(Received for publication, December 22, 1995)
From the
D-Glucose is absorbed across the proximal tubule of the
kidney by two Na/glucose cotransporters (SGLT1 and
SGLT2). The low affinity SGLT2 is expressed in the S1 and S2 segments,
has a Na
:glucose coupling ratio of 1, a K
for sugar of
2 mM, and a K
for Na
of
1 mM.
The high affinity SGLT1, found in the S3 segment, has a coupling ratio
of 2, and K
for sugar and Na
of
0.2 and 5 mM, respectively. We have constructed a
chimeric protein consisting of amino acids 1-380 of porcine SGLT2
and amino acids 381-662 of porcine SGLT1. The chimera was
expressed in Xenopus oocytes, and steady-state kinetics were
characterized by a two-electrode voltage-clamp. The K
for
-methyl-D-glucopyranoside (0.2 mM) was
similar to that for SGLT1, and like SGLT1 the chimera transported D-galactose and 3-O-methylglucose. In contrast, SGLT2
transports poorly D-galactose and excludes
3-O-methylglucose. The apparent K
was 3.5 mM (at -150 mV), and the Hill coefficient
ranged between 0.8 and 1.5. We conclude that recognition/transport of
organic substrate is mediated by interactions distal to amino acid 380,
while cation binding is determined by interactions arising from the
amino- and carboxyl-terminal halves of the transporters. Surprisingly,
the chimera transported
-phenyl derivatives of D-glucose
as well as the inhibitors of sugar transport: phlorizin,
deoxyphlorizin, and
-D-glucopyranosylphenyl
isothiocyanate are transported with high affinity (K
for phlorizin was 5 µM). Thus, the pocket for
organic substrate binding is increased from 10
5
5
(Å) for SGLT1 to 11
18
5 (Å) for the
chimera.
Reabsorption of D-glucose from the glomerular filtrate
along the proximal tubules of the kidney occurs by two apparently
distinct cotransport systems(1, 2, 3) . In
the S1 and S2 segments of the renal convoluted tubule, reabsorption is
mediated to 90% by a low affinity Na/glucose
cotransporter (SGLT2) (
)with an apparent K
for sugar of 6 mM and a Na
:glucose
coupling ratio of 1. The reabsorption process is completed in the S3
segment by another Na
/glucose cotransporter (SGLT1)
with significantly higher apparent affinity for sugar (K
0.35 mM) than SGLT2 and a
Na
/glucose coupling ratio of 2.
The pig renal cell
line LLC-PK1 (4) has often been used as a model system to study
glucose transport in kidney proximal epithelia. LLC-PK1 cells express
Na/glucose transport activity (apparent
K
0.28 mM; (5) and (6) ) located at the apical
surface(7) . The presence of SGLT1 (2:1
Na
/glucose stoichiometry) in these cells was shown by
Misfeldt and Sanders (8) and Moran et al.(9) .
In 1990, Ohta et al.(10) isolated a SGLT1 cDNA clone
(pSGLT1) from LLC-PK1 cells, with 84% and 87% identity to the high
affinity rabbit and human SGLT1. There are three electrophysiologically
characterized isoforms of the high affinity
Na
-dependent glucose transporter, the rat, human, and
rabbit
SGLT1s(11, 12, 13, 14, 15, 16) .
All three isoforms show an apparent K
between 0.17 and 0.49 mM, an apparent
K
of 2-7 mM, and a Hill
coefficient >1.5.
Recently a low affinity
Na/glucose cotransporter (pSGLT2) from LLC-PK1 cells
was also cloned and functionally
characterized(17, 18) . While the amino acid sequence
of pSGLT2 was highly homologous to pSGLT1 (75% identical; 88% similar
amino acid residues), the apparent K
for
MDG was
2 mM. Na
/glucose
stoichiometry for SGLT2 was 1, and the apparent K
4-5 mM (at -150 mV). Phlorizin inhibited the
MDG-evoked currents with an apparent K
of 18 µM, and D-galactose and
3-O-methylglucose were poor substrates for SGLT2.
Given the
high similarity of the primary sequences and secondary structure of
SGLT1 and SGLT2, what accounts for these differences in functional
properties? Which parts of the proteins are involved in Na and sugar binding? To begin to answer these important questions,
we generated a chimeric DNA-construct corresponding to amino acids
1-380, which form the putative transmembrane helices 1-8 of
pSGLT2, and amino acids 381-662 (which form putative helices
9-14) of pSGLT1 (19) and compared its functionality with
the parental proteins.
