(Received for publication, April 29, 1997, and in revised form, June 10, 1997)
From the Departments of Physiology and § Neurobiology, UCLA Medical Center, Los Angeles, California 90095-1751
To test the hypothesis that the C-terminal half
of the Na+/glucose cotransporter (SGLT1) contains the
sugar permeation pathway, a cDNA construct (C5) coding
for rabbit SGLT1 amino acids 407-662, helices 10-14, was expressed in
Xenopus oocytes. Expression and function of C5
was followed by Western blotting, electron microscopy, radioactive
tracer, and electrophysiological methods. The C5 protein was synthesized in 20-fold higher levels than SGLT1. The particle density in the protoplasmic face of the oocyte plasma membrane increased 2-fold after C5-cRNA injection compared with
noninjected oocytes. The diameters of the C5 particles were
heterogeneous (4.8 ± 0.3, 7.1 ± 1.2, and 10.3 ± 0.8 nm) in contrast to the endogenous particles (7.6 ± 1.2 nm).
C5 increased the -methyl-D-glucopyranoside (
MDG) uptake up to 20-fold above that of noninjected oocytes and
showed an apparent K0.5
MDG
of 50 mM and a turnover of ~660 s
1.
Influx was independent of Na+ with transport
characteristics similar to those of SGLT1 in the absence of
Na+: 1) selective (
MDG > D-glucose > D-galactose
L-glucose
D-mannose), 2) inhibited by
phloretin, KiPT = ~500 µM, and 3) insensitive to phlorizin. These
results indicate that C5 behaves as a specific low affinity
glucose uniporter. Preliminary studies with three additional
constructs, hC5 (the human equivalent of C5),
hC4 (human SGLT1 amino acids 407-648, helices 10-13), and
hN13 (amino acids 1-648, helices 1-13), further suggest
that helices 10-13 form the sugar permeation pathway for SGLT1.
Cotransporters are a major class of membrane proteins that use cation (Na+, H+, and Li+) gradients to drive the uphill transport of solutes and water into cells. Although many of these proteins have been cloned and subjected to structure/function analyses, it has been problematic to draw definitive conclusions about their structure or to identify ligand binding sites and permeation pathways. This is largely due to the difficulty in obtaining structural information at the atomic level. Today, most of our understanding comes from indirect methods such as the comparison of the primary and secondary structure and function of homologous proteins, and functional analysis of chimeric proteins. Here we have studied the functional properties of protein truncations to probe the pathway for sugar transport through the Na+-dependent glucose transporter, SGLT1 (1, 2).
SGLT1 is a protein of 662 residues that contains 14 transmembrane helices (3). The structure and function of cloned SGLT1 isoforms and homologs, such as SMIT1 and SNST1, have been compared (4, 5), and the major differences were found to be concentrated toward the C termini. Functional analysis of a SGLT2-SGLT1 chimera (6) suggested that a C-terminal fraction of SGLT1 (residues 410-662, helices 10-14) determines both the sugar affinity and selectivity. To test if the terminal five helices of SGLT1 actually form the pathway for sugar transport, we expressed and functionally analyzed the truncated protein (residues 407-662) in Xenopus oocytes. It behaves as a specific low affinity glucose uniporter with properties comparable with those of native SGLT1 in the absence of Na+.
The recombinant construct of
plasmid pGEM3Zf+ containing the full-length coding sequence of rabbit
SGLT1 (7) was digested with NcoI to remove a ~1220-base
pair DNA fragment. The fragment encodes amino acids 1-406, which cover
the nine N-terminal -helices of the SGLT1 transporter in the
secondary structure model. Recirculation of the remaining large
fragment (C5 construct) recreated the methionine start
codon in frame. The truncated protein consisted of amino acids
407-662, which include transmembrane helices 10-14. A human equivalent of C5 (hC5) was made by the same
strategy. A second human SGLT1 truncation lacking only the 14th
transmembrane span (hN13) was made by mutagenic
incorporation of a stop codon after Asn-648, which thereby deleted the
16 C-terminal hydrophobic residues, and the mutated domain was verified
by sequencing. A BstXI fragment of hN13 bearing
the new stop codon was swapped into construct hC5 to
produce a third truncated human construct, hC4, amino acids 407-648 (see Fig. 1). Template DNA was linearized with
EcoRI (C5) or XbaI (hC5,
hC4, hN13) and used for in
vitro transcription and capping with SP6 RNA polymerase
(MEGAscript transcription kit, Ambion, Austin, TX).
