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INTRODUCTION |
The human high affinity Na+/glucose cotransporter
(hSGLT1)1 is a
member of a family of secondary transporter proteins encompassing more
than 55 homologues from archaea, bacteria, yeast, insects, and mammals
(1, 2). This family uses electrochemical Na+ gradients to
drive the coupled uphill transport of a variety of substrates (sugars,
amino acids, vitamins, osmolytes, ions, myo-inositol, urea,
and water). The expression of hSGLT1 in Xenopus laevis
oocytes has resulted in a comprehensive study of both steady-state and
pre-steady-state kinetics (3-5). A six-state ordered binding model has
been proposed in which transport results from ligand-induced conformational changes (6, 7). In this model Na+ binds
before sugar, with a coupling ratio of 2 Na+:1 glucose, and
voltage influences both Na+ binding and the conformational
states of the unloaded transporter. Functional analysis of SGLT
chimeras and truncated proteins strongly suggests that the sugar
pathway is located in the C-terminal domain of the protein (8, 9).
Site-directed thiol labeling of a residue in the proposed sugar pathway
indicates that conformational changes are responsible for the coupling
of Na+ and sugar transport (10). We suggest that these
conformational alterations are induced by cation binding in the
N-terminal domain of the protein.
Although the functional importance of the N terminus in cation
binding/translocation was shown for another SGLT family member (the
Na+/proline transporter (PutP) of Escherichia
coli) (11-13), there is little information on the role of the
N-terminal domain in hSGLT1. We have initiated a study to explore the
role of N-terminal residues in hSGLT1 in cotransport. In the present
study, we have targeted a conserved residue, Asp204,
located in a short cytoplasmic loop of hSGLT1 connecting transmembrane domains V and VI, which has been implicated in cation selectivity in
PutP (12). Replacing Asp204 in hSGLT1 with Asn, Cys, or Glu
dramatically modulated the steady-state and pre-steady-state kinetics
of the transporter. Remarkably, although a transporter with a negative
amino acid (Asp or Glu) exhibited cation/glucose cotransport with a
stoichiometry of 2 (Na+ or H+) to 1, neutralization of Asp204 (by Asn or Cys) resulted in the
activation of a glucose-activated H+ channel.
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EXPERIMENTAL PROCEDURES |
Molecular Biology--
A plasmid containing human SGLT1 (hSGLT1)
cDNA was used as template for site-directed mutagenesis.
Replacement of Asp204 with Asn, Cys, and Glu was performed
using a two-step polymerase chain reaction protocol (14). For each pair
of mutagenic oligonucleotides the sequence of the sense primer is
presented with the altered nucleotide(s) underlined:
D204C, 5'-GATTTACACGTGCACCTTGC-3'; D204E, 5'-GATTTACACGGAAACCTTGC-3'; D204N,
5'-GATTTACACGAACACCTTGC-3'. Polymerase chain reaction
products were digested with BglII and Eco47III,
and the resulting 428-bp fragments were ligated into a similarly
treated wild-type hSGLT1-containing plasmid. The fidelity of the
inserted DNA fragments was confirmed by sequencing double-stranded DNA
(Sequenase version 2.0, DNA sequencing kit, United States Biochemical,
Cleveland, OH) after alkaline denaturation (15). Each mutagenized DNA
template was linearized with XbaI, transcribed, and capped
in vitro using the T3 RNA promoter (MEGAscript kit, Ambion,
Austin, TX). X. laevis oocytes were injected with 50 ng of
mRNA and were incubated in Barth medium containing gentamicin (5 mg/ml) at 18 °C for 3-7 days (4).
Transport Assays and Electrophysiological Techniques--
For
transport and electrophysiological experiments, oocytes were bathed in
an assay buffer composed of 2 mM KCl, 1 mM
MgCl2, 1 mM CaCl2, 10 mM HEPES-Tris, pH 7.5, and a combination of Na+
or choline+ chloride salts to give a final concentration of
100 mM. For H+ activation experiments the pH of
100 mM choline buffer was varied between 8.0 and 4.5 by
titration with Tris or Mes. Uptake of
methyl-
-D-[U-14C]glucopyranoside (293 Ci/mol, Amersham Pharmacia Biotech) and electrophysiological
measurements using the two-microelectrode voltage clamp technique were
performed as described (16, 17). The stoichiometry of cation-coupled
D-[U-14C]glucose (316 Ci/mol, ICN
Radiochemicals) uptake was determined under voltage clamp conditions
(18).