All two-microelectrode voltage clamp studies
were controlled by the Clampex computer program of pCLAMP software
(Axon Instruments, Foster City, CA) as described in (12) .
During experiments measuring steady-state kinetics, the oocyte membrane
voltage was first held at V = -50 mV.
Then step changes to 11 different test potentials (V
) in 20-mV intervals between +50 and
-150 mV (each of a duration of 100 ms) were applied. The voltage
pulse to the test potential was followed by return of the membrane
voltage to the holding potential. Currents were averaged over three
sweeps and low pass-filtered at 500 Hz by an eight-pole Bessel filter.
Sugar-induced steady-state currents at different substrate
concentrations [S] were fitted to by nonlinear
regression analysis using Sigma Plot (Jandel, San Rafael, CA):
I represents the substrate (sugar or cation)-induced
current, I is the calculated maximal current, n is the coupling coefficient, and K
is
the substrate concentration for 50% I
.
Sugars and phenylglucosides were purchased from Sigma. Three-dimensional images of these were created by computational modeling based on the program Hyperchem 2.0 (Autodesk, Sausalito, CA) as described in (23) .
Multiple sequence alignments of the amino acid sequences were performed using the program PILEUP (Genetics Computer Group, Madison, WI).
Figure 1:
Na inward currents
generated by the chimeric protein. A, current records for the
chimera obtained 5 days post-cRNA injection in 100 mM NaCl, in
the absence (left panel) and presence (right panel)
of 20 mM
MDG. The oocyte membrane potential V
was held at -50 mV and stepped to
various test potentials V
between
+50 and -150 mV. Shown are the traces for voltage jumps to
+50, +30, -10, -110, and -150 mV. B, steady-state current-voltage (I-V)
relationships. Currents mediated by the chimera at 100 ms after
applying the voltage jump are presented as a function of the test
potential in the presence of 100 mM choline chloride, 100
mM NaCl, or 100 mM NaCl + 20 mM
MDG (left panel). The right panel shows the
net
MDG-induced Na
currents, after subtraction of
the steady-state currents recorded in 100 mM NaCl from these
induced by the addition of sugar substrate. C, steady-state I-V relationship of the inward currents induced by
100 µM phlorizin. The protocol is as described in B. The left panel shows the I-V curves in 100 mM choline chloride, 100 mM NaCl,
and after the addition of 100 µM phlorizin for the same
oocyte as presented in B. The right panel shows the I-V curve of the current induced by
phlorizin.
The sugar-induced current
is the difference in the steady-state currents in the presence and
absence of sugar. Addition of the saturating concentration of MDG
to the bath solution evoked an increase of the inward current of
-187 nA at -150 mV and completely eliminated the
pre-steady-state current (Fig. 1A, right
panel). The right panel of Fig. 1B shows
the
MDG-induced current, measured on another oocyte, as a function
of voltage. The current increased as the membrane potential increased
from -50 to -150 mV, but did not reach saturation at the
most negative voltage applied. The steady-state current at -150
mV was -89 nA.
When the choline chloride solution was replaced
by NaCl, the chimera generated an inward current in the absence of
sugar (Fig. 1B). This sugar-independent current, or
Na-leak, has also been observed for SGLT1 (24) and SGLT2 (18) . The sugar-independent
Na
current of the parent proteins can be estimated
either as the difference in the currents when external Na
is replaced by choline (Fig. 1B, left
panel) or as the current blocked by phlorizin in the absence of
sugar. Phlorizin has been shown to block the sugar-independent current
in SGLT1 (24, 14) and SGLT2(18) .
Surprisingly, when phlorizin was added to the 100 mM NaCl
buffer in the absence of sugar (on the same oocyte presented in Fig. 1B), there was an increase in inward current
instead of a block (Fig. 1C, left panel). The
current-voltage relationship of the current induced by 100 µM phlorizin is shown in the right panel of Fig. 1C and was similar in amplitude (-108 nA at
-150 mV) as well in its voltage dependence to the current induced
by 20 mM
MDG (Fig. 1B, right
panel).
For the chimera, the ratio between the
sugar-independent current (estimated by choline substitution as
described above) and the sugar (MDG)-mediated current was
considerably higher than observed for both SGLT1 and SGLT2. Both
parental transporters show a sugar-independent current (the
Na
- leak current) that is
10% of the
sugar-mediated current(15, 18) . In contrast, the
Na
-leak current mediated by the chimera was relatively
higher, 44% of the maximal current induced by 20 mM
MDG.
