(Received for publication, May 31, 1995; and in revised form, August 9, 1995)
From the
The rat Na/glucose cotransporter (SGLT1) was
expressed in Xenopus oocytes and steady-state and transient
currents were measured using a two-electrode voltage clamp. The maximal
glucose induced Na
-dependent inward current was
300-500 nA. The apparent affinity constants for sugar
(
-methyl-D-glucopyranoside;
MDG) (K
) and sodium (K
) at a membrane potential of
-150 mV were 0.2 mM and 4 mM. The K
increased continuously with
depolarizing potentials reaching 40 mM at -30 mV. K
was steeply voltage dependent,
0.46 mM at -30 mV and 1 mM at -10 mV.
From all tested monovalent cations only Li
could
substitute for Na
, but with lower affinity. The
relative substrate specificity was D-glucose >
MDG
D-galactose > 3-O-Me-Glc
-naphthyl-D-glucoside uridine. Phlorizin (Pz), the
specific blocker of sugar transport, showed an extremely high affinity
for the rat cotransporter with an inhibitor constant (K
) of 12 nM. SGLT1 charge
movements in the absence of sugar were fitted by the Boltzmann equation
with an apparent valence of the movable charge of
1, a potential
for 50% maximal charge transfer (V
) of -43
mV, and a maximal charge (Q
) of 9 nanocoulombs.
The apparent turnover number for the rat SGLT1 was 30
s
. Model simulations showed that the kinetics of the
rat SGLT1 are described by a six-state ordered nonrapid equilibrium
model, and comparison of the kinetics of the rat, rabbit and human
cotransporters indicate that they differ mainly in their
presteady-state kinetic parameters.
Cotransporters are membrane proteins that use the
electrochemical potential gradient for ions to accumulate sugars, amino
acids and osmolytes into cells. Using the electrochemical potential
gradient for Na, the Na
/glucose
cotransporter (SGLT) (
)accumulates glucose across the brush
border membrane of the epithelial cells of the intestine and the
proximal tubule of the kidney.
Several members of the SGLT family
have been cloned, and these include the high affinity glucose
cotransporters (SGLT1, K
0.2
mM) from rabbit small intestine (1) and kidney (2) , pig(3) , and rat kidney(4) , human
intestine(5) , as well as the low affinity glucose
cotransporter (pSGLT2, K
2
mM) from pig kidney(6) . Mapping the genomic
arrangement of the human SGLT1 gene, Turk et al.(7) showed that SGLT1 is a single-copy gene, so that the
amino acid sequences from various tissues of a given species are
identical. Comparison of the amino acid sequences from the rat, human
and rabbit clones reveal 86-87% identity and 93-94% similarity. How does this high degree of homology between the three
clones affect their kinetic properties? In this study, we characterized
the presteady-state and steady-state kinetics of the rat SGLT1 clone
with a view to understand the relationship between structure and
function of members of the SGLT1 family.
The pBluescript II SK plasmid containing the coding sequence
for rat SGLT1 (4) was linearized with SalI and
transcribed in vitro with T3 RNA polymerase(11) . The
cRNA was overexpressed in Xenopus oocytes and protein function
studied 5-10 days after injection using the two-microelectrode
voltage clamp(8, 11) . To obtain a current-voltage
(I-V) relationship, the membrane voltage was stepped for 100 ms to
various test values (V) between 50 and
-150 mV in 20-mV decrements and returned to the holding potential
(-50 mV). Averaged currents from three sweeps were low-pass
filtered at 500 Hz by an 8-pole Bessel filter and digitized at 100
µs/point. In experiments to study the substrate and cation
specificity (see Fig. 3and Fig. 4) the currents were
continuously monitored on a chart recorder.
Figure 3:
Substrate specificity of the rat SGLT1. A, sugar specificity. Continuous current record from a single
rat SGLT1 cRNA-injected oocyte showing the sugar-induced inward
currents as the membrane potential was held at -50 mV. The dashed
line represents the base-line in 100 mM NaCl. At the time,
indicated by the arrows 20 mM of each of the potential sugar
substrates were added. The sugars were: D-glucose, L-glucose, 3-O-methyl-D-glucopyranose,
-methyl-D-glucopyranoside, D-galactose, myo-inositol, and
-naphthyl-
-D-glucopyranoside. Prior to the addition
of the sugar the oocyte was equilibrated in 100 mM NaCl. After
the sugar exposure it was rinsed in 100 mM choline chloride. B, nucleoside specificity. Uridine was tested at 50 mM alone, or in the presence of 400 µM
-methyl-D-glucopyranoside. Formycin B was tested at 10
mM.
