(Received for publication, August 9, 1996, and in revised form, October 23, 1996)
From the Department of Physiology, UCLA School of Medicine, Los Angeles, California 90095-1751
Cation-driven cotransporters are essential
membrane proteins in procaryotes and eucaryotes, which use the energy
of the transmembrane electrochemical gradient to drive transport of a
substrate against its concentration gradient. Do they share a common
mechanism? Cation selectivity of the rabbit isoform of the
Na+/glucose cotransporter (SGLT1) was examined using the
twoelectrode voltage clamp and the Xenopus oocyte
expression system. The effect of H+, Li+, and
Na+ on kinetics of SGLT1 was compared to the effects of
these cations on the bacterial melibiose. In SGLT1, substitution of
H+ or Li+ for Na+ caused a kinetic
penalty in that the apparent affinity for sugar (K0.5sugar) decreased by an order of
magnitude or more (from 0.2 to 30 mM) depending on the
membrane potential and cation. The effect of the cation on the
K0.5sugar/V profiles was
independent of the sugar for glucose and
-methyl-
-D-glucose; this profile was maintained for
galactose in Li+ and Na+, but was 2 orders of
magnitude higher in H+, but the
Imax for glucose, galactose, and
-methyl-
-D-glucose in a given cation were identical.
Li+ supported a lower maximal rate of transport
(Imax) than Na+ (~80% of
ImaxNa), while the
Imax in H+ was higher than
Na+ (
180% of ImaxNa).
Our interpretation of these results and simulations using a six-state
mathematical model, are as follows. 1) Binding of the cation causes a
conformational change in the sugar binding pocket, the exact
conformation being determined by the specific cation. 2) Once the sugar
is bound, it is transported at a characteristic rate determined by the
cation. 3) Mathematical simulations suggest that the largest
contribution to the kinetic variability of both cation and sugar
transport is associated with cation binding. Similarity to the effects
of cation substitution in MelB suggests that the mechanism of energy
coupling has been evolutionarily conserved.
Cotransporters are found in bacteria, plants and animals, and the driving cations are either Na+ (e.g. Na+/glucose; Ref. 1), K+ (e.g. insect K+/amino acid; Ref. 2) or H+ (e.g. Lac-permease; Ref. 3). Do all of these cotransporters share a common mechanism? All of these integral membrane proteins use the energy of the transmembrane electrochemical ion gradient to drive the accumulation of a substrate against its concentration gradient into the cell. This is commonly described as an ordered process, in which the binding of the first substrate (e.g. Na+) increases the affinity of the transporter for its co-substrate (e.g. sugar): the essential activator model. The transporter then undergoes another conformational change(s), which results in release of the substrates into the cell. Cotransporters are characterized as being highly specific for one cation above all others, but in earlier studies (e.g. Refs. 4 and 5), it was reported that Li+, for example, could partially substitute for Na (6, 7), and the bacterial melibiose cotransporter MelB can use gradients of H+, Li+, or Na+ (8). In this study we investigated the effects of cation substitution on function of the Na+/glucose cotransporter (SGLT1) in the steady state using electrophysiological methods and compared effects of substitution of Na+, H+, and Li+ on SGLT1 with those described for MelB (8). Fitting the results to a six-state kinetic model suggests that the largest part of the cation substitution effect can be attributed to cation binding on the internal and external faces of SGLT1. The similarity of the effects of cation substitution on sugar transport by SGLT1 and the melibiose cotransporter (8) suggests that the cotransporters share a common mechanism.
Mature oocytes from Xenopus laevis were injected with
cRNA encoding the rabbit isoform of SGLT1 (9). Ionic currents, which are stoichiometrically coupled to Na+ and sugar fluxes
(10), were measured using the two-electrode voltage-clamp and a pulse
protocol as described previously (100-ms pulses over the range +50 to
150 mV from a holding potential of
50 mV). Sugar-induced currents
were the difference between the records taken in cation + sugar and the
preceding record taken in cation alone (11). Oocytes were held in a
perfusion chamber and bathed in (in mM) 100 NaCl (or LiCl
or choline chloride), 2 KCl, 1 MgCl2, 1 CaCl2,
10 HEPES-Tris, pH 7.5. The pH of the choline buffer was varied between
pH 5.5 and pH 7.5 by using mixtures of
MES,1 Tris, and HEPES buffers.
