Department of Physiology, University of California Los Angeles, School of Medicine, Los Angeles, California 90095-1751
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ABSTRACT |
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Sodium-glucose
cotransporters (SGLTs) mediate active transport of sugar across cell
membranes coupled to Na+, by using the electrochemical
gradient as a driving force. In the kidney, there is evidence for two
kinds of cotransporters, a high-affinity, low-capacity system, and a
low-affinity, high-capacity system, with differences in substrate
specificity and kinetics. Three renal SGLT clones have been identified:
SGLT1 corresponding to the high-affinity system, and SGLT2 and SGLT3
with properties reminiscent of the low-affinity system. We have
determined the stoichiometry of pig SGLT3 (pSGLT3) by using a direct
method, comparing the substrate-induced inward charge to
22Na or
[14C]-methyl-D-glucopyranoside uptake in
the same oocyte. pSGLT3 stoichiometry is 2 Na+:1 sugar, the
same as that for SGLT1, but different from SGLT2 (1:1). The
Na+ Hill coefficient for SGLT3 is ~1.5, suggesting low
cooperativity between Na+ binding sites. Thus SGLT3 has
functional characteristics intermediate between SGLT1 and SGLT2, so,
whereas SGLT3 stoichiometry is the same as that for SGLT1 (2:1), sugar
affinity and specificity are similar to SGLT2.
sodium-glucose cotransporter; electrophysiology; ion-substrate coupling
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INTRODUCTION |
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SODIUM-DEPENDENT GLUCOSE COTRANSPORTERS (SGLTs) mediate the "active" transport of glucose across the brush border membranes of renal and intestinal epithelia by using the Na+ electrochemical gradient across the membrane as a driving force. It has been commonly accepted that there is a single high-affinity cotransporter in the intestine and two cotransporters in the proximal renal tubule, a low-affinity, high-capacity SGLT in the convoluted tubule, and a high-affinity, low-capacity SGLT in the straight tubule (1). There is compelling evidence that SGLT1 is the high-affinity transporter expressed in both the intestine and straight proximal tubule (6, 19, 23), but there is some ambiguity about the gene coding for the major renal SGLT (commonly referred to as SGLT2).
So far, two SGLTs have been cloned from renal tissue, and there are at least six other SGLT family members expressed in the kidney whose functions are unknown. The first SGLT to be cloned from rat and human kidney was SGLT2 (28, 29). Although poorly expressed in heterologous systems, its function resembles those traditionally attributed to the transporter in the rabbit proximal renal tubule, i.e., low affinity for sugars, highly selective for glucose over galactose, and, on the basis of indirect experiments, a Na+-to-sugar stoichiometry of 1:1 (12, 28, 29). Furthermore, in the intact rat kidney, the distribution of SGLT2 mRNA mirrors that expected for the high-capacity cotransporter (29). The second renal SGLT to be identified was a clone originally thought to be a neutral amino acid transporter and called SAAT1 (13). This pig clone was later expressed in Xenopus laevis oocytes, permitting functional characterization with a high degree of precision, and it was found that SAAT1 was a low-affinity Na+/sugar cotransporter (15, 16). This transporter was called pig SGLT2 (pSGLT2) and renamed as SGLT3 (3) after the cloning of the human ortholog (4).
In common with SGLT2, SGLT3 has a low affinity for sugars, and is highly selective for D-glucose; i.e., unlike SGLT1 it has a very low affinity for D-galactose and 3-O-methyl-D-glucopyranoside (3OMDG) apparent affinity [(K0.5)>> 20 mM; 3, 16]. A major difference between SGLT3 and SGLT2 is that SGLT2 has a high affinity for Na+ under the same sugar concentration (2 mM) (12, 15).
In Na+-coupled cotransporters, a knowledge of the ion-substrate coupling stoichiometry is important because it reveals insights into the concentrative capacity of the transport system under investigation. The higher the degree of coupling (one or more Na+ per substrate), the greater the substrate concentration that can be achieved in the cell by the transporter, and this becomes particularly critical when large concentration gradients need to be overcome. On the basis of analysis of the Na+ Hill coefficient it was suggested that the SGLT3 coupling was 1 Na+:1 sugar (15). In this work, we have directly determined the ion-substrate coupling stoichiometry of SGLT3 by using radioactive tracer uptakes into voltage-clamped oocytes (14).