Data Analysis--
Sugar-evoked steady-state currents were
fitted to Eq. 1,
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(Eq. 1)
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where IGlc and
ImaxGlc represent
D-glucose-induced current and maximal
D-glucose-induced current, respectively, at saturating
[cation], [Glc] is the concentration of D-glucose, and
K0.5Glc is [Glc] at 0.5 ImaxGlc. Kinetic parameters for
the phlorizin-inhibited cation leak or cation-activated glucose
transport were determined by using Eq. 2,
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(Eq. 2)
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where Ication and
Imaxcation is the cation-evoked
(leak) current and the maximal cation (leak) current at saturating
cation concentration [C], respectively,
K0.5cation is [C] at 0.5 Imaxcation, and n
represents the Hill coefficient. Charge-voltage (Q-V) relations for each membrane voltage (Vm) were
obtained by integrating pre-steady-state current transitions (after
subtracting the capacitive and the steady-state currents from the total
currents) with time and were fitted to Eq. 3 (17, 19),
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(Eq. 3)
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with Qhyp and Qdep
for Q at hyperpolarizing and depolarizing limits,
respectively. V0.5 represents
Vm at which 50% of the total charge in the
membrane electric field has moved, z is the apparent valence
of the moveable charge, and F, R, and
T have their usual meanings. All experiments were repeated
at least three times with oocytes from different donor frogs. Data fits
were performed using the non-linear regression algorithm in SigmaPlot (version 5.0, SPSS Inc., Chicago, IL). Unless otherwise noted, figures
are based on data obtained from a typical experiment on a single
oocyte, and errors represent S.E. of the fit.
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RESULTS |
In these studies all comparisons between Na+ and
H+ kinetics were performed in the same oocyte expressing
WT, D204C, D204E, or D204N cRNA. For comparison of WT kinetics and the
kinetics of a transporter with a substitution at position 204, oocytes of the same batch were analyzed on the same day within 10 h.
Uptake Experiments--
The most conservative substitution
Asp204
Glu resulted in a reduction of
Na+-dependent
MDG uptake by 87%, whereas
the uptake rates of D204C and D204N were reduced by ~50% (Fig.
1). In the absence of Na+
ions, the rate of
MDG uptake in oocytes expressing D204E was about 1 pmol/h, a value comparable to oocytes with the native transporter (1.7 pmol × h
1 × oocyte
1). However,
replacing Asp204 with Cys or Asn increased the uptake rate
by 7- and 16-fold. This increase may reflect the enhanced apparent
affinity for sugar (see Table I).

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Fig. 1.
Uptake of
[14C] MDG by oocytes 5 days after
injection with hSGLT1, D204C, D204E, or D204N cRNA. Transport of
50 µM [14C] MDG (293 µCi/µmol) was
assayed in the presence of 100 mM NaCl
(Nac) or 100 mM choline chloride buffer,
pH 7.5, for 60 min at 22 °C. H2O-injected oocytes
(NI) served as a control. Data are from the same batch of
oocytes, and errors represent the S.E. (n = 7).
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Table I
Summary of kinetic parameters in presence of Na+ and H+
Steady-state kinetic parameter and turnover rates for cation-coupled
sugar transport (kcatcation) are shown at
Vm = 110 mV. ImaxGlc,
K0.5Glc, and Qmax were
determined at 100 mM Na+ and 3.2 µM
H+.
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Steady-State Currents--
Fig. 2
shows representative sugar-induced steady-state current-voltage
(I-V) relationships for WT, D204C, and D204E in
the presence of Nac and Hc at saturating
[Glc]. For WT both curves were sigmoidal and saturated at
hyperpolarizing voltages. A similar I-V relation
was observed for D204E in Nac, but in Hc the
sugar-induced currents exhibited a supralinear increase with
hyperpolarization. D204C and D204N (data not shown) exhibited
essentially identical I-V relations with no saturation of
the sugar-evoked currents at the most negative potential (
150 mV) in
either Nac or in Hc. The glucose-evoked currents in
Hc for these transporters were much greater than the
Na+ currents (see Table I). In Hc for the latter
transporters, glucose activated outward currents at potentials more
positive than ~+20 mV (~+100 nA at +50 mV). A comparison of the
sugar-induced currents at Vm =
150 mV revealed
similar values in the presence of Hc for WT (
1220 nA), D204C
(
1120 nA), and D204N (
870 nA), and a reduction of about 75% for
D204E (
300 nA). In Nac, WT and D204E showed comparable
currents generated by 100 mM D-glucose at
150 mV (
1480 and
930 nA). Under the same test conditions, D204C and
D204N exhibited less than 10% of the current observed for WT.