Kinetic constants were determined by fitting the data to the Hill equation: I = (Imax)([S]n)/([S]n + K0.5n) using the non-linear fitting algorithm in Sigmaplot (Jandel, Foster City, CA). Here [S] is the substrate concentration, I is current, Imax = maximal current, the apparent affinity, K0.5, is the concentration of S which gives 0.5 Imax, and n is the Hill coefficient. For sugar activation, n = 1. Cation activation was from 0.5-100 mM for Na+ and Li+. For H+ activation the pH range was from 5.5 (3.2 µM) to 7.5 (0.032 µM). Figures are from individual oocytes, unless noted in the legend; however, all experiments were repeated at least three times on different oocytes taken from different animals with similar results. Statistics for the kinetics are either the standard error for the fit or standard error of the mean. Error bars are not shown if they are smaller than the symbol.
All chemicals were purchased from Sigma, Aldrich, or Research Organics (Cleveland, OH) and were of the highest grade available.
The effects of cation substitution on transport were modeled by fitting
the experimental data to the six-state kinetic model of SGLT1 (12-14).
As this is a simplified model of SGLT1 function, a conservative
approach was used. Rate constants were initially adjusted to match a
representative data set for Na+. After this, only the rate
constants affecting affinity for cation (k12o,
k21o,
k56o, and
k65o) were altered to match the data
from H+ or Li+. For simulations of cation
activation the MG concentrations were: Na+, 10 mM; Li+, 100 mM; H+, 50 mM. For simulations of sugar activation,
[Na+] and [Li+] were 100 mM,
and [H+] was 3.2 µM (pH 5.5).
Fig. 1 shows the sugar-induced current recorded
from an oocyte expressing SGLT1 to show the consistency of the
transport characteristics for Na+, Li+, and
H+; sugar is transported with the cation, which causes an
inward cationic current, and this transport is sensitive to the classic inhibitor of SGLT1, phlorizin.
Activation of 100 mM sugar transport by [Li+]
(0.5-100 mM) is shown in Fig. 2.
Representative sugar-dependent currents for 5, 25, and 100 mM Li+ (Fig. 2A) are
[Li+]- and voltage-dependent, and show
saturation with 25 and 100 mM Li+ at 150 mV.
The data at each voltage were fit to the Hill equation to determine the
kinetic parameters (Fig. 2B). Values for the Hill
coefficient (n) ranged from 1.3 ± 0.1 to 1.8 ± 0.1 at Vm =
50 mV (n = 3).
Fig. 3 compares current-voltage curves for
sugar-dependent currents for the three cations. The
sugar-dependent Na+ current showed the
characteristic curve (11); there was no transport at +50 mV, and a
substantial current at 0 mV (about 40% of the maximum), which
approached saturation by 150 mV. Both the Li+ and
H+ traces were shifted toward hyperpolarizing potentials,
relative to the Na+ curve. Like Na+, neither
supported transport at +50 mV. In choline, with 3.2 µM
H+ (pH 5.5) in the bath,
MG induced a small current at 0 mV (~10% of the Na+ current), but a gradient of
Li+ was unable to support transport in the absence of an
electrical driving force. As the membrane potential increased to
50
mV, the transport rate for both H+ and Li+
increased, but were only 30% and 10% of the Na+-driven
current, and, while the Li+ supported transport saturated
by
150 mV, the H+ current curve did not show saturation
at
150 mV.
We measured cation and sugar kinetics to determine the origin of these
functional differences. Fig. 4A shows the
effect of the cation on the apparent affinity for MG
(K0.5
MG). The points are data from
one oocyte (used in panels A-D). The K0.5
MG (Vm =
150 mV) in Na+ was 0.15 ± 0.03 mM
(n = 3) and essentially voltage-insensitive. The
K0.5
MG in H+ was
3.8 ± 1.0 (n = 3) mM and increased
about 2-fold (to 6.8 ± 0.4 mM) as the membrane
depolarized to
50 mV. When the driving ion was Li+, the
K0.5
MG was similar to that in
H+ (1.7 ± 0.6 mM, n = 4),
but the apparent affinity decreased by almost 15-fold (to 28 ± 8 mM) as the membrane depolarized from
150 to
50 mV.
