From the Medical Research Council Group in Membrane Biology, Department of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada
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ABSTRACT |
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An alanine to cysteine mutation at position 166 has been introduced by site-directed mutagenesis into the rabbit
sodium/glucose transporter (rSGLT1). When expressed in Xenopus
laevis oocytes, this mutant transporter (A166C rSGLT1)
demonstrates a significantly lower apparent affinity for -methyl
glucoside (
MG) compared with the wild-type transporter (apparent
Km = 0.8 versus 0.15 mM).
Using the two-electrode voltage clamp technique, transient currents
have also been measured, and for the mutant transporter, the transients
induced by large depolarizations exhibit longer time constants than
those for wild type. Moreover, the substitution of Ala-166 with a
cysteine allows the sulfydryl specific reagent, methanethiosulfonate
ethylamine (MTSEA), to react with and alter the function of the
transporter. Whereas the wild-type transporter is unaffected by
reaction with MTSEA, A166C rSGLT1 has its steady-state currents induced
by 1 mM
MG inhibited 83% within a minute of exposure to
MTSEA. Furthermore, the pre-steady-state transients of the A166C mutant
after MTSEA exposure demonstrate much shorter time constants than
before while the total amount of charge transferred is only slightly
diminished. These results together provide evidence that position 166 is situated in a region critical to the functioning of rSGLT1.
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INTRODUCTION |
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The sodium/glucose cotransporter is one member of a family of sodium-dependent transport proteins found in the brush border membrane of intestinal and kidney epithelia (1). It uses the sodium electrochemical gradient to drive the transport of sugar into epithelial cells and is specifically inhibited by phlorizin. Since being cloned in 1987 from a rabbit small intestinal cDNA library (2), the high affinity sodium/glucose transporter (SGLT1)1 has been extensively characterized. The expression of SGLT1 in Xenopus laevis oocytes has allowed for the application of electrophysiologic techniques to measure in detail the kinetics of the transporter. The two-electrode voltage clamp method and the cut open oocyte technique have been used to measure the sodium currents mediated by the transporter, and from such measurements, substrate affinities as well as the sodium:glucose coupling stoichiometry have been determined (3, 4). In addition, electrophysiology has been used to demonstrate that in the absence of sugar or phlorizin, SGLT1 exhibits pre-steady-state currents in response to rapid changes in membrane potential. These transient currents have been hypothesized to be due to charge movements associated with conformational changes of the transporter, and by studying them, estimates have been made of the rate constants for sodium association/dissociation and the reorientation of the empty transporter from an outside facing to inside facing conformation (5).
A number of studies have combined site-directed mutagenesis with the oocyte expression system to identify residues that contribute to the functioning of SGLT1 and its proper trafficking to the plasma membrane (6, 7). A set of glycosylation mutants has yielded information on topology, and a chimera of the high affinity and low affinity isoforms has suggested that sugar binding is determined by the C-terminal half of the protein (8). From comparisons of SGLT1 homologues cloned from different species, certain residues have been hypothesized to be important in determining the kinetic differences between these isoforms (9). Direct experimental evidence elucidating specific structure/function relationships is, however, lacking, and the exact residues that make up the substrate binding sites or determine the coupling of sodium binding to glucose transport remain unknown.
In this study, we report on the characterization of a single cysteine mutant of rSGLT1 with properties distinct from wild type that was identified during a cysteine scanning mutagenesis project. The alanine at position 166, which was replaced with cysteine to generate this mutant, had not previously been hypothesized to be important, although in one glucose galactose malabsorption patient, it was found mutated to a threonine (10). Ala-166 is conserved across the rabbit/rat/human isoforms of SGLT1 as well as SGLT2 and the sodium myoinositol transporter. This is not surprising given the high degree of sequence identity for these members of the sodium cotransporter family. What was unexpected was the degree to which some of the steady-state and pre-steady-state kinetics of the transporter was changed by the relatively conservative alanine to cysteine substitution. To date, there have been no other single-site mutations reported that produce dramatic changes to both the steady-state and transient currents mediated by SGLT1.