Oocyte Preparation, Western Blot Analysis, Freeze Fracture, and Functional Assays
Mature Xenopus laevis oocytes were
manipulated as described (7). All experiments were repeated 2-3 times
on oocytes from different donors, usually 3-5 days after cRNA
injection. Solubilization of the oocytes for Western blot analysis,
SDS-polyacrylamide gel electrophoresis, and immunoblotting were done as
described (8), using antibody 8821 (9) at a 1:3000 dilution.
Immunoreactive proteins were detected by chemiluminescence
(SuperSignalTM Kit, Pierce) and exposure on HyperfilmTM ECLTM
(Amersham Life Science Inc.). Freeze fracture of oocyte plasma
membranes and sugar influxes were performed by standard procedures (10,
11). 7-10 control or cRNA-injected oocytes were incubated in the
presence of
[14C]MDG1
(specific activity, 293 mCi/mmol; Amersham Life Science Inc.) alone or
in the presence of different nonradioactive substrates/inhibitors. Sugars, phlorizin, and phloretin were from Sigma. Two-electrode voltage
clamp measurements were performed as described (7).
The truncated rabbit SGLT1
transporter (C5) includes the last five transmembrane
domains of wild-type SGLT1 (Fig.
1). Western blot analysis (Fig.
2A) showed that oocyte
expression of C5 was 20-fold higher than that of SGLT1.
C5 appeared as a major band at ~29 kDa and a minor band
at ~50 kDa. Full-length SGLT1 appeared as two bands; the lower band
at ~55 kDa corresponds to the core glycosylated protein, and the
higher band corresponds to the complex glycosylated protein. All
signals were completely blocked with the antigenic peptide.
Freeze fracture and morphometric techniques were used to quantify the
proteins in the plasma membranes of C5-expressing oocytes and noninjected controls. Fractured plasma membranes expose
complementary protoplasmic and external faces. Intrinsic membrane
proteins appeared as particles and complementary pits in the fracture
faces. We found that the density of particles in the protoplasmic face
of C5-expressing oocytes (726 ± 292 µm2) was twice as high as in noninjected oocytes
(353 ± 54 µm
2, Fig. 2B). In contrast,
particle density in the plasma membrane of oocytes expressing SGLT1 was
~5000 µm
2 (10). Electron micrographs (Fig.
2B) showed heterogeneity in the size of C5
intramembrane particles: analysis of 916 particles revealed three
populations: one with a diameter of 7.1 ± 1.2 nm (mean ± S.D.), representing 90% of all C5 particles, a second with
a diameter of 4.8 ± 0.3 nm (2%), and a third with a diameter of
10.3 ± 0.8 nm (8%). In contrast, noninjected oocytes showed a
homogeneous distribution with diameter of 7.6 ± 1.2 nm
(n = 875).
Can the
truncated protein function as an active sugar transporter? Electrical
properties of C5 expressed in oocytes were examined by the
two-electrode voltage clamp technique (7). In experiments over several
months on C5-expressing oocytes from different donors, none
of the following currents characteristic of full-length SGLT1 (12) were
observed: 1) transient currents in the voltage range 150 to +50 mV;
2) phlorizin-sensitive "leak" currents in the absence of sugar; or
3) Na+-specific sugar-induced currents, even at high (100 mM) sugar concentrations. The sugar uptake in 50 µM [14C]
MDG mediated by C5
was Na+-independent; there was no difference in transport
between C5-expressing oocytes incubated for different time
periods in 100 mM Na+ or choline chloride.