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EXPERIMENTAL PROCEDURES |
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In our experiments, we made a direct determination of
Na+-to-sugar-coupling stoichiometry and also investigated
the ion-protein binding interaction of SGLT3. The cloned
pSGLT3 (SAAT1) protein was functionally expressed in X. laevis oocytes. For all experiments the membrane potential was
controlled and sugar-induced currents were measured by using the two
electrode voltage-clamp technique. Our experimental strategy was first
to directly compare unidirectional ligand uptakes
(22Na+ or
[14C]-methyl-D-glucopyranoside;
-MDG)
into voltage-clamped cells to the cotransporter currents over the same
time course in single cells (14), and second to determine
the Na+ activation of cotransport for a Hill analysis.
Oocytes expressing pSGLT3. Stage VI oocytes from X. laevis (Nasco, Fort Atkinson, WI) were defolliculated and injected with pSGLT3 cRNA (pSAAT1, 13,16) and maintained at 18°C in modified Barth's medium (21) containing gentamycin (5 mg/ml) and penicillin (100 units/ml)/streptomycin (100 µg/ml). Experiments were performed at 22 ± 1°C, 5-15 days after the injection.
The oocyte was placed in the chamber, impaled with electrodes, and continuously superfused with the required medium. Na+ medium contained (in mM) 100 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES (pH 7.5 with Tris). In Na+-activation experiments, where the Na+ concentration was changed, ionic strength and chloride concentration were maintained by replacing NaCl with choline chloride.Hill analysis.
Na+ activation experiments were carried out at 11 different
voltages (from 150 to +50 mV). The oocyte was bathed in a
Na+ solution, its concentration varying from 50 µM to 10 mM. At each Na+ concentration, sugar-dependent currents
were measured with 3, 25, or 100 mM
-MDG. At each voltage the data
were fitted to Eq. 1
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(1) |
Charge-to-Na+ stoichiometry.
Uptake of 22Na was optimized for specific activity and
transport rate by using a saturating concentration of -MDG (100 mM) and a 22Na concentration of 3 or 10 mM (>2 times the
Na+ K0.5; see below). The
voltage-clamped oocyte (at
50 or
100 mV) was superfused with 3 mM
(or 10 mM) Na+ solution. A stable baseline current was
recorded, and then the bath solution was changed to 100 mM
-MDG with
3 mM (or 10 mM) 22Na solution (2.2-4.5 µCi/ml) for
2-10 min. The solution was changed back to the Na+
medium until the current returned to baseline (see Fig. 2). The oocyte
was recovered from the chamber, rinsed three times in ice-cold choline
medium (5 ml each) and solubilized with 10% SDS for liquid scintillation counting.
Charge-to-sugar stoichiometry.
The oocyte was clamped at 100 mV and superfused with 100 mM
Na+ solution. When the baseline was stable, 0.5, 1, or 2 mM
[14C]
-MDG was added to the Na+ solution.
After 10 min the sugar was removed from the bathing solution, and the
current was returned to the baseline. The oocyte was washed,
solubilized, and counted as described above. Sugar-induced current was
obtained as the difference between baseline and the
-MDG current.
The [14C]
-MDG uptakes were optimized by using a sugar
concentration close to the sugar K0.5 (2 mM) at
a high Na+ concentration, 100 mM.
[14C]
-MDG uptake in noninjected oocytes was used to
correct for endogenous
-MDG uptake. The current was converted to its
molar equivalent as described above.
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RESULTS |
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Na+ activation kinetics.
The Hill equation is often used as an indirect method to estimate
substrate coupling ratios of transporters. In pSGLT3-expressing oocytes, the steady-state dose-response activation curve for
Na+ was fitted to the Hill equation. Figure
1 is an example of the data for one sugar
concentration and its fit to the Hill equation. The Hill coefficient
(n) at 90 mV was 1.5 ± 0.1.
-MDG concentration was 25 mM, and the Na+ concentration varied between 50 µM
and 10 mM. The n was independent of the sugar concentration
(from 3 to 100 mM) and membrane potential (from
10 to
150 mV) and
ranged between 1.3 and 1.8 (mean = 1.6 ± 0.03, n = 18). The K0.5 and
Imax at each voltage and sugar concentration were consistent with the previously published values (15).