The Imax/V curves for MG are
plotted in Fig. 4B. The influence of membrane potential on
Imax in each cation is similar to Fig. 2
(Imax
MG curves for H+
and Li+ are shifted toward hyperpolarizing values, relative
to Na+, and the H+-driven transport does not
saturate by
150 mV). Imax
MG is
determined by the driving cation; at all voltages
Imax
MG in Li+ is lower
than Imax
MG in Na+, and
Imax
MG in H+ is
greater. In this oocyte, where the
Imax
MG in Li+ was
70-90% of Imax
MG in
Na+; Imax
MG in
H+ was 170-200% of
Imax
MG Na+.
Fig. 4C shows that the effect of the cation on the
K0.5Glu for glucose was similar to
MG: When Na+ was the driving ion the
K0.5Glu was 0.1 ± 0.01 mM and voltage-independent; when the cation was H+ the K0.5Glu increased
by an order of magnitude (1.7 ± 0.3 mM) and was about 2-fold higher (4.1 ± 0.6 mM) as the membrane
potential decreased to
50 mV; and when the cation was Li+
the K0.5Glu increased about 15-fold as
the voltage depolarized from
150 to
50 mV (0.9 ± 0.2 to
13.2 ± 1.2 mM).
This pattern changed for galactose (Fig. 4D). The
K0.5Gal at 150 mV was 2 orders of
magnitude higher in H+ compared to the
K0.5Gal in Na+ (16 ± 1.6 mM versus 0.2 ± 0.02 mM,
n = 3). The pattern for Na+ and
Li+ was similar to that measured for the other two sugars.
The highest affinity was measured when SGLT1 used Na+ and
was insensitive to voltage. When SGLT1 used Li+, the
K0.5Gal at
150 mV was an order of
magnitude higher (4.9 ± 2.1 mM, n = 3) and highly voltage sensitive (at
70 mV, 57 ± 16 mM, n = 3). The 2-fold sensitivity of
K0.5Gal in H+ to
depolarization (45 ± 18 mM at
50 mV) was similar to
that measured when SGLT1 used H+ to transport
MG and
glucose.
The effect of the individual cation on the
Imax/V relationship was
quantitatively identical for all three sugars. Fig. 4B shows
the curves for MG, but the curves for the
Imax/V relationship for the other
sugars, measured in the same oocyte, were identical.
Na+-driven sugar transport has been described by an ordered
six-state kinetic model, shown in Fig. 5 (12-14).
Transport is envisaged as a series of conformational changes induced by
ligand binding. The states are: the empty transporter, [C]; the
transporter bound to Na+, [CNa2]; and the
sugar-cation complex, [SCNa2]. The empty transporter binds 2 Na+ before the sugar. The sugar-cation complex then
undergoes a conformational change, which results in transport of the
cosubstrates into the cell. The substrates are released on the inside,
and the substrate binding sites again become accessible at the external
surface. The steps that are sensitive to the membrane voltage are the
conformational change of the empty transporter between the internal and
external membrane surfaces ([C]
[C]") and the cation
binding/dissociation steps ([C]
[CNa2]). The
internal cation binding step in the simulation is insensitive to
voltage (11).
By simply changing the rate constants
k12o,
k21o,
k56o, and
k65o, as shown in Table
I, we could reproduce both the apparent cation and sugar
affinity, the influence of membrane potential on affinity, and the
magnitude of the change in Imax
(ImaxLi = 75%
ImaxNa;
ImaxH = 200%
ImaxNa, data not shown), although the
present model does not reproduce the shape of the Li+ and
H+ Imax/V curve. The
activation of sugar transport by Na+, H+, and
Li+ is shown in Fig. 6A as
symbols, and the prediction using the simulation are the smooth curves.
We used the same values to predict the effect each cation had on
affinity for MG. The model predicts that when SGLT1 uses
Li+ the K0.5
MG will be
the most sensitive to Vm, that H+ will
be intermediate to Li+ and Na+, and suggests
that K0.5
MG approaches a constant
value at extremely hyperpolarizing values, independent of the identity
of the cation.
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The H+-driven transport did not saturate under the
conditions used for these experiments. This is at least partially due
to the K0.5H of ~pH 5.2 and our
maximum experimental pH of 5.5, so we could not saturate the cation
binding site by 150 mV. We could therefore expect non-saturating
I/V curves in H+, consistent with the
data from Na+ (11) and Li+ (i.e.
