390:
191-197,
1981[Medline].
5.
Busch, A. E.,
S. Waldegger,
T. Herzer,
J. Biber,
D. Markovich,
G. Hayes,
H. Murer,
and
F. Lang.
Electrophysiological analysis of Na+/Pi cotransport mediated by a transporter cloned from rat kidney in Xenopus oocytes.
Proc. Natl. Acad. Sci. USA
91:
8205-8208,
1994[Abstract].
6.
Busch, A. E.,
C. A. Wagner,
A. Schuster,
S. Waldegger,
J. Biber,
H. Murer,
and
F. Lang.
Properties of electrogenic Pi transport by NaPi-3, a human renal brush border Na+/Pi transporter.
J. Am. Soc. Nephrol.
6:
1547-1551,
1995[Abstract].
7.
Chen, X.-Z.,
M. J. Coady,
F. Jackson,
A. Berteloot,
and
J.-Y. Lapointe.
Thermodynamic determination of the Na+:glucose coupling ratio for the human SGLT1 cotransporter.
Biophys. J.
69:
2405-2414,
1995[Abstract].
8.
Chen, X.-Z.,
C. Shayakul,
U. V. Berger,
W. Tian,
and
M. Hediger.
Characterization of a rat Na+-dicarboxylate cotransporter.
J. Biol. Chem.
273:
20972-20981,
1998
9.
Eskandari, S.,
D. D. F. Loo,
G. Dai,
O. Levy,
E. M. Wright,
and
N. Carrasco.
Thyroid Na+/I symporter. Mechanism, stoichiometry, and specificity.
J. Biol. Chem.
272:
27230-27238,
1997
10.
Forsén, S.,
and
S. Linse.
Cooperativity: over the Hill.
Trends Biochem. Sci.
20:
495-497,
1995[Medline].
11.
Forster, I. C.,
C. A. Wagner,
A. E. Busch,
F. Lang,
J. Biber,
N. Hernando,
H. Murer,
and
A. Werner.
Electrophysiological characterization of the flounder type II Na+/Pi cotransporter (NaPi-5) expressed in Xenopus laevis oocytes.
J. Membr. Biol.
160:
9-25,
1997[Medline].
12.
Forster, I. C.,
N. Hernando,
J. Biber,
and
H. Murer.
The voltage dependence of a cloned mammalian renal type II Na+/Pi cotransporter (NaPi-2).
J. Gen. Physiol.
112:
1-18,
1998
13.
Hartmann, C. M.,
C. A. Wagner,
A. E. Busch,
D. Markovich,
J. Biber,
F. Lang,
and
H. Murer.
Transport characteristics of a murine renal Na+/Pi cotransporter.
Pflügers Arch.
430:
830-836,
1995[Medline].
14.
Hoffmann, N.,
M. Thees,
and
R. Kinne.
Phosphate transport by isolated renal brush border vesicles.
Pflügers Arch.
362:
147-156,
1976[Medline].
15.
Loo, D. D. F.,
B. A. Hirayama,
E. M. Gallardo,
J. T. Lam,
E. Turk,
and
E. M. Wright.
Conformational changes couple Na+ and glucose transport.
Proc. Natl. Acad. Sci. USA
95:
7789-7794,
1998
16.
Mackenzie, B.,
D. D. F. Loo,
and
E. M. Wright.
Relationships between Na+/glucose cotransporter (SGLT1) currents and fluxes.
J. Membr. Biol.
162:
101-106,
1998[Medline].
17.
Magagnin, S.,
A. Werner,
D. Markovich,
V. Sorribas,
G. Stange,
J. Biber,
and
H. Murer.
Expression cloning of human and rat renal cortex Na/Pi cotransport.
Proc. Natl. Acad. Sci. USA
90:
5979-5983,
1993[Abstract].
18.
Murer, H.,
and
J. Biber.
A molecular view of proximal tubular inorganic phosphate (Pi) reabsorption and of its regulation.
Pflügers Arch.
433:
379-389,
1997[Medline].
19.
Murer, H.,
D. Markovich,
and
J. Biber.
Renal and small intestinal sodium dependent symporters of phosphate and sulphate.
J. Exp. Biol.
196:
167-181,
1994
20.
Parent, L.,
S. Supplisson,
D. D. F. Loo,
and
E. M. Wright.
Electrogenic properties of a cloned Na+/glucose cotransporter. I. Voltage-clamp studies.
J. Membr. Biol.
125:
49-62,
1992[Medline].
21.
Segel, I. H.
Biochemical Calculations. New York: Wiley, 1976, p. 309-310.
22.
Sonders, M. S.,
and
S. G. Amara.
Channels in transporters.
Curr. Opin. Neurobiol.
6:
294-302,
1996[Medline].
23.
Stein, W. D.
Transport and Diffusion Across Cell Membranes. Orlando: Academic, 1986, p. 410.
24.
Strévey, J.,
S. Giroux,
and
R. Beliveau.
pH gradient as an additional driving force in the renal reabsorption of phosphate.
Biochem. J.
271:
687-692,
1990[Medline].
25.
Umbach, J. A.,
M. J. Coady,
and
E. M. Wright.
Intestinal Na+/glucose cotransporter expressed in Xenopus oocytes is electrogenic.
Biophys. J.
57:
1217-1224,
1990[Abstract].
26.
Weiss, J. N.
The Hill equation revisited: uses and misuses.
FASEB J.
11:
835-841,
1997
27.
Werner, A.,
H. Murer,
and
R. K. H. Kinne.
Cloning and expression of a renal Na-Pi cotransport system from flounder.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F311-F317,
1994