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Charge transfer and -MDG uptake are linearly correlated (2:1).
Figure 2 shows the sugar-dependent
current in an oocyte-expressing pSGLT3 clamped at
100 mV. Initially,
the oocyte was superfused with 100 mM Na+ solution, and the
addition of 2 mM [14C]
-MDG induced an inward current
of ~330 nA. When sugar was removed from the bath, the current
returned to the baseline. The current was integrated with time to
determine the sugar-dependent net charge influx
(Q
-MDG). This charge was converted
to its molar equivalent and compared with the sugar
([14C]
-MDG) uptake measured in this same oocyte. The
inward charge transported was 1.7 × 10
4 C of
positive charge, which corresponds to 1.77 nmol, and sugar uptake was
0.8 nmol, resulting in a charge-to-sugar ratio for this oocyte of
2.2:1. The same process was repeated with 12 oocytes and
[14C]
-MDG concentrations of 0.5, 1, or 2 mM. All the
data could be fitted to a single regression line, indicating that the
stoichiometry was independent of the sugar concentration. For all
oocytes tested, the value of Q
-MDG/
-MDG
uptake was 2.2 ± 0.1 (Fig.
3).
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Charge transfer and Na+ uptake are
linearly correlated (1:1).
The relationship between charge influx and 22Na uptake was
measured in 11 oocytes clamped at 50 or
100 mV. Each oocyte was bathed in 3 or 10 mM 22Na solution and, after stable
baseline, 100 mM
-MDG was added. The sugar-dependent inward current
was recorded, and 22Na uptake was measured in the same
oocyte. Figure 4 shows the relationship
between the net charge transported (Q) and the radiolabeled Na+ uptake. The QNa/Na+
uptake ratio was independent of voltage and Na+
concentration. All data were fitted to a regression line with a slope
of 1.2 ± 0.1. Open circles represent the
sugar-independent Na uptake (zero current), obtained from six
pSGLT3-expressing oocytes in experiments carried out in the absence of
sugar (indistinguishable from 22Na uptake in noninjected
oocytes).
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DISCUSSION |
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There have been three SGLT genes identified in the human genome: SGLT1 (9) and SGLT3 (4) on human chromosome 22 and SGLT2 (27) on chromosome 16. They are functionally distinct, having different sugar specificities and, as we have determined in this paper, different Na+/sugar coupling ratios. The SGLT3 stoichiometry is 2 Na+:1 sugar, identical to that of SLGT1 (14), but different from the 1 Na+:1 sugar stoichiometry of SGLT2 (12, 29).
We used the fact that transport of sugar by SGLT3 generates an
electrical current (Fig. 2), indicating that a positive ion is
transported into the cell with sugar, to normalize our data. We
correlated uptake of both sugar and Na+ to the translocated
charge by using simultaneous electrophysiological measurements and
unidirectional radioactive tracer fluxes (14). These
measurements indicated that 1.2 ± 0.1 net inward positive charges
were transported for every Na ion cotransported (Fig. 4), verifying
that the sugar-dependent current is carried by Na ions. In comparison,
2.2 ± 0.1 inward charges were generated for each sugar
transported (Fig. 3), resulting in a Na+/sugar ratio of
1.8 ± 0.1:1. This indicates a stoichiometry of 2:1, identical to
that for rabbit SGLT1 measured by using the same methods
(14), but different from the 1:1 stoichiometry reported
for human (12) and rat (29) SGLT2. Those
studies (12) compared the initial rate of
[14C]-MDG uptake with that of the Na+
influx calculated from the
-MDG-evoked inward current in a second oocyte, but they did not directly measure the Na+ influx.
Assuming the current is completely carried by Na+, they
concluded that the Na+-to-glucose coupling ratio for SGLT2
is 1:1. The Na-to-sugar stoichiometry of 2:1 means that SGLT3 has the
same ability to concentrate sugar in the cell as SGLT1.
Once the stoichiometry is known, application of the Hill analysis can
provide insights into the cooperativity of binding between the first
and second Na+ sites (6, 22, 26). In our
experiments the Hill coefficient (n) for SGLT3 was 1.6 ± 0.03, and it was voltage and substrate concentration independent, in
agreement with the previous studies (1.3 at 70 mV; 15). In studies on
SGLT1, n was found to range from 1.7 to 2.3 (21). This suggests that Na+ binding in SGLT1
has a higher degree of cooperatively between the Na+
binding sites than in SGLT3.