Fig. 2A). (The H+/sugar I/V curves approach
saturation under other experimental conditions, unpublished
observations.) The model predicts that the cation binding steps,
k12o and
k65o, are greatly increased for
H+ (104 and 2 × 103-fold
compared to Na+) and that
k21o and
k56o are similar to those for
Na+. For Li+ K21 was
increased 500-fold over Na+. More than one set of
parameters was found to fit the H+ data. The parameter set
in Table I was selected based on our assumption that the H+
affinity would increase symmetrically.
These experiments show that under hyperpolarizing membrane
potentials SGLT1 can use the electrochemical gradient of
Na+, Li+ and H+ to drive sugar
transport using a common mechanism. The effects of cation binding are
to: 1) induce a conformational change in SGLT1, which enhances the
cotransporter's affinity for sugar; and 2) alter a rate-limiting step
in the transport cycle. Using these criteria, the favored cation was
Na+ at physiological values of the membrane potential
(Vm = 50 mV) where Na+ bestows the
highest sugar affinity (K0.5
MG = 0.15 mM) and transport capacity. Use of either
Li+ or H+ imposed a kinetic penalty on apparent
sugar affinity (by at least an order of magnitude) and altered the
Imax (~80% of
ImaxNa in Li+, ~190% of
ImaxNa in H+). The maximal
rate in Li+ was lower than Na+ for all sugars,
and was highly regulated by the voltage. The fact that H+
was capable of supporting a significantly higher maximal rate of
transport may play a physiological role in the very proximal parts of
the gut where the pH of the chyme is acidic and the lumenal sugar
concentration will be high.
The original experiments that defined the cationic requirements for
intestinal Na+/glucose cotransport (4, 5, 7) did not detect
cotransport supported by other cations. From the present experiments
the reason becomes clear; transport kinetics, both affinity and maximal
rate, are highly modulated by the membrane potential. Since in tissue or vesicle experiments the membrane potentials are nominally between 60 and 0 mV, it is expected that in these classic experiments transport energized by H+ should be small, and that by
Li+ should be barely detectable.
The cation functions as an essential activator (Fig. 5), so when the
cation binds it increases the affinity of SGLT1 for sugar. The highest
sugar affinity will be measured when the cation binding sites are
saturated, and the lowest sugar affinity when the cation sites are
empty. Therefore, anything that affects cation affinity will also have
an effect on the apparent affinity for sugar. When SGLT1 used
Na+ it had the highest affinity for sugar, in the order
glucose (0.1 mM at 150 mV) <
MG (0.15 mM) < galactose (0.25 mM), and sugar affinity was essentially
unaffected by the membrane potential (9, 11). This is consistent with
Na+ serving as an essential activator, as the
Na+ concentration was at least 5 times higher than the
lowest Na+ affinity measured. In this case the
Na+ binding site is essentially saturated at all values of
Vm, and so the
K0.5sugar should be constant (Fig. 4,
A, C, and D). All three cations appear to activate SGLT1 using the same mechanism; all increase the apparent K0.5sugar with increasing [cation],
and have a Hill coefficient > 1, suggesting that the
stoichiometry (cation:sugar) remains constant at 2:1 (9, 11, 14).
When Li+ was the activator the apparent sugar affinity was
an order of magnitude lower than in Na+, but the same
pattern of affinity was maintained; highest affinity for glucose (0.9 mM) < MG (1.7 mM) < galactose (4.9 mM). The K0.5Li was very
sensitive to the Vm (Fig. 6A) so when
Vm depolarized the cation binding site became
progressively less saturated. Since cation binding is required to
increase sugar affinity, and Li+ affinity is much more
sensitive to Vm than Na+ or
H+, we would expect that the apparent sugar affinity in
Li+ would greatly decrease as Vm
depolarized (Fig. 4, A, C, and D).
The apparent affinity for sugar in H+, however, showed a
different pattern. The profiles of MG and glucose affinity followed the expected order: glucose (1.7 mM) <
MG (3.8 mM), but for galactose sugar affinity was decreased by a
factor of about 10 (16 mM). As expected from the
K0.5H/V relationship (Fig.
6A), the voltage sensitivity of the apparent affinity for
all three sugars remained slight. This effect suggests that the binding
of each cation induces a unique conformational change in the sugar
binding pocket. In the case of galactose, since the only difference
between it and glucose is the orientation of the hydroxyl at C-4, we
expect that H+ binding produces a conformational change
that results in misalignment of a residue, which is important in
recognition of the C-4 hydroxyl.