In addition to the kinetic changes, we also observed that the A166C mutation introduced sensitivity to the cysteine-specific reagent methanethiosulfonate ethylamine (MTSEA). Belonging to a class of compounds known as MTS derivatives, MTSEA has been used in combination with cysteine site-directed mutagenesis in the study of proteins such as ACh receptor channel, GABA receptor channel, lactose permease, and voltage-gated ion channels (11-14). We present here experimental evidence that such an approach can also be taken with SGLT1, providing unique insight into certain structure/function relationships for this transporter.
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MATERIALS AND METHODS |
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Molecular Biology--
The multicloning site of the eukaryotic
expression vector PMT3 (kindly provided by the Genetics Institute,
Boston, MA) was removed by digestion with PstI and
KpnI, and the cDNA of rSGLT1 (kindly provided by M. Heidiger) was subcloned into the remaining EcoRI site. The
A166C mutation was introduced into this construct using the megaprimer
method of polymerase chain reaction mutagenesis (15) with
5-TCGGAGCCTCTCTGTTTG-3
as the sense primer,
5
-TACCCAGACAGAATCGAGCCGGCCTT-3
as the antisense primer, and
5
-GGATGAAGATGCATCCGGAAAAGATG-3
as the mutagenic primer. These primers
were used to make a mutation-containing polymerase chain reaction
product that was digested with BclI and then ligated to
BclI-digested PMT3-rSGLT1 using a cycle ligation protocol
(16). The mutation and the stretch of DNA between the two
BclI sites was verified by dideoxy chain termination DNA
sequencing using the Pharmacia Biotech Inc. T7 polymerase sequencing
kit. The DNA used for the oocyte injections was prepared using the QIAprep Spin Plasmid Kit (Qiagen, Chatsworth, CA) without further purification.
Oocyte Preparation and Injection-- X. laevis frogs were anaesthetized with a 0.17% solution of 3-aminobenzoic acid ethyl ester in water. Stage V or VI oocytes were then surgically removed and digested with 2 mg/ml of type IV collagenase (Sigma) prepared in modified Barth's saline (MBS) for 60-90 min. The composition of the MBS was 0.88 mM NaCl, 1.0 mM KCl, 2.4 mM NaHCO3, 15.0 mM HEPES-NaOH (pH 7.6), 0.3 mM CaNO3, 0.41 mM CaCl2, 0.82 mM MgSO4, 10 mg/ml penicillin, and 10 mg/ml streptomycin. After the collagenase digestion, oocytes were kept in MBS overnight at 18 °C before being injected with the DNA.
Using a Drummond Nanoject (Drummond Scientific, Broomall, PA), 4.7 nl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) containing 0.15 ng of PMT3-rSGLT1 or PMT3-A166C-rSGLT1 and 0.15 ng of PMT3-SEAP was injected into the animal pole of the defolliculated oocytes as described previously by Swick et al. (17). The injected oocytes were kept in MBS supplemented with 2.5 mM sodium pyruvate for 2-3 days before transfer to 96-well plates and incubation individually another 16-24 h. The incubation solution from each oocyte was then tested for alkaline phosphatase activity following the protocol of Tate et al. (18). Oocytes that were positive according to this assay were then selected for the electrophysiology that was conducted over the next 2 days.Two-electrode Voltage Clamp--
In all experiments, the oocyte
currents were measured with the two-electrode voltage clamp technique
(19). We used an Axoclamp-2A amplifier, TL-2 data acquisition system,
and pCLAMP software (Axon Instruments, Foster City, CA) to generate
voltage pulses and measure the current responses. Oocytes with resting
membrane potentials less negative than 30 mV were discarded. During
an experiment, the voltage clamped oocyte was under the constant
perfusion of buffer at approximately 2 ml/min. The composition of this
uptake buffer was 100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2,
10 mM HEPES-Tris base (pH 7.4). Current responses were
recorded at a sample rate of 2.5 ms
1 as the average of
the responses to three consecutive trials and were subjected to a 500 kHz, 5 point gaussian filter prior to curve fitting or the calculation
of steady-state parameters. Curve fitting was done either using the
Simplix method with the pCLAMP software or using the Levenberg
Marquardt algorithm (Origin 4.0, Microcal Software, Northampton, MA).