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Fig. 2.
Voltage dependence of sugar-induced
steady-state currents. Net currents induced by 100 mM
D-glucose (Inet = Isubstrate Ino
substrate) were measured in the presence of Nac
(open symbols) or Hc (solid symbols) and
are plotted as a function of the membrane potential
(Vm) for WT ( , ), D204C ( , ), and
D204E ( , ). Experiments were carried out in single oocytes from
one batch.
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We measured the sugar kinetics of each transporter by varying external
[Glc] from 0.05 to 100 mM in the presence of Nac
or Hc. The apparent affinity constants for
D-glucose (K0.5Glc)
at Vm =
110 mV are shown in Table I. As
described earlier (20), hSGLT1 exhibited a
K0.5Glc of 0.3 mM in
Nac. When Hc substituted for Nac the apparent
affinity for Glc was reduced by 20-fold
(K0.5Glc = 6 mM). A
similar reduction of the apparent Glc affinity in Hc was also
observed for each transporter with a replacement of Asp204.
A closer examination of K0.5Glc
revealed two classes of mutations. First, Glu in place of
Asp204 reduced the apparent affinity for Glc in either
Nac or Hc about 4-fold. The second class encompasses
transporters with a neutral side chain at position 204 (Asp204
Asn or Cys), which showed increased apparent
affinities for Glc. Although the
K0.5Glc of D204C in Hc
was only slightly decreased (3 mM), a ~5-fold reduction
of K0.5Glc was observed in
Nac (0.07 mM). D204N exhibited a more dramatic increase of the apparent affinity for Glc with a 10- and 15-fold reduction of K0.5Glc in
Hc (0.6 mM) and in Nac (0.02 mM), respectively. Over a range from
150 to
30 mV, the
K0.5Glc determined in Nac
was essentially voltage-insensitive for all transporters. From
150 to
50 mV, WT exhibited an approximate 4-fold increased
K0.5Glc in Hc, but no
voltage dependence of the K0.5Glc
was observed for the other transporters.
The ImaxGlc values for each
transporter increased (more negative) with hyperpolarizing potentials
and were comparable to the currents generated by 100 mM
D-glucose (see Fig. 2). The cation had no impact on the
magnitude of ImaxGlc for WT
(Table I). WT and D204E exhibited a sigmoidal
ImaxGlc-V relation in
Nac and thus, the similar
ImaxGlc values of these two
transporters shown at
110 mV (Table I) were not significantly changed
by more hyperpolarizing potentials. With the exception of WT, no
saturation of the
ImaxGlc-V relationship
was observed for each transporter at hyperpolarizing potentials in
Hc.
To elucidate whether the substitution of Asp204 in hSGLT1
changed the sugar specificity of the transporter, oocytes were held at
50 mV and currents generated by 10 mM sugar were
monitored in the presence of Nac or Hc. Under either
test condition the selectivity pattern for each transporter was in the
order Glc
MDG
D-galactose > 3-O-methyl-D-glucose (data not shown). Consistent with the effect of the cation on the apparent Glc affinity for WT and D204E, each sugar induced ~10-fold lower currents in the
presence of Hc than in Nac. The effect on the apparent
Glc affinity of D204E was reflected by ~4-fold lower sugar-induced currents in Nac and Hc than with WT. In Hc,
D204C and D204N exhibited sugar-induced currents similar to WT, but the
currents generated by each sugar in Nac were ~50-fold lower
than the sugar-evoked currents observed for WT.
We determined the apparent half-maximal cation concentration for
Na+- and H+-activated glucose transport
(K0.5cation). Table I
summarizes K0.5cation for all
transporters at Vm =
110 mV. For WT the
K0.5H+ (7 µM, pH 5.2) was about three orders of magnitude smaller
than the K0.5Na+ (6 mM). Again, the nature of the substitution of
Asp204 grouped transporters with a neutral amino acid side
chain (Asp204
Asn or Cys) in one group. These
transporters exhibited a 10-fold reduced
K0.5Na+, but the
K0.5H+ was not
significantly altered. On the other hand, Glu in place of
Asp204 reduced
K0.5Na+ by about 3-fold
(2 mM). However, this conservative substitution reduced
K0.5H+ of this
transporter by a factor of >20, shifting the pH of the apparent
half-maximum concentration for H+-activated glucose transport
from pH 5.2 to ~6.5. In general, for each transporter
K0.5Na+ was slightly
voltage-dependent and increased about 4-fold from
150 to
50 mV, while K0.5H+
over the same voltage range increased by a factor of about 2.