Since fluorescence studies suggest that the cation binding site is remote (~35 Å) from the sugar binding site (15) the question arises "Can binding of these monovalent cations produce conformational changes in distant parts of the protein?" Hohenester et al. (16) have shown that the monovalent cation-dependent enzyme dialkylglycine decarboxylase undergoes specific conformational rearrangements, both at the reaction center (11 Å from the cation; Ref. 17) and even small ternary alterations in the arrangements of the dimers. These changes appear to be solely due to the mechanism by which the protein compensates for the differences in cationic radius and coordination number between the activators, the large cations K+ and Rb+, and the smaller inhibitors, Li+ and Na+. This mechanism centers on how the rigidity of the cation binding site is compensated by addition of a single water molecule, which both accommodates a reduction in coordination number (from 6 to 4 or 5) and replaces ion-ligand bonding. This leads to "an altered local protein structure around metal binding site 1 that, in turn, leads to changes in both the dialkylglycine decarboxylase active site structure and the quaternary structure of the dialkylglycine decarboxylase tetramer" (16). Such a mechanism may account for the cation selectivity as well as the effect of the cation on sugar recognition by SGLT1.
We have simulated the effect of cation substitution on the apparent affinity for both cation and sugar (Fig. 6) using the six-state model (12, 13). The simulations suggest that the greatest influence of cation substitution is at the cation binding steps, as changing only the rate constants for these steps, k12o, k21o, k56o, and k65o, can simulate not only the observed K0.5 values, but how the membrane potential influences affinity for both cation and sugar. The model predicts that SGLT1 will have the highest affinity for sugar when it uses Na+, followed by H+ and then Li+. Using the same rate constants, it also correctly predicts the apparent affinity and voltage dependence of the cations: highest affinity for H+ and lowest for Li+.
The fact that the maximal transport rate, while set by the cation
(highest in H+; lowest in Li+), is independent
of the transported sugar (quantitatively identical for all three sugars
in a given cation regardless of
K0.5sugar), in turn suggests that the
mechanism of translocation is separate from the process of sugar
binding. In this scheme the molecular mechanisms underlying the kinetic
model might be described as follows. 1) Binding of the cation causes a
conformational change in the sugar binding site, which results in a
increase in affinity for sugar. 2) When sugar binds it is transported
to the inside, at a rate independent of the identity of the cation. 3)
Maximal transport rate is determined by recycling of the empty
transporter to the "outside-facing" conformation, and this is
controlled by cation binding on the inside. Note that our experiments
measure the overall kinetics, and all 14 rate constants influence the kinetics, so the translocation event (k34 k43) can remain constant even though the
Imax increases.
The effect of cation substitution on SGLT1 function is similar to that described for the bacterial melibiose cotransporter (MelB) (for example, Refs. 8 and 18), which can also use Na+, H+, and Li+ to drive sugar transport. Table II is a comparison of the kinetic parameters for these transporters (MelB data taken from Ref. 8). In both cotransporters the order of decreasing cation affinity is H+ > Na+ ~ Li+, and both transporters preferred H+ by several orders of magnitude over Na+ or Li+. The same order follows for maximal velocity of transport; H+ supported a higher transport rate than Na+ or Li+, which were similar. And, like SGLT1, the driving cation determined the preferred substrate in MelB, presumably by differences in conformation of the sugar-binding site (18, 19).
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On the other hand, there is a difference in how the membrane potential affected MelB transport kinetics in Na+ and H+ (20). If MelB used Na+ for melibiose transport, depolarization caused a decrease in Vmax but no change in Ktsugar. If the driving cation was H+, however, the same decrease in membrane potential resulted in a decreased affinity for sugar, and no change in Vmax. In SGLT1 depolarization caused an increase in K0.5sugar in all three cations, and there was a voltage-sensitive range of Imax for Na+ and Li+, as well as a voltage-independent range as the membrane hyperpolarized; in H+, transport was voltage-sensitive over the entire range. These similarities suggest that the basic mechanism of activation and transport has been conserved in evolution from bacteria to mammals. We anticipate that further investigations of the effects of cation substitution, using lanthanides (21, 22), for example, will provide further insights into the characteristics of the cation binding site and mechanism of activation of cotransporters, and the sources of divergence of eucaryotic and procaryotic transporters.
We are indebted to Manoli Contreras for excellent technical assistance and Debra Moorehead for computer graphics. We also thank our colleagues for helpful comments and discussions.