Time constants describing the transient currents were obtained only
from data collected in which the voltage clamp was sufficiently fast.
Specifically, by fitting the current responses collected in the
presence of 0.2 mM phlorizin to a single exponential, the
time constant of the voltage clamp for these experiments was estimated
to be between 0.4 and 0.7 ms.
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RESULTS |
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Fig. 1 shows the current responses
to a series of 100-ms voltage steps (150 to 90 mV in 20 mV
increments) from a
50 mV holding potential before (panel
A) and after (panel B) the addition of 10 mM
MG for an oocyte expressing A166C rSGLT1. As
described previously for wild-type SGLT1 (3), the presence of
MG in the bath solution induces an inward sodium current that is not observed
in water-injected oocytes. Moreover, A166C rSGLT1 expression results in
current responses which consist of a capacitive transient followed by a
slow decay to steady state (most clearly seen for voltage steps to
positive potentials). The slow decay has been extensively characterized
in wild-type SGLT1 as a pre-steady-state current associated with sodium
binding and the reorientation of the empty carrier (5). As with
wild-type SGLT1, this pre-steady-state current is abolished by the
addition of
MG. Fig. 1C shows, for the same
representative oocyte expressing A166C, the I-V relationship of the
current induced by the 10 mM
MG and the I-V relationship of the sodium leak. In this and all other experiments, the
MG-induced current is defined as the calculated difference in a
measurement made in the absence of
MG and a second measurement made
immediately afterward in the presence of
MG; the sodium leak is
defined experimentally as the current inhibited by saturating phlorizin
concentrations in the absence of
MG. In Fig. 1D, we
compare the sodium leak of the A166C mutant and wild-type SGLT1 by
normalizing with respect to Imax (Fig.
2A). Clearly, the A166C mutant
exhibits a much smaller sodium leak at negative membrane
potentials.
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Since the MG-induced currents for A166C rSGLT1 were, generally
speaking, comparable in magnitude with those observed for wt SGLT1, we
were able to proceed with a characterization of the steady-state
transport kinetics of the mutant. The data from a series of I-V curves
for A166C rSGLT1 are replotted as functions of
MG concentrations in
Fig. 2A and fitted to the Michaelis-Menten equation to
obtain values for an apparent Km. The results from
fitting data from a number of different oocytes expressing either A166C
rSGLT1 or wt SGLT1 are presented in Fig. 2B. Clearly, the
apparent affinity of
MG has been lowered by the alanine to cysteine
substitution. In experiments where the
MG-induced currents were
determined for a series of sodium concentrations ranging from 0 to 100 mM, data were obtained which showed that the mutation did
not appear to affect stoichiometry or the apparent sodium affinity. In
Fig. 2C, a representative experiment is shown in which the
sodium dependence of a 1 mM
MG-induced current at
30,
50, and
70 mV is fitted to the Hill equation.
An interesting consequence of the introduction of the cysteine at
position 166 was the effect of allowing MTSEA to react with the
transporter and alter its function. In Fig.
3, the steady-state MG-induced
currents for A166C rSGLT1 are shown to be inhibited by the addition of
1 mM MTSEA to the bath solution. The inhibition by the
MTSEA exhibited the same rapid kinetics in the presence of saturating
concentrations of
MG (10 mM) as at lower concentrations. Although we could not directly monitor the time course of inhibition in
the absence of
MG, MTSEA exposure under such conditions resulted in
the same degree of inhibition. Moreover, saturating concentrations of
the competitive inhibitor phlorizin had no effect on the degree of the
MTSEA inhibition nor did replacing the 100 mM NaCl with 100 mM choline-Cl (data not shown).
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The inhibition by the MTSEA did not eliminate the MG-induced
currents although the kinetics of the curve in Fig. 3 indicate that the
reaction has gone to completion. Analysis of the
MG currents
remaining after exposure to MTSEA (Fig.