The Hill coefficient (n) of WT for cation-activated glucose
transport was larger than unity for both Na+
(nNa+ > 1.5) and H+
(nH+ > 1.2) and was not
affected by voltage (
50 to
150 mV).
nNa+ of D204E was >1.3 but for
H+/sugar cotransport
nH+ was <1. Independent on the
cation, D204C and D204N exhibited a
nNa+ or nH+ of < 1.
Whereas at
110 mV
ImaxNa+ for each
transporter was essentially indistinguishable from
ImaxGlc in Na+, this
similarity in Hc was only observed for WT and D204E (note that
the ImaxNa+-V
relation of D204C and D204N and the
ImaxH+-V
relation of D204E did not saturate at hyperpolarizing potentials). On
the other hand, at Vm =
110 mV, a transporter
with a neutral amino acid side chain at position 204 exhibited a
~3-fold larger ImaxH+
(D204C,
1630 nA; D204N,
2450 nA) than
ImaxGlc in Hc (D204C,
675 nA; D204N,
769 nA) and
ImaxH+ increased
supralinearly with more negative potentials.
To determine the net Na+ and H+ leaks through each
transporter, [Na+] or [H+] was varied between 0.1 and 100 mM Na+ or between 0.03 and 32 µM H+ and phlorizin-sensitive currents and
choline currents at pH 8.0 were subtracted from the total currents. The
kinetics of the cation leak were calculated at
Vm =
110 mV by fitting the data to Eq. 2. In
general, for each transporter
K0.5Na+ for glucose
cotransport and leak were essentially identical, but the
K0.5H+ value for the leak
was ~10-fold smaller than
K0.5H+. WT exhibited a
leak ImaxNa+ of
97 ± 4 nA and replacement of Asp204 with Asn (54 ± 5 nA), Cys (18 ± 2 nA), or Glu (47 ± 3 nA) reduced the leak
ImaxNa+ ~2- to 5-fold.
The leak ImaxH+ for WT
(430 ± 4 nA) was 2.5 to 3 times larger than for D204C (
172 ± 16 nA) and D204N (
142 ± 7 nA), but there was apparently no
H+ leak through D204E. Although the Hill coefficient
(n) for the Na+ or the H+ leak pathways was
>1.2 only for WT, the value of n for each modified transporter was
1.
Pre-Steady-State Charge Movement--
Fig.
3A shows representative
current records of oocytes injected with WT or D204E cRNA in the
presence of Nac or Hc after stepping the membrane
potential from the holding potential (
50 mV) to the test potential
(Vm =
150 to +50 mV in 20-mV decrements). After the initial fast membrane capacitive transients (
< 1 ms) each
transporter exhibited currents that relaxed to a steady-state with a
single time constant (see Fig. 4). These
relaxations were abolished after addition of saturating [sugar]
and/or [phlorizin] (data not shown). The removal of a negative amino
acid side chain at position 204 caused a dramatic reduction of the
current transients (compare Qmax in Table
I).

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Fig. 3.
Pre-steady-state kinetics associated with WT
and D204E. A, current traces in Nac and
Hc were obtained by a pulse protocol in 20-mV decrements
(Vm = +50 to 150 mV from a holding potential
of 50 mV). To determine the relaxation time constant of D204E (see
Fig. 4), the pulse duration was increased from 100 to 200 ms.
B, Q-V relationship for WT and D204E at various
[Na+] or [H+] in the absence of substrate or
inhibitor. Smooth lines represent fits to Eq. 3 (see
"Experimental Procedures"). For comparison, curves were normalized
(Qnorm) and aligned vertically with respect to
Qdep in Nac or Hc.
Symbols represent [Na+] (in mM) of 10 ( ), 50 ( ), and 100 ( ), and [H+] (in
µM) of 0.032 ( ), 0.316 ( ), and 10 ( ).