With the determination of the stoichiometry, the basic functional
characterization of the three SGLTs is fairly complete and shows that
SGLT3 has characteristics intermediately between SGLT1 and SGLT2 (Table
1). The sugar specifity is the same for
SGLT2 and SGLT3 and different for SGLT1; so all three proteins
transport D-glucose, but only SGLT1 also transports
D-galactose and 3OMDG.
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One of the criteria that has been used to distinguish the renal glucose
cotransporters is sodium affinity. In 1982, Turner and Moran
(24), in renal brush-border vesicles, were able to determine that for the high-affinity renal system the
K0.5Na was 60 mM at 0.1 mM glucose,
and for the low-affinity renal system the
K0.5Na was 230 mM at 1 mM glucose,
both at membrane voltage = 0 (25). We now know that
the Na+ affinity for SGLT cotransporters is highly voltage
and sugar concentration dependent (15, 21) and also
depends on species (Table 2). Given
the species, voltage-, and sugar-concentration dependence of
the apparent sodium affinity, it is therefore not possible at present
to definitively assign the low-affinity sugar transporter in the kidney
to either SGLT2 or SGLT3 based on Na+ affinities. Given
that inherited renal glycosuria, which is thought to be caused by a
defect in the major renal SGLT (5, 6), has been assigned
to human chromosome 6 by linkage analysis (2), it is
likely that the gene coding for the renal low-affinity SGLT has yet to
be identified.
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SGLT1 is similar to SGLT3 in its tissue distribution: SGLT1 is found in the rabbit intestinal epithelium (8) and in the inner renal cortex/outer medulla, by Northern analysis (20) and in situ hybridization (29). This tissue distribution suggests that the renal distribution of SGLT1 is confined to the S3 segment of the proximal tubule. Low levels of mRNA were detected in other tissues. SGLT2 mRNA was found in greatest abundance in the outer parts of the rat renal cortex and a weak signal in the intestine, but not in the liver, spleen, muscle, or brain (28), and in situ experiments indicated localization of SGLT2 to the S1 segment of the rat proximal tubule (12, 29). Functional assays also suggest a renal distribution of a low-affinity SGLT favoring the early proximal tubule in rabbits (20, 24, 25). On the other hand, SGLT3 RNA, which was cloned from the renal LLC-PK1 line, was most abundant in pig intestine followed by spleen, liver, kidney, and muscle (13).
Identification of the specific genetic defects in the SGLT1 protein that are the cause of the disease glucose-galactose malabsorption suggests a single functional SGLT exists in human intestine. Because genetic defects in SGLT1 are the cause of glucose and galactose malabsorption (19), it is unlikely that another glucose/galactose absorption pathway (e.g., SGLT3) with enough functional significance is present in the gut to compensate for the loss of SGLT1, so the functional significance of SGLT3 is unclear. However, there have been studies that have reported multiple human intestinal SGLTs. Malo (17, 18) described both high-affinity, low-capacity and low-affinity, high-capacity SGLTs in human fetal intestinal brush border. The differences in substrate specificities between these two cotransporters and their kinetics showed that in human fetal intestine, SLGT1 and a transporter similar to SGLT2 and SGLT3 coexist. This second transporter is developmentally regulated and is not expressed at birth. The identity of this transporter and its physiological role are still not known. When the human SGLT3 (4) becomes available for study, we anticipate new insights into the regulation, developmental biology, and function of glucose cotransporters. Comparison of the functional differences and similarities of these highly homologous proteins will help to elucidate the mechanism of cotransport.
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ACKNOWLEDGEMENTS |
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We thank Mary L. Bing for the isolation and preparation of the oocytes. This work was supported by National Institutes of Health Grants DK-19567, DK-44602, and GM-52094.
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FOOTNOTES |
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A. Díez-Sampedro is a recipient of a postdoctoral fellowship from the Departamento de Educación, Universidades e Investigación del Gobierno Vasco.
Address for reprint requests and other correspondence: B. A. Hirayama, Dept. of Physiology, UCLA School of Medicine, Los Angeles, CA 90095-1751 (Email: Bhirayama{at}mednet.ucla.edu).
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.
Received 11 August 2000; accepted in final form 19 October 2000.
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