4) demonstrates that the effect of MTSEA
is both on the
MG apparent affinity as well as the
Imax of the transporter.
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To further examine the effect of the cysteine substitution and the MTSEA inhibition, we undertook a series of experiments to examine the pre-steady-state currents of the mutant transporter before and after MTSEA exposure. Fig. 5 (panels A and B) show the transient currents observed for an oocyte expressing A166C rSGLT1 prior to and subsequent to reaction with MTSEA. The traces are the difference in the currents obtained in the presence and absence of 200 µM phlorizin. Phlorizin is a specific inhibitor of the transporter that has been shown to effectively eliminate the transients associated with the transporter and provides a means to measure and subtract the nonspecific capacitive currents (3, 5). For purposes of clarity and comparison, in Fig. 5C, we overlay the transient currents for the +90 mV voltage step from panels A and B.
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MTSEA affected the rate of decay of the transient currents exhibited by the A166C mutant. Qualitatively, the decay of the on currents is faster after MTSEA exposure. When the on currents are fitted to a first order exponential decay equation, time constants can be obtained that demonstrate the effect quantitatively (Fig. 5D).
The transient currents for each voltage pulse (Vt) can be integrated to give a value for the quantity of charge transferred (Q) as a result of the change in membrane potential. Q as a function of Vt has been demonstrated previously to be adequately described by the Boltzmann equation (5). Fig. 6A shows how the Q versus Vt relationship for A166C is changed by exposure to MTSEA and panel B compares the normalized Q versus Vt relationship for wt A166C before and after MTSEA inhibition.
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The Qmax obtained from fitting data to the
Boltzmann relation correlates with the number of transporters expressed
in a given oocyte. It has been shown that
Imax/Qmax can provide an
estimate of the maximal rate of transporter turnover (assuming a
stoichiometry of 2:1 and that 2 charges per transporter are
translocated by the transient currents) (5, 20). Using the
Imax for 150 mV, we have found that wt SGLT1
has a maximal turnover rate of 22.8 ± 0.5 s
1
(n = 3), whereas the A166C mutant exhibits a turnover
of 12.3 ± 0.3 s
1 (n = 5). Since the
Imax of A166C before and after MTSEA inhibition can be determined in the same oocyte, the change in turnover rate caused by the MTSEA can be determined directly. Assuming that the
number of transporters has not changed during the MTSEA exposure, the
decrease in turnover is directly proportional to the decrease in
Imax. Hence, MTSEA results in a 3-fold decrease
in turnover rate
(Imax, before MTSEA/Imax, after MTSEA is ~3).
To explore further the nature of the MTSEA effect on the mutant, we
tested a related compound, methanethiosulfonate ethylsulfonate (MTSES).
Like MTSEA, MTSES reacts specifically and rapidly with thiols to form
mixed disulfides. However, the unreacted MTSES in solution posesses a
negative charge instead of a positive charge, and the mixed disulfide
it forms is the negatively charged thiol ethylsulfonate instead of the
positively charged thiol ethylamine. We first confirmed that MTSES at 1 mM and 10 mM had no effect on wt SGLT1
steady-state or pre-steady-state kinetics (data not shown). Then
interestingly, we found that MTSES at these concentrations likewise had
no effect on the kinetics of the mutant (data not shown). Incubation
with 10 mM MTSES did however protect against a subsequent
inhibition with MTSEA. Fig. 7 shows that
for progressively longer pre-treatments with MTSES followed by washout
of the MTSES, there is a decreasing degree to which MTSEA exposure can
inhibit the MG-induced steady-state currents. These findings
indicate that MTSES can also react with the cysteine at position 166, but that the mixed disulfide it forms does not perturb transporter function.