C, V0.5 of WT (rectangle)
and D204E (triangle) as a function of [cation].
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Fig. 4.
Voltage dependence of relaxation time
constant. Time constants ( ON and
OFF) were determined from the charge transfer
in Nac (open symbols) and Hc (solid
symbols) shown in Fig. 3A. ON
of WT (rectangle) and D204E (triangle) was
plotted as a function of membrane potential
(Vm). Smooth lines represent a
Gaussian fit to ON.
OFF for both transporters was
voltage-independent and shown as the average over the test range (+50
to 150 mV) for WT (square with black diamond,
in Nac; square with white diamond, in
Hc) and D204E (triangle with black
diamond, in Nac; triangle with white
diamond, in Hc).
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The charge movement was obtained by integration of the
transporter-mediated relaxation. At each Vm, a
plot of the charge (Q) versus the membrane
voltage (Vm) yielded a sigmoidal charge-voltage (Q-V) curve (Fig. 3B). Data were
fitted to Eq. 3 to obtain the Boltzmann parameters
Qmax (maximal charge),
V0.5 (Vm at which 50% of
the charge has moved in the membrane electric field), and z
(the apparent valence of the charge). Qmax of WT
and D204E were comparable in Nac and Hc (~23
nanocoulombs (nC)), and Qmax of D204C and
D204N were reduced about 5-fold (Table I). The substitution of
Nac by Hc shifted V0.5 of WT
from
36 ± 1 mV to
74 ± 4 mV. Glu in place of
Asp204 exhibited a V0.5 in
Nac of
50 ± 2 mV. However, the
V0.5 of this transporter in Hc was
shifted to more positive potentials (V0.5 ~+40 ± 5 mV).
In Fig. 3B the effect of varying [cation] on the
charge-voltage (Q-V) relationship is shown for WT
and D204E. At [Na+] or [H+] below 25 mM
or 3.2 µM, respectively, the Q-V
curve for WT didn't become saturated at negative potentials.
D204E exhibited a sigmoidal Q-V curve over the
entire [Na+] range tested. At [H+] higher than 3.2 µM, the Q-V relationship of the
latter transporter did not become saturated at +50 mV.
Fig. 3C summarizes V0.5 as a function
of [cation]. Reducing [Na+] from 100 to 25 mM
shifted V0.5 of WT from
36 ± 1 mV to
91 ± 2 mV. Plotting V0.5 as a function
of log[Na+] revealed a linear relation with a slope of
98 ± 3 mV per 10-fold change in [Na+]
(
V0.510).
V0.5 for D204E was apparently not dependent on
[Na+]
(
V0.510 of
8.7 ± 0.2 mV). In H+ WT
(
V0.510 = 92 ± 7 mV), and D204E
(
V0.510 = 87 ± 11 mV) exhibited a similar slope of
V0.510.
However, V0.5 of D204E was shifted by about 110 mV toward positive potentials. Over the concentration range shown in
Fig. 3C, WT and D204E exhibited a z value of
~1.
Relaxation Time Constants--
Substraction of capacitive and
steady-state currents from the total currents (see Fig. 3A)
revealed a monoexponential time constant (
) of the pre-steady-state
relaxation currents.
was voltage-dependent in the
ON response but not dependent on voltage in the
OFF response (Fig. 4). In Nac the shapes of the
ON-V curves for the transporters tested were
bell-shaped and described a Gaussian fit. The maximum
(
max) for WT and D204E was ~20 ms and ~27 ms at
~
100 mV. In contrast, in Hc the
-V curve of
WT didn't reach
max over the applied
Vm (+50 mV to
150 mV) but the bell-shaped
-V relation of D204E was dramatically shifted to the
right (
max of ~32 ms at ~
22 mV). As reported
above, D204C and D204N exhibited very small pre-steady-state currents
that were at the border of resolution and precluded us from determining
reliable
max values over the entire voltage range.
Stoichiometry--
The stoichiometry of ion/sugar cotransport was
determined by simultaneously measuring the unidirectional flux of
D-[14C]glucose into oocytes expressing WT or
D204N and monitoring the sugar-evoked inward current. Integration of
the inward current with time revealed the net positive charges that
entered the oocyte. A plot of the net charge against glucose uptake by
oocytes expressing WT revealed a linear relation in both Nac
and Hc with a slope of ~2 inward charges per glucose molecule
transported (2.1 ± 0.1 in Nac; 2 ± 0.3 in
Hc) (Fig. 5). In the presence of
Nac the slope of the inward charge/glucose relation of D204N
was 2.1 ± 0.4. Replacing Nac with Hc changed the
slope for D204N to 39 ± 3. By reducing the final
D-[14C]glucose concentration in Hc
from 5 to 0.1 mM, the slope for D204N was 144 ± 7 (Fig. 5, inset), but no significant effect on the
H+/glucose stoichiometry was observed for WT (data not shown).