Figure 4:
Cation specificity of the sugar transport.
Continuous current record showing the effects of Na and Li
in
MDG on the inward currents
mediated by the same rat SGLT1 cRNA-injected oocyte, described in Fig. 1. V
was -50 mV and the
oocyte was first perfused with choline (base line is indicated as a dashed line), followed by a 1-min equilibration in 100 mM solution of the appropriate cation (large arrows). At the
time indicated by the small arrows, 25 mM
MDG
was added. The substrate was continuously washed out until currents
returned to the base line. The record for V
= -50 mV is shown. The sugar-induced currents
in the presence of lithium and sodium were -25 nA and -130
nA, respectively, and the leak currents were -8 and -20
nA.
Figure 1:
Steady-states kinetics of the MDG
induced transport. A, membrane current records obtained before
and after the addition of 400 µM
MDG to a cRNA rat
SGLT1 injected oocyte. The oocyte was clamped at -50 mV.
Presented traces are the response to voltage steps (30, -10,
-50, -90, and -150 mV) applied for 100 ms. The traces
were averaged from three sweeps. B, current-voltage (I-V) relationships of the steady-state currents induced by
MDG. The external Na
concentration
[Na
]
was fixed at 100
mM while the [
MDG]
was
varied (in mM: 0.015, 0.031, 0.062, 0.125, 0.250, 0.5, 1, 5,
and 20). The I-V relationships were obtained as the difference
of the steady-state currents in the presence and absence of
MDG.
The
symbols represent the calculated maximal current (I
). C, voltage
dependence of the apparent affinity for
MDG (K
). At each membrane potential (V
) the
MDG-induced inward currents (I) were fitted to the equation: I = I
[
MDG]/((K
) + [
MDG]) where I
is
the apparent maximal current at saturating
MDG concentrations and K
is the sugar concentration at
50% I
. K
was 0.2 mM at
-150 mV, increased to 0.46 mM at -30 mV and 1
mM at -10 mV. I
was
-265 nA at -150 mV. Similar results were obtained in two
other experiments.
Nonlinear regression analyses were performed using the software ENZFITTER (Elsevier-Biosoft, Cambridge, UK), and the fitting routines in Sigmaplot (Jandel Scientific, San Rafael, CA). The Marquardt-Levenberg algorithm was used by both programs.
Data presented in Fig. 3, Fig. 4, and Fig. 5A were carried out on the same oocyte. Similar results were obtained by repeating the experiments 2-4 times on oocytes from different donors.
Figure 5:
Inhibition of the sugar induced currents
by phlorizin. A, continuous current record illustrating the
inhibition of the MDG-induced currents by two different phlorizin
concentrations. The dashed line represents the base line in
100 mM NaCl, the arrows show the time when the
substrate or inhibitor were added. V
was
-50 mV. Steady-state currents were measured in 100 mM NaCl containing 5 mM
MDG. Addition of 5 µM phlorizin inhibited the maximal sugar-induced current 84%
(-170 to -27 nA), whereas 50 µM phlorizin
completely blocked the sugar-induced current. B, voltage
independence of K
. Shown are the
calculated K
in the presence of two
concentrations of
MDG. For the inhibition at 5 mM
MDG the following concentration of inhibitor were added (in
µM): 0.05; 0.1, 0.5, 1, 5, 10, and 20, and for the
inhibition at 250 µM (in µM): 0.025, 0.05,
0.1, 0.5, 1, and 5. The current differences between the measured
steady-state currents in 5 mM
MDG and those measured in
each concentration of the inhibitor in 5 mM
MDG at each V
were fitted to the equation described
in Fig. 1C. Same calculations were repeated for the
currents measured at 250 µM
MDG. Note that on
reducing the sugar concentration from 5 mM to 250
µM, K
decreased from
0.9 to
0.09 µM. For both curves error bars are
mean of three oocytes. C, Dixon plots of the phlorizin
inhibition of
MDG induced currents as function of different
phlorizin concentrations. The obtained reciprocal currents
(1/I) at each V
for 0.4 mM or 5 mM
MDG at the following phlorizin
concentrations (in µM: 0.01, 0.02, 0.05, 0.1, 0.25, and
0.5) were plotted against these phlorizin concentrations
[Pz]. The straight lines fit the equation: 1/I = K
[Pz]/I
[
MDG]K
+ 1/I
(1+K
/[
MDG]),
where K
is the apparent binding constant
for substrate, I
is the maximal current at the
applied substrate concentration [
MDG], [Pz] is
the applied concentration of the inhibitor. Lines obtained for
different fixed concentrations of substrate have a different positive
slope and cross at the point equivalent to the K
for a competitive inhibitor. As an example we show the plot
at V
= -150 mV where the K
was 12
nM.