Similar values were obtained in four additional experiments.
To
confirm that the phlorizin- or MDG-induced currents were
accompanied by uptake of phlorizin or
MDG into the oocytes, we
measured the uptake of radiolabeled phlorizin or sugar in 100 mM NaCl or 100 mM choline chloride. Fig. 2shows that
oocytes expressing the chimera transported eight times more
[
C]
MDG and
[
H]phlorizin in 100 mM NaCl than
noninjected oocytes. No phlorizin transport could be measured in
SGLT1-cRNA injected oocytes, and 100 mM choline chloride did
not support any uptake of phlorizin into oocytes injected with chimeric
cRNA (not shown).
Figure 2:
Transport of radioactive tracer
substrates. Uptakes of 50 µM [C]
MDG or 5 µM [
H]phlorizin were performed in 100 mM NaCl 5-6 days post cRNA injection. Oocytes expressing the
chimera in [
C]
MDG transported: 58 ±
12 pmol/h/oocyte, whereas noninjected oocytes transported 8 ± 2
pmol/h/oocyte. Phlorizin transport could be measured only in oocytes
injected with chimeric cRNA (13 ± 1 pmol/h/oocyte), but not
SGLT1 cRNA (1.5 ± 0.5 pmol/h/oocyte) or noninjected oocytes (1.5
± 0.4 pmol/h/oocyte). Shown is the mean of four independent
experiments performed with noninjected oocytes or oocytes injected with
chimeric cRNA. The error bars are
S.E.
The current induced by MDG was strongly
dependent on membrane voltage and increased with more negative test
voltages. Fig. 3A shows that the current-voltage
relationship at 20 mM
MDG did not saturate in the voltage
range from +50 to -150 mV and reached values in different
oocytes between -80 and -300 nA (at -150 mV). To
obtain the apparent affinity constant for
MDG
(K
) in 100 mM NaCl, currents
induced by different sugar concentrations were fitted for each test
voltage to . The obtained K
of 0.25 mM was close to the value for the high affinity
transporter SGLT1 and voltage independent (Fig. 3B).
Dependent on the level of expression the maximal sugar-induced current
I
was between 90 and 250 nA (at
-150 mV).
Figure 3:
Steady-state kinetics of the
MDG-induced currents. To obtain the maximal induced current (I
) and the K
for
MDG for the chimera, the sugar-induced steady-state current at
various [
MDG] concentrations (in mM: 0.01,
0.02, 0.05, 0.20, 0.50, 1, 2, 5, and 20) were fitted to ,
while the external Na
concentration was fixed at 100
mM. Data for the high (SGLT1) and low affinity (SGLT2)
Na
/glucose cotransporters were obtained from Refs. 21
and 18, respectively. A, comparison of the current-voltage
(calculated I
/V
)
relationship of the
MDG-induced Na
inward
currents for the chimera, the high affinity and the low affinity
Na
/glucose cotransporters. To compare the three I/V curves, the data have been normalized to the
currents at -150 mV. B, comparison of the apparent K
for
MDG between the chimera, the high
affinity and the low affinity Na
/glucose
cotransporters.
To obtain the kinetic description of phlorizin
transport in 100 mM Na, the phlorizin-induced
currents were measured as a function of different external phlorizin
concentrations (0-100 µM). Similar to the I/V curve of the current induced by sugar (
MDG),
the I/V curve of the current induced by phlorizin did
not saturate in the voltage range from +50 mV to -150 mV (Fig. 1C, right panel). I
reached between -100 and -300 nA (at -150 mV)
and was comparable to the I
. The
apparent K
for phlorizin transport
(K
) in 100 mM NaCl obtained from
two experiments was 4.5 ± 0.8 µM and voltage
independent (not shown).
Figure 4:
Substrate specificity of the chimera. Each
of the substrates was added to the bath solution for 2 min after
the oocyte has been equilibrated in 100 mM NaCl. The exposure
to different substrates was followed by washes in 100 mM choline chloride prior to re-equilibration in NaCl. The oocyte
membrane potential was held at V
=
-50 mV. For comparison of data from different oocytes and
substrates, we expressed the steady-state substrate-induced current (at
-150 mV) as a ratio to the
MDG-induced current (at
-150 mV), where the D-glucose-induced current was taken
as 100%. All glucose analogues were tested at 20 mM and are
indicated by the diagonal hatch. The applied concentrations of
the different phenylglucosides were as follows:
- and
-PG and
arbutin, 10 mM;
-Naph-Glc, 2.5 mM; Pz and
deoxy-Pz, 0.1 mM; GPITC, 1 mM.