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DISCUSSION |
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Based on theoretical considerations and data from glycosylation
mutants, position 166 is hypothesized to be part of a short 16 amino
acid loop connecting transmembrane helices IV and V of SGLT1 (21). To
date, the only experimental data ascribing function to any part of this
loop comes from a study by Panayotova-Heiermann, et al. (6)
in which the aspartic acid at position 176 (located at the
transmembrane V end of the loop) was mutated to an alanine. In this
study, the D176A mutation was shown to result in 1) an increased rate
of decay for the on current transients corresponding to
membrane depolarizations and 2) a shift of the Q versus
Vt curve along the voltage axis toward more
hyperpolarizing potentials. In contrast, we have shown that an A166C
mutation results in these pre-steady-state parameters changing in the
opposite direction, namely in 1) a decreased rate of decay for these
same current transients and 2) a shift of the Q versus
Vt curve along the voltage axis toward more depolarizing
potentials. Since an aspartic acid to alanine change is from an
electronegative to neutral amino acid and an alanine to cysteine change
is from a neutral to relatively electronegative amino acid, these
results are consistent with the hypothesis that this putative
extracellular loop is sensitive to changes in charge and/or polarity.
In fact, the direction in which the polarity is adjusted seems to
determine the direction in which the time constants describing these
on transient currents (on) change and the
Q versus Vt curve shifts.
Referring to the modelling of SGLT1 current transients by Loo and
Wright (5), the above changes in pre-steady-state parameters caused by
the mutations may be interpreted in terms of conformational transitions. These conformational transitions are induced by changes in
membrane potential and involve the movement of charged residues and
sodium ions through the membrane electric field. For membrane depolarizations, the transition would be the dissociation of sodium followed by reorientation of the unloaded transporter from outside to
inside facing (i.e. NaCout Cout
Cin, where
Cout and Cin represent
the outside and inside facing conformations of the cotransporter). Therefore, the observation that the A166C mutant exhibits a slower
on (for Vt = +90 mV) than wild type
implies that the cysteine substitution has caused a decrease in the
rate of this NaCout
Cin transition. Moreover, the change in the
Q/Vt relationship may be interpreted as a
change in the voltage-sensing capacity of the transporter.
It is useful to apply the same kind of interpretation to the
pre-steady-state data relating to the effect of MTSEA on the A166C
mutant. Here were have shown that reaction with MTSEA reduces the value
for on (for Vt = +90 mV) and shifts
the Q versus Vt curve along the voltage axis toward
more depolarizing potentials such that the normalized Q versus
Vt curve is indistinguishable from that of wild type.
Assuming that the MTSEA is reacting with the cysteine introduced at 166 and therefore placing an electropositive ethyl amine group at this
location, these results are consistent with the notion presented above, namely that the region around 166 is sensitive to changes in charge and/or polarity. Futhermore, they lend support to the idea that this
region is important in defining the energetics of the
NaCout
Cin transition
and the transporter's voltage-sensing capacity.
In addition to alterations in pre-steady-state parameters, the A166C
mutation also results in changes in the steady-state operation of the
transporter. Most notably, the A166C mutant demonstrates a
significantly lower MG apparent affinity, an observation which indicates that the mutation has affected either the true
MG affinity or a transition that is directly coupled to sugar binding. We consider
two possibilities, 1) that position 166 is located in the sugar binding
site and has a direct influence on that event, and 2) that position 166 is in a region that can influence the sodium activation of sugar
binding, perhaps sodium binding itself. The detailed characterization
of the transient currents exhibited by the A166C mutant, unfortunately,
do not distinguish between these two possibilities, although analysis
of the MTSEA effect argues against the first one. Since the inhibition
by the MTSEA appears to be unaffected by saturating concentrations of
MG and phlorizin, the cysteine at 166 reacting with the MTSEA is
unlikely to be located within either the
MG or the phlorizin binding
sites. The decrease in the
MG affinity upon substituting alanine 166 for cysteine and the further decrease in affinity upon reaction with
MTSEA, we believe, are due to an influence on the ability of sodium
binding to activate
MG binding and then transport.