The steady-state current-voltage (I-V) relationship of the
sugar-induced current is the difference in steady-state current in the
absence, and in the presence of MDG. Fig. 1B shows
a family of sigmoidal I-V curves obtained as
[
MDG]
increased from 31 µM to
20 mM. At each test potential (V
),
increasing [
MDG]
increased the sugar-induced
current until saturation was reached at 5 mM. For each
MDG concentration, as the test potential was made more negative,
the current increased for V
between 0 and
-100 mV, and then became independent of membrane voltage.
The
voltage dependence of the apparent K for sugar (K
) is shown in Fig. 1C. Between -150 and -50 mV, K
was relatively insensitive to
membrane voltage. However, between -30 and -10 mV, K
increased steeply with
depolarizing potentials, from 0.46 ± 0.03 at -30 mV to 1.0
± 0.2 mM at -10 mV. The calculated maximal
current I
at -150 mV for
this oocyte was -265 nA.
To determine the
Na-dependence of the sugar-evoked currents, the
steady-state inward currents were measured as
[Na
]
was varied from 0 to 100
mM while [
MDG]
was maintained at 5
mM. Fig. 2A shows the
Na
-dependent sugar-evoked current at V
= -30, -50, and -70 mV.
At each V
the current was described by the Hill
equation (see legend to Fig. 2B). There was a steep
voltage dependence of the apparent affinity for sodium (K
). K
increased from 4 ± 0.6 mM at -150 mV to 40
± 2 mM at -30 mV. The Hill coefficient (1.8
± 0.3) was independent of voltage for V
between -150 to -50 mV, and the I
was -318 nA at -150 mV.
Figure 2:
Na-activation of the
steady-state sugar induced currents. A, dependence of the
steady-state currents on the Na
concentration.
[Na
]
was varied between
0 and 100 mM (0, 5, 10, 20, 50, 70, and 100),
[
MDG]
was 5 mM. The
MDG-induced steady-state currents were measured as a function of
[Na]
. The measured inward currents
corresponding to three different membrane voltages are shown. Curves
were drawn according to the Hill equation (see B). B,
for each test potential (V
), the currents
shown in A were fitted to the equation: I = I
[Na]
/((K
)
+ [Na]
). I
is the maximal current at saturating
Na
concentrations, K
is
the value of [Na]
at 50% I
, and n is the apparent
coupling coefficient for Na
. K
was more voltage-dependent at
depolarizing membrane potentials. The values for K
were 4 ± 0.6 mM at
-150 mV and 40 ± 2 mM at -30 mV, but n
remained independent on voltage. As an example, at V
= -50 mV all three parameters were: I
= -221 ± 2 nA; K
= 26 ± 2 mM; n = 1.8 ± 0.3. The errors are errors of the fit.
Almost identical findings were obtained from two additional
oocytes.
Consistent with
reports about the rat intestinal absorption of glucose-conjugated
compounds (15) we observed -naphthyl
-D-glucopyranoside induced Na
inward
currents. Addition of 20 mM of this compound induced about
18-20% (-50 nA at -150 mV) of the recorded currents
induced by D-glucose (Fig. 3A).
The ability
of the monovalent cations Li, K
,
Rb
, and Cs
to substitute for
Na
was also examined. NaCl in the 100 mM NaCl
buffer was replaced isoosmotically by LiCl, KCl, RbCl, and CsCl and the
currents induced by addition of 25 mM
MDG was measured.
In the presence of 100 mM KCl, RbCl or CsCl, no detectable
inward currents were generated by 25 mM
MDG, indicating
that they cannot support sugar transport by rat SGLT1. Li
was found to be able to support sugar transport (Fig. 4).
There was an inward current upon substitution of LiCl for choline.
Similar to the Na
leak, this current was also blocked
by 50 µM phlorizin (not shown), and indicates that there
is a leak of Li
by rat SGLT1 in the absence of sugar.
This Li
leak was
50% of the Na
leak. The current carried by Li
in 25 mM
MDG was about 25% (-25 versus -130 nA, V
= -50 mV) of the current carried by
Na
, suggesting lower affinity for sugar in LiCl, as
detected for rabbit SGLT1(16) . At V
= -150 mV, the
MDG-induced currents were
-150 nA in 100 mM Li
and -250 nA
in 100 mM Na
.