2-(Methylamino)isobutyrate (MeAIB) was tested at 2
mM, uridine at 20 mM, and their currents were
indicated by the black bars.
Hager et al.(26) showed that for the
high affinity rabbit SGLT1 the myo-inositol-induced current is
about 10% of the MDG-induced current and the apparent
K
is
500 mM. The currents
generated by the chimera in the presence of 20 mMmyo-inositol were higher than the myo-inositol-induced currents on SGLT1 and were 21 ± 7%
of the control currents, suggesting a higher chimera affinity for myo-inositol.
Fig. 5shows the dependence of the MDG-induced
Na
-current on external Na
concentration at -150 mV. The curve was drawn according to . The fit was poor as indicated by the large errors of the
parameters, in part due to small inward currents at low external
Na
concentrations: I
= -76 ± 12 nA, K
= 3.4 ± 1.8 mM, and n =
0.8 ± 0.4. Similar results were obtained in 3 additional
experiments. The apparent K
was independent
of membrane voltage in the range -150 and -70 mV. The mean
value of 3.3 ± 0.8 mM (n = 4, S.E.) at
-150 mV, indicated a remained high affinity for Na
at hyperpolarizing voltages. But in contrast to SGLT1 and SGLT2,
the Hill coefficient for Na
varied from 0.8 ±
0.4 (n = 4, S.E.) at -150 mV to 1.5 ± 0.6 (n = 4, S.E.) at -70 mV. For voltages more
positive than -70 mV, we were unable to obtain estimates of
K
and n for Na
because of the large parameter errors.
Figure 5:
Na activation of the
steady-state current. The Na
concentration of the bath
solution [Na
]
was
varied between 0 and 100 mM (0, 0.5, 1, 5, 10, 20, 50, 70, and
100), whereas [
MDG] was maintained at 20 mM.
Shown is the dependence of the steady-state current at -150 mV
with increasing [Na
]
.
The curve is drawn according to the fit.
To date, little information is available on domains or
residues responsible for ion and organic substrate recognition and
binding in the family of Na/glucose cotransporters. In
this study we show that functional chimeras can be obtained between
homologous members of this family. We expressed a protein which
according to the predicted topology for SGLT (19) consisted of
putative membrane helices 1-8 of SGLT2 and putative membrane
helices 9-14 of SGLT1.
The phenylglucoside phlorizin is the most
potent inhibitor of both SGLT1 and SGLT2, but it was transported by the
chimera with high affinity. Dependent on the pH, the conformation of
the aglucone of phlorizin (phloretin) varies between two tautomeric (keto-enol) forms. At pH 5.5, the absorption peak at 285 nm
corresponds to the keto form, and, at pH 8.4, the maximal
absorption at 320 nm is due to the enol form(31) .
Between pH 5.5 and 8.5, we observed that maximal transport of phlorizin
was at pH 5.5. This suggests that the chimera distinguishes between the
two forms and transports the keto better than the enolic form.
The keto form is always preferred whether it is in NaCl,
choline chloride, or at various pH values. The most effective
inhibition of sugar transport by phlorizin for SGLT1 is due also by the keto form. ()
In addition to phlorizin, other
phenylglycosides which have been shown to be inhibitors of sugar
transport in SGLT1 were transported by the chimera ( Fig. 4and Fig. 6A). Substituting the NCS group (in GPITC) for the
OH group in para-position of the phenyl ring of arbutin
converts this substrate for the high (23) and low affinity
cotransporters into an inhibitor. ()GPITC inhibits the
Na
leak current of SGLT1, maybe by covalently binding
to a lysine residue 0.8 nm away from the sugar binding
pocket(32) . In contrast, this phenylglucoside is a good
transport substrate for the chimera.
Figure 6:
Superimposition of predicted
three-dimensional structures of some phenylglucosides. The
three-dimensional images of phenylglucosides were obtained by
conformational search and energy minimizations as described in (23) . A, transported substrates by the chimera.
Presented are the -phenylglucosides phlorizin, GPITC, and the
-phenylglucoside (
-PG). The phenyl ring of
-phenylglucoside (
-PG) superimposes with the phenyl
ring of GPITC and is not explicitly indicated in the figure. B, transported substrates by the high-affinity SGLT1 isoforms.