Another piece of evidence that supports the hypothesis that 166 is located in a region important to sodium binding and the coupling of this event to sugar transport is the order of magnitude lower sodium leak exhibited by the A166C mutant as compared with that for wild type. The comparison holds for the case where the leak was normalized to Imax and also for the case where the leak was normalized to Qmax. The interpretation that the sodium leak represents an uncoupled pathway for sodium transport therefore suggests that the introduction of a cysteine in place of an alanine at 166 has altered the coupling characteristics of the transporter.
The importance of the molecular character of the residue at 166 to the
function of the transporter is also supported by the estimates of
turnover rate for the A166C mutant and the mutant following MTSEA
exposure. Compared with wild type, the turnover rate was halved by the
cysteine substitution (~23 s1 versus ~12
s
1) and the reaction with MTSEA caused an additional
3-fold decrease in the rate (~4 s
1). Due to the
complexity of the kinetics for a system that couples the transport of
two sodium ions to one glucose molecule, it is difficult to explain
these changes in turnover rate in terms of the changes observed for the
pre-steady-state data. Nonetheless, it is reasonable to speculate that
the changes in the nature of the reorientation of the empty carrier
along with sodium binding in response to membrane potential changes
would be evidence that the same transition occurring during a transport
cycle, in which membrane potential is held constant, has also changed.
Since this transition is believed to be one of the rate-limiting steps
in the transport cycle, such speculation is consistent with the
turnover data.
Assuming that the effects of the MTSEA are due to reaction with
cysteine 166, the results presented also provide information about the
topology of the protein at position 166. Since MTSEA is a water-soluble
compound that would not be expected to permeate across membranes, the
residue at 166 must be placed either extracellularly or in some pore
that is accessible from the extracellular compartment. This is
important confirmation of the results of Turk et al. (21) which show that a 42-amino acid loop containing the native
glycosylation site, when inserted between positions 169 and 170, is
glycosylated by the Xenopus oocyte expression system. That
particular mutant was reportedly non-functional while the A166C mutant
we have shown is still able to mediate substantial MG-induced
currents.
One important question we have been able to address is whether the effect of the MTSEA on A166C rSGLT1 is due to steric effects or charge effects introduced by the ethyl amine group. Our data suggest that the positive charge on the amine is responsible for altering both the steady-state and pre-steady-state kinetics of the mutant transporter. Although we cannot determine directly whether MTSES is reacting with the transporter, the results showing that MTSES preincubation can protect against MTSEA incubation demonstrate that MTSES and MTSEA react with the same cysteine. Since MTSES had no effect on any of the functional characteristics of the transporter and given that ethyl sulfonate is negatively charged and bulkier than ethyl amine, we conclude that the MTSEA effect on transport kinetics and charge transfer are due to the addition of a positive charge in what must be a key location.
In conclusion, this study presents an application of cysteine mutagenesis coupled with reaction to MTS compounds to the elucidation of SGLT1 structure function relationships. We have provided evidence favoring the hypothesis that changes in the polarity of the molecular group at position 166 can bring about significant changes in both the pre-steady-state and steady-state kinetics of the transporter. A significant advantage to the approach with the MTSEA is that it allowed the comparison of functional parameters perturbed by a specific and rapid covalent reaction, with those same parameters measured in the same oocyte for presumably the same population of transporters before the perturbation took place. We speculate that application of this approach to other residues in SGLT1, particularly the region around position 166, can continue to provide valuable information about the mechanism of sodium-coupled transport.
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ACKNOWLEDGEMENTS |
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We thank R. Reithmeir, G. Gristein, and P. Back for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by a Medical Research Council Membrane Biology Group Grant (to M. S.).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.
Supported by a studentship from the Medical Research Council of
Canada.
§ To whom correspondence should be addressed: Medical Science Building, Rm. 7207, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-7189; Fax: 416-971-2132.
1
The abbreviations used are: SGLT1,
sodium/glucose transporter; MG,
-methyl glucoside; MTSEA,
methanethiosulfonate ethylamine; MBS, modified Barth's saline; wt,
wild type; MTSES, methanethiosulfonate ethylsulfonate; SEAP, secreted
alkaline phosphatase.
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REFERENCES |
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