To determine the
inhibitor constant K for phlorizin inhibition (K
) we performed a series of Dixon plots.
We plotted the reciprocal of the currents (1/I) against the phlorizin
concentration. The lines in Fig. 5C were obtained by
linear regression on phlorizin inhibition of the steady-state currents (V
= -150 mV) generated by 1 mM and 0.4 mM
MDG. The lines intersect at a phlorizin
concentration of -0.012 µM. Thus the inhibitory
constant K
is 0.012 µM (at
-150 mV). It remained slightly voltage dependent in the range
-150 mV to -50 mV, increasing to 0.053 ± 0.003
µM and 0.030 ± 0.010 µM at -70
mV and -50 mV. The errors are S.E. from three oocytes.
Figure 6:
Characterization of the presteady-state
currents in the absence of sugar. A, presteady-state current
records. The presteady-state current records were obtained by
subtracting the capacitive (Ie
)
and the steady-state currents (I
) from the total
current as described in B, and in Loo et
al.(8) . V
was -100
mV. The traces at V
= 50,
-10, -50, and -150 mV are presented beginning 1 ms
after applying the pulse. The inset shows the pulse protocol. B, kinetics of the presteady-state current relaxation. The
time constants of relaxation for the ON and OFF current transients
(
) for each tested membrane potential (V
) were obtained by fitting the measured
current (I) to the equation: I = I
e
+ I
e
+ I
, where I
is the
oocyte capacitive current with time constant
, I
is the rat SGLT1 transient current with time
constant
, before decaying to the steady-state
currents (I
). V
was
-100 mV. C, charge-voltage relationship of the current
transients. The integrals of the ON and OFF transient currents (Q) due to rat SGLT1 were plotted as a function of the applied
test voltage V
. The smooth curve was
obtained by fitting the average (
) of these charges to the
Boltzmann equation: (Q - Q
)
= Q
/[1 +
exp(V
- V
)zF/RT]. Q
= (Q
- Q
) is the maximal charge transfer, Q
and Q
are the charge
movements at the depolarizing and hyperpolarizing limits, F is
the Faraday's constant, R is the gas constant, T is the absolute temperature, V
is the
potential for 50% Q
, and z is the
apparent valence of the movable charge. Shown are the data from a
single oocyte (V
= -50 mV)
with parameters: z = 0.85; V
= -46 mV, and Q
= 11
nanocoulombs.
The dependence of the relaxation
time constant of the ON transients () on test voltage V
is presented in Fig. 6B.
decreased monotonically from 13.5 ± 2 ms at -50 mV to 2.6
± 0.1 ms at 50 mV. In the OFF response,
was independent of
the test voltage V
and was 53 ± 2 ms over
the voltage range -50 to 50 mV. Error bars are S.E. from
three oocytes.
of the oocyte capacitive current
(
) was independent of the membrane potential (0.6
- 0.8 ms).
Fig. 6C shows the dependence of the
total charge (Q, integral of the current transients) on
membrane voltage. The curve was drawn according to the Boltzmann
relation (see legend to Fig. 6C) to estimate the
parameters Q (maximal charge transferred), z (apparent valence of the movable charge), and V
(voltage for 50% Q
). The maximal charge
transferred is: Q
= Q
- Q
, where Q
and Q
are the charges transferred at the
tested voltage limits. Since Q
depends on the
level of expression, to compare oocytes with differing levels of
expression, the data were normalized between 0 and 1 using the
relation: (Q - Q
)/Q
. Mean values from
five different oocytes were V
= -43
± 3 mV, z = 1.0 ± 0.15, and Q
= 9.0 ± 2.5 nanocoulombs.
The archetypical member of the Na-dependent
family of transport proteins is SGLT. SGLTs have been cloned from
rabbit, rat, human, and pig. This family also includes the transporters
for myo-inositol and nucleosides(18) . In this study,
we characterized the kinetics of the Na
/glucose
cotransporter cloned from rat kidney. Our goal is to understand the
structure-function relations of Na
-dependent glucose
transport by comparing and contrasting the kinetics of highly
homologous proteins of this gene family.
Lee et
al.(4) found that the K was 397 µM at -60 mV and our value of 300
µM is in agreement with the value reported. In this study,
the steady-state current induced by
MDG was three times higher
than the study of Lee et al.(4) , and we were able to
obtain the voltage dependence of the K
for sugar
and sodium. The stoichiometry from the Hill analysis was 2
Na
:1 sugar molecule, and is similar to that of the
rabbit and human(9, 10) .