No Nac- or Hc-dependent glucose uptake or
sugar-evoked current was observed in control (H2O-injected)
oocytes.

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Fig. 5.
Stoichiometry of cation/sugar cotransport of
WT and D204N. Glucose-dependent charge
(Qglc) and D-[14C]glucose
uptake were simultaneously determined at Vm = 90 mV in the presence of Nac ( , WT; , D204N) or
Hc ( , WT; , D204N) for time periods ranging from 5 to 15 min. The final concentration of
D-[14C]glucose was 1 mM (1 µCi/µmol) in Nac and 5 mM (1 µCi/µmol) or
0.1 mM (100 µCi/µmol) (inset) in Hc,
respectively. Integration of the glucose-evoked inward current with
time was used to calculate the net cation influx by converting
nanocoulombs to nanomoles using the Faraday constant. Uptake of
D-[14C]glucose by oocytes expressing given
cRNA was corrected for basal uptake by control
(H2O-injected) oocytes.
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Outward Currents--
We next studied the effect of intracellular
acidification on glucose-induced H+ currents. Acidification of
the intracellular compartment was produced by rapid replacement of
external choline chloride with potassium acetate (21). Fig.
6 shows representative I-V
curves on the effect of internal acidification of oocytes expressing D204N or WT in the presence of 31.6 µM H+ (pH
4.5). In the absence of acetate, the glucose-induced currents for D204N
increased with hyperpolarization and reversed at +23 mV (Fig.
6A). Internal acidification increased the magnitude of outward currents with depolarization (+800 nA at +50 mV) but had only
minor effects on currents at potentials more negative than
70 mV. No
outward currents were observed after internal acidification of oocytes
expressing WT under either condition (Fig. 6B), despite the
fact that the amount of WT in the plasma membrane was about four times
that of D204N (Qmax for WT = 14 nC, for
D204N = 4 nC). Addition of 0.2 mM phlorizin reversibly
blocked the inward and outward glucose-induced currents for D204N
completely (Fig. 6C).

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Fig. 6.
Effect of intracellular acidification on
sugar-induced steady-state currents. A and
B, net currents induced by 10 mM
D-glucose in the presence or absence of 50 mM
potassium acetate (equimolar replacement for choline chloride) at pH
4.5 are plotted as functions of the membrane potential
(Vm) for (A) D204N (in the presence
(inverted triangle with black diamond) or absence
( ) of acetate) and (B) WT (in the presence
(square with black diamond) or absence ( ) of
acetate) as described in the legend of Fig. 2. C, phlorizin
inhibition of glucose-evoked inward and outward currents for D204N in
the presence of 50 mM potassium acetate at pH 4.5. Net
currents were presented as the difference of total currents induced by
10 mM glucose in the absence ( ) or presence ( ) of 0.2 mM phlorizin minus H+ currents in the presence of
0.2 mM phlorizin. Data for D204N (A and
C) are from the same oocyte.
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DISCUSSION |
The present study confirms and extends our previous reports on the
kinetics of SGLT1 (4, 5): First, the steady-state and pre-steady-state
kinetics of the transporter in Na+ are in close agreement with
previous observations on hSGLT1 (10, 17, 20, 22-24). Second, we
confirm and extend our findings on rabbit SGLT1 (25, 26) that
H+ can drive sugar cotransport through hSGLT1. The kinetics of
H+ and Na+ sugar cotransport are quite similar, even
though the apparent affinity of hSGLT1 for H+ is ~1000-fold
greater than for Na+, and the apparent affinity for sugar is
about 10-fold lower in Hc than in Nac (see Table I). In
fact, the maximum rates of cotransport, the cation-to-sugar
stoichiometry (2:1), the Hill coefficients (>1), turnover numbers
(~50 s
1), and voltage dependence of transport are
virtually identical for Na+ and H+ cotransport when
measured in the same oocyte. Furthermore, the pre-steady-state kinetic
parameters Qmax, V0.5,
and
max are comparable in Nac and Hc.