Therefore all following experiments, unless indicated, were in 100 mM choline chloride. To estimate the initial rates of
MDG transport, noninjected and C5-expressing oocytes were incubated for different times in 100 mM choline
chloride (Fig. 3A). Whereas
the 50 µM [14C]
MDG sugar uptake by
noninjected oocytes increased linearly with time (3 ± 1 pmol/oocyte/h), C5 uptake increased linearly for 5-10 min,
plateaued in 15-20 min, and amounted to 27 ± 3 pmol/oocyte after
1 h. Therefore, sugar uptakes of C5 were measured
after 7 min in subsequent experiments. C5 transported
15 ± 1.4 pmol/oocyte in choline chloride and 13 ± 1.6 pmol/oocyte in Na+, where control uptakes were 1.7 ± 0.1 pmol/oocyte in choline and 2.4 ± 0.1 pmol/oocyte in
Na+. In general, C5 initial uptake rates in
choline were 10-20-fold greater than rates in noninjected oocytes
(Figs. 3, C and D, and 4).
The C5-mediated influx of 50 µM
[14C[MDG was reduced 50% by the addition of 50 mM
MDG (Fig. 3C), suggesting an apparent
Km
MDG of
about 50 mM. The maximal velocity
(Vmax) for C5 was estimated from the
net initial rates (v) of sugar uptake in the presence of 50 mM cold substrate ([S]). At any substrate concentration the initial velocity is related to Vmax by the
equation: v/Vmax = [S]/(Km + [S]). The calculated
Vmax was ~1500 pmol/oocyte/min. Knowing the
number of C5 particles in the plasma membrane and the
maximal velocity for
MDG, we estimated the C5 turnover
number (Vmax/number of particles). The number of
C5 plasma membrane particles is approximately the product
of the oocyte surface area (6 × 107
µm2; Ref. 10) and the C5 particle density
(375 µm
2; Fig. 2B). The difference in the
particle density between C5-injected oocytes and
noninjected oocytes (legend of Fig. 2B) was ~373
µm
2. Therefore the C5 turnover number is
~660 s
1 (Vmax/number of
particles = 1500 × 10
12 × 6.02 × 1023/6 × 107 × 375/oocyte × 60 s/min).
D-galactose reduced the uptake by 20% at 100 mM and by 90% at 200 mM (not shown). 100 mM D-glucose inhibited the
C5-mediated uptake by 44%, whereas L-glucose
and D-mannose were without effect (Fig. 3D). The
apparent affinity of C5 for D-glucose is
therefore about 100 mM. The SGLT1-mediated uptake in the
absence of Na+ was blocked by 100 mM
MDG and D-glucose and to a lesser extent by
L-glucose and D-mannose (Fig.
3B).
Phlorizin is a potent high affinity competitive
inhibitor of Na+-dependent sugar transport by
SGLT1 (KiPZ = ~0.03-1.4 µM; Ref. 5). The MDG uptake mediated by
rbSGLT1 in the absence of Na+ was unaffected by 100 µM phlorizin but was inhibited 47 and 60% by 500 and
1000 µM phlorizin, respectively (Fig.
4A). The apparent KiPZ for SGLT1
in the absence of Na+ is therefore between 500 and 1000 µM. Sugar transport by C5 was unaffected by phlorizin (Fig. 4A). Phloretin, the aglycone
of phlorizin, had a strong inhibitory effect on sugar uptake mediated by C5 and by SGLT1 in the absence of Na+ (Fig.
4B). In both cases the apparent phloretin
Ki was ~500 µM.