Lee et al.(4) also observed a K two
orders of magnitude less than the value of 10 µM for
rabbit SGLT1(9) . The K
based on
inhibition of 50 µM [
C]
MDG
uptake was 0.17 µM. Our estimate of the real inhibitory
constant K
was 0.012-0.03
µM. It was recently observed(19) , that the
Na
-dependent glucose transport system in sheep
tracheal epithelium also has a high affinity for phlorizin (K
0.020 µM). The
species differences in the affinity to phlorizin observed here are
almost certainly due to differences in the amino acid sequence. For the
rat SGLT1, the estimated real K
can also
be regarded as binding/dissociation constant and used in future
determination of the number of phlorizin molecules binding per
cotransporter molecule. The rat, rabbit, and human clones all exhibit a
phlorizin sensitive Na
leak current, which is about
15-20% of the maximal
MDG-induced current.
Figure 7:
Comparison of the presteady-state current
due to rat, rabbit and human SGLT1 transporters. Data for rabbit SGLT1
() are based on the estimates shown in Fig. 3, A and B, of Panayotova-Heiermann et
al.(11) , data for human SGLT1 (
) are taken from Fig. 3B of Loo et al.(8) . Data for
rat SGLT1 (
) were obtained from a single oocyte as described in Fig. 6.
The maximal charge Q depends on the level of expression of SGLT1 in the membrane since Q
= qzC
, where C
is the total number of transporters. The maximal
steady-state inward Na
current induced by saturating
sugar concentrations (I
) is proportional to Q
(8, 21) . I
= kqzC
, where k is the
apparent turnover number of the transporter. k for rat SGLT1
was 30 s
and comparable to that of the human and
rabbit (Table 1).
Computer simulations resulted in a set of rate constants which
account quantitatively and qualitatively for the observed presteady-
and steady-state kinetics. The results suggest that differences in the
kinetics between the rabbit and rat/human cotransporters are due to
differences in k and k
(Table 2). Such changes in the rate constants must be due
to differences in structure between the isoforms. Aligning the primary
amino acid sequences show that there are different residues at 129 out
of 665 positions, and, when conservative substitutions were taken into
account (K = R; S = T; D = E; Y = F
= W; and I = V = L = M) this reduces to
differences at 76 positions. These 76 are evenly distributed between
the N- and C-terminal halves of the protein (Fig. 8), and are
mostly confined to hydrophobic loops between the putative transmembrane
helices. The cytoplasmic hydrophilic N-terminal and the external loops
between helices 5/6 and 11/12 contain 43 of the 76 nonconserved
residues.
Figure 8:
Sequence alignment of the human, rat, and
rabbit SGLT1 cotransporters. The full sequence is given for the human
SGLT1, for the rat and rabbit cotransporters only the nonconserved
residues are included. Identical and residues similar to that in human
(D = E, R = K, S = T, I = V = L, and
Y = F = W) are replaced by dashes(-). The rabbit residues
that are significantly different from those in rat and human, i.e. are polar in either rabbit or rat and human, are shown in bold
italics. The location of the putative transmembrane domains is
indicated by the lower case letters and underlined (). The N- and C-terminals in this secondary structure
model (7) are placed on the cytoplasmic side of the plasma
membrane.
A clue about the residues that may be important in
determining kinetic differences comes from consideration of the
residues that are identical in pairs of the three transporters. There
are 25 residues shared between human and rat, 17 shared between human
and rabbit, and 21 between rabbit and rat. Overall, there are 37
residues that are different between the rabbit and the rat and human,
and 25 of these are polar (indicated in bold type in Fig. 8). At 10 positions the residues are charged in either the
rabbit or in the human and rat, at 7 positions the residues are serines
or threonines in either rabbit or human and rat, and at 5 other
positions the residues are polar in either rabbit or human and rat. The
polar residues are mostly in hydrophilic segments, and half are
clustered in external loops between helices 5/6 and 11/12. There are no
significant differences between the residues in putative transmembrane
domains of the cotransporters (in transmembrane helix 4 the
substitution 176DN is of no functional
significance)(11) . This suggest that the polar residues in
hydrophilic domains of the protein play an important role in
determining differences in kinetics between species, k
and k
, by determining the three-dimensional
protein structure through electrostatic interactions. This could be
tested by examining functional properties of clones after either
mutating the polar residues or swapping hydrophilic loops between
species.
Our conclusion is that the kinetic differences between the
human, rat, and rabbit transporters primarily are due to two partial
reactions involving binding/dissociation of Na ions
and translocation of the empty carrier. These differences are probably
due to polar residues clustered between helices 5/6 and 11/12.