Arbutin is transported by all three SGLT1 isoforms from rat, rabbit,
and human, whereas
-Naph-Glc is transported by the rat and human
isoforms, but not by rabbit
SGLT1(16) .
-Phenyl derivates of D-glucose are not transported by SGLT1(23) . Similar
to GPITC, the chimera accepted the
phenyl-
-D-glucopyranoside as well as the
phenyl-
-D-glucopyranoside (17% and 11% transport,
respectively).
The superimposed three-dimensional geometry of the
phenylglucoside substrates of the chimera (Fig. 6, upper
panel) indicated outer dimensions of 11 18
5
(Å). The transport pocket for substrate in the case of the high
affinity (SGLT1) human and rat isoforms is significantly smaller: 10
5
5 (Å), and the bulkiest known transported
substrate is
-Naph-Glc (Fig. 6B). The rabbit SGLT1
isoform does not transport this substrate(16) . Thus, we
conclude that the chimera provides a less restrictive selectivity,
compared to SGLT1 (comparable data are not yet available for SGLT2). In
view of the transport of phlorizin, deoxyphlorizin, and GPITC by the
chimera, its inability to transport
-Naph-Glc is probably caused
by the loss of specific interactions with the naphthyl moiety, rather
than by a steric hindrance due to the bulky aglucone. In addition,
since the chimera does not transport arbutin (see structural formulas
in Fig. 6B), we speculate that in this case there is a
lack of electrostatic interactions in the area surrounding the para-position of the phenyl ring.
Comparisons of the pre-steady-state (12) and the steady-state (16) kinetic parameters of the SGLT1s from rat, rabbit, and human have given initial ideas about residues that may account for the functional differences between these isoforms. Since the amino terminus (residues 1-27) and two hydrophilic loops located in the center (residues 229-271) and the carboxyl terminus (residues 548-644) involve most of the nonconserved polar residues between the three species, it has been proposed that they possibly contribute for charge movement and/or substrate specificity differences. The present study locates the sugar recognition domain distal to amino acid 380. Therefore, a comparison of the primary sequences distal to amino acids 380 of all cloned high affinity transporters from rat(11) , human(37) , rabbit(38) , and porcine(10) , with the corresponding sequence of the low affinity transporter from porcine(17, 18) , should specify individual amino acids that modulate sugar recognition (Fig. 7). According to the recently proposed topology for SGLT (19) and taking conservative substitution into account (K = R, S = T, D = E, Y = F = W, and I = V = L = M), our comparison localized differences in the loops between M10/M11, M12/M13, and M13/M14. In particular, in the loop M10/M11, two conserved serines (Ser-446, Ser-449) in the high affinity subfamily were substituted in SGLT2 by Val-446 and Asn-449; the conservative aspartic acid Asp-455 was substituted by a countercharge in SGLT2 (His-455), and, in the loop between M12/M13, two adjacent conservative residues Glu-514 and Pro-515 were both replaced in SGLT2 by a neutral alanine (Ala-514, Ala-515). The largest loop between M13/M14 contained most of the substitutions: a conservative serine (Ser-562) in the SGLT1s was replaced by an alanine (Ala-562) in SGLT2, and both conserved acidic residues (Glu-577) and aspartic acid (Asp-614) were missed in SGLT2. In the same loop, aspartic acid (Asp-580), isoleucine (Ile-581), and glutamine (Gln-582) were all replaced by positively charged residues in SGLT2: Lys-580, Arg-581, and His-582. A lysine (Lys-599) was converted into a threonine (Thr-599) in SGLT2, a methionine (Met-629) to a glutamine (Gln-629) and leucine (Leu-631) to an arginine (Arg-631). Any of these amino acids could contribute to the organic substrate binding. This easily could be tested by site-directed mutagenesis.
Figure 7: Amino acid sequence alignment of the SGLT family members. Shown are the sequences from the carboxyl-terminal halves of the high affinity cotransporters (SGLT1s) from porcine, human, rat, mouse, ovine, rabbit, and the low affinity cotransporter (SGLT2) from porcine. The dashes(- - -) represent identical residues and conservative substitutions (see text), and the shaded regions are putative membrane domains(19) . The residues in the SGLT1 family that are significantly different from residues in SGLT2 (dark regions) are depicted by bold letters.
The presented structure-functional analysis of a chimeric protein between two members of the SGLT family initiates a powerful strategy for further studies in localizing the structural determinants for cotransporter function.