The only quantitative differences are that the turnover number for the
leak pathway (uniport) (27) is about 5-fold higher in Hc (22 s
1) than in Nac (4.4 s
1) and that
K0.5H+ is about one order
of magnitude smaller for the leak than for the cotransport. The latter
observation is consistent with the results for hSGLT1 reported by Chen
et al. (24). In contrast to their work (they reported a Hill
coefficient for Na+ uniport = 1 and for H+
uniport > 1), the present study clearly emphasizes that the Hill coefficients for both Na+ and H+ uniport are >1.
Together with the concentration dependence of V0.5 (95 mV per 10-fold change in
[Na+] or [H+]), this finding supports our
hypothesis that two cations, Na+ or H+, bind to SGLT1
before sugar. This is in direct contradiction to the Na+ (or
H+)-sugar-Na+ binding sequence model proposed by Chen
et al. (24) based on their experiments. However, Chen's
model is inconsistent with their reported results, because it does not
take into account that the Hill coefficient for the H+ leak
current was >1 and that the currents for the Na+ leak were no
greater than 15 nA (compared with 100 nA in the present study).
Furthermore, their conclusion that Na+ is the last substrate
bound to the transporter cannot account for the steady-state kinetics,
because the maximum rate of Na+/glucose cotransport is
independent of [Na+] (4, 5).
To explore the role of the N terminus of hSGLT1 in cation/sugar
cotransport, we have analyzed the effect of replacing a conserved amino
acid residue, Asp204. This residue is aligned with
Asp187 in the Na+/proline cotransporter (PutP),
which is functionally important for cation selectivity and
Na+-dependent proline binding and transport (12).
Replacing Asp204 revealed two functional classes of
mutations, depending on the presence or absence of a negative-charged
amino acid side chain at position 204. This stands in contrast to PutP,
where a polar rather than a charged residue at position 187 is
essential for function.
The first class of mutations consists only of a transporter with a Glu
in place of Asp204. Independent of the coupling cation, the
apparent affinity for glucose is increased by ~4-fold, indicating a
cation species-independent effect on the glucose translocation through
D204E. The apparent affinity of the transporter for Na+ is
increased by 3-fold, whereas the I-V relation and
ImaxGlc in Nac+,
ImaxNa+, and the leak
ImaxNa+ are all
comparable to WT. These minor kinetic differences are reflected by
similar turnover numbers by these transporters. On the other hand,
D204E exhibits an increased apparent H+ affinity. This implies
that lengthening the side chain at position 204 by only one methylene
group (about 1.5 Å) dramatically influences, directly or indirectly,
the geometry of the cation site(s). A similar observation was reported
for the glutamate transporter (GLT-1 or EAAT-2) (28). The increased
apparent H+ affinity of D204E is mirrored by the alkaline shift
in the Q-V relation (Fig. 3B), suggesting that,
even at very low [H+], binding of H+ induces
conformational changes that are the basis for coupled transport (10).
This fact is most likely the reason for the apparent independence of
the Q-V relation of D204E on [Na+], because
H+ is the preferred cation species. At [H+] larger
than 3 µM no saturation at depolarizing potentials is detectable, precluding reliable calculations in this [H+]
range. However, between 0.1 and 3 µM H+
V0.510of D204E
is similar to
V0.510for WT
from 1 µM to 32 µM H+. The
~20-fold increase in apparent affinity is also reflected by a
~110-mV shift of V0.5 toward positive
potentials and a
max of D204E at
25 mV (no
max is detectable for WT in Hc from
150 mV to
+50 mV). According to computer simulations of the 6-state kinetic model
for SGLT1 (5), this right shift of
max and the generally
higher
values are due to higher binding and lower dissociation rate
constants for the cation. This prediction is consistent with the
observation that no H+ leak through D204E is observed and may
explain why the glucose-evoked H+ currents (Fig. 2) were about
~5-fold smaller for D204E than for WT. Because the I-V
relation of D204E in H+ does not saturate at hyperpolarizing
potentials, the turnover number for H+/glucose cotransport for
D204E at
110 mV must be greater than 9 s
1.
The second class of mutations encompasses transporters with a neutral
side chain at position 204 (Asp
Asn or Cys). By using thiol
labeling, the predicted cytoplasmic location of Asp204 (1,
29) is confirmed, because Cys204 is only accessible from
the cell inside (data not shown). Furthermore, introduction of a
positive charge (by 2-aminoethyl methanethiosulfonate) doesn't have a
significant effect on the kinetics of the transporter. Based on this
observation, we may exclude a possible role of Asp204 in
salt-bridge formation with a positive amino acid as proposed for Asp
residues in the lactose permease (LacY) (30-32). However, as indicated
by the 5-fold reduction of Qmax for D204C and
D204N, removal of the negative charge at position 204 reduces the
number of transporters in the plasma membrane. These data imply a
trafficking defect of the protein to the plasma membrane: a common
problem observed with the expression of a membrane protein in a
eukaryotic expression system (33, 34). This trafficking problem
precludes us from a detailed analysis of the pre-steady-state kinetics
of D204C or D204N.
Analysis of steady-state kinetics of D204C and D204N shows a >10-fold
reduction of K0.5Glc in
Nac and Hc. The finding that this single mutation also
produces a 10-fold reduction in
K0.5Na+ raises the idea
that cation and sugar sites are in close proximity or may overlap. A
similar hypothesis is proposed for the melibiose permease of E. coli, where an N-terminal domain of the transporter plays a
fundamental role in connecting cation- and sugar-binding sites in terms
of coupling (35, 36). Although the apparent affinity parameters of the
(co)substrates for D204C and D204N were altered and the steady-state
activation of the currents revealed a Hill coefficient < 1, neither the coupling ratio of Na+ to glucose transport (Fig. 5)
nor the turnover number for Na+/glucose cotransport
(ImaxNa+/Qmax,
see Table I) was significantly altered.
Unexpectedly, all experimental evidence indicates that neutralization
of Asp204 (D204C and D204N) in hSGLT1 results in the
activation of a glucose-activated H+ channel. This conclusion
is based on the observations that: 1) The glucose-evoked H+
currents are an order of magnitude greater than the Na+
currents (Fig. 2). Because the H+ currents do not become
saturated within the applied voltage range, this would imply
that the turnover number
(ImaxH+/Qmax)
increases by more than one order of magnitude over that in Na+
(Table I). 2) The H+ currents reverse at positive voltages,
unlike either the Na+ currents for D204C or D204N or the
H+ and Na+ currents for WT or D204E (Fig. 2). These
outward H+ currents are enhanced by internal acidification of
the oocyte (Fig. 6A) and are blocked by the addition of
phlorizin (Fig. 6C). 3) The H+ currents greatly
exceed those expected for the strict coupling of H+/sugar
cotransport. The H+/sugar stoichiometry increases from the
expected value of 2 to as high as 144 (Fig. 5). The Na+/glucose
stoichiometry for both WT and D204N was 2:1. 4) There is no change in
the H+ leak currents for these mutants. The turnover numbers
for the H+ leak currents for D204C and D204N (
172 nA/5
nC = 34.5 s
1;
142 nA/4.4 nC = 32 s
1) are comparable to that for the wild-type protein (22 s
1).
These results imply that minor structural changes in hSGLT1,
i.e. replacing a carboxyl side chain with an amine or
sulfhydryl group, are sufficient to open a glucose-activated H+
channel. This channel activity is intimately associated with sugar
transport, because the competitive blocker phlorizin is unable to
activate the H+ channel but is only able to block the effect of
glucose. Clearly, based on our results we cannot distinguish whether
neutralization of this conserved acidic residue generates a new
"artificial" H+ pore or affects interactions with the
coupling cation and/or other parts of the protein, thereby modifying
the "original" H+ pathway. Because Asp204 is
located in a cytoplasmic loop, it seems unlikely that this residue is
part of the membrane-spanning H+ pathway. However, with the
lack of high resolution structural data of this transporter, this
question will remain an enigma.
Our observations lend support to the suggestion that
cotransporters and channels share features in common. For example, the glutamate cotransporters also behave as ligand-induced chloride channels (37, 38, see also Ref. 39 for review). However, this report
shows that hSGLT1 appears to be unique in that: 1) ion channel activity
is only observed in the mutagenized protein; 2) the ligand (sugar)
opens a channel for the driving cation (H+) for cotransport,
and 3) SGLT1 functions as a monomer (40). The glutamate cotransporter
EAAT3 occurs as a homopentamer in oocyte plasma membranes, and it has
been suggested that the chloride channel is formed by the oligomer
(41).