To further define the pathway for sugar transport through SGLT1, three
additional truncations were made: 1) hC5, the human counterpart of rabbit C5; 2) hN13, which lacks
the 16 C-terminal hydrophobic residues comprising most of the 14th
span; and 3) hC4, the protein sequence common to both
hN13 and hC5 (spans 10-13). Both
hC5 and hN13 transported amounts of MDG
similar to C5: 38 ± 1 pmol/oocyte/h and 16 ± 2 pmol/oocyte/h, respectively (incubation was done for 1 h in 100 mM NaCl). Noninjected oocytes in these experiments
transported 3-4 pmol/oocyte/h, so the hC5- and
hN13-mediated uptakes were 5-8-fold above controls. The
hC4 uptake (4.7 ± 0.1 pmol/oocyte/h) was 30% above
the noninjected level of transport (3.4 ± 0.1 pmol/oocyte/h,
p < 10
4).
Expression of the C5 protein in Xenopus
oocytes resulted in a 20-fold increase in the rate of
Na+-independent glucose uptake; the half-time of sugar
equilibration with the cell was 5-7 min for C5 and 180 min
for control oocytes (Fig. 3A), and the initial rate of sugar
uptake was 10-20-fold greater than in controls (Figs. 3, C
and D, and 4). Sugar uptake by C5 was
independent of the cation species (Na+ or choline) and was
equilibrative, i.e. the steady-state uptake, 20 pmol/oocyte,
was that expected for the equilibration of 50 µM sugar
with an intracellular space of 0.5 µl. The apparent affinity of
C5 for MDG
(Km
MDG) was
~50 mM), and sugar transport was similar to the uptake
mediated by SGLT1 in the absence of Na+. This includes: 1)
the higher initial rate of sugar uptake than in control oocytes (Figs.
3 and 4); 2) the sugar specificity,
MDG > D-glucose > D-galactose
L-glucose
D-mannose (Fig. 3); 3) the
low sensitivity to phlorizin (Fig. 4), which is not unexpected as
phlorizin inhibition of SGLT1 is Na+-dependent;
and 4) the similar sensitivity to phloretin,
KiPT ~500
µM (Fig. 4). Because the facilitated sugar transport
mediated by the GLUT family members also shows sensitivity to phloretin (13), it is possible that the GLUT family members and C5
interact by a common mechanism with the sugar substrate. Our results
suggest that the C5 truncated protein retains sufficient
tertiary structure to transport sugar in a fashion similar to the
wild-type protein in the absence of Na+
consistent with
the previous report (6) that affinity and substrate specificity of
Na+/sugar cotransport are determined by the C-terminal half
of the protein. Although it is perhaps surprising that the
truncated protein retains function, it should be noted that a truncated lactose permease (helices 7-12) mediates downhill lactose
transport (14), suggesting the localization of essential structures for high affinity binding and substrate translocation within the last six
helices of lac permease.
Injection of C5 cRNA into oocytes resulted in high levels
of accumulation (10-20 times higher than SGLT1) of a protein with the
expected molecular mass of ~30 kDa (Fig. 2A). This higher synthesis may be due to a prolonged half-life of the cRNA or changed secondary structures of the cRNA favoring easier initiation of C5 translation, or it could simply be a reflection of a
more effective transfer to nitrocellulose of the smaller C5
protein. Freeze fracture electron microscopy of the plasma membrane
(Fig. 2B) showed that only a small fraction of the protein
was inserted into the plasma membrane and that the density of the
protoplasmic face intramembrane particles in C5 expressing
oocytes was about twice that of noninjected oocytes. The lower amount
of C5 molecules in the plasma membrane is probably due to
inefficiency of C5 trafficking to the plasma membrane (8).
The estimated Vmax for C5 was 1500 pmol/oocyte/min, and the turnover of MDG by C5 was 660 molecules/s/particle. In contrast, the turnover of wild-type SGLT1 is
30-60 s
1 (5), an order of magnitude lower. This lower
turnover of Na+-driven sugar transport by SGLT1 presumably
reflects constraints imposed on the conformational changes in the
C5 domain, which normally accompany sugar transit through
intact SGLT1. These constraints are likely the natural consequences of
the coupling of sugar transport with Na+ transport.
Interestingly, the C5 turnover number is in the range of
the turnover of facilitated glucose carrier proteins (GLUTs), like the
human erythrocyte glucose transporter (330-660 s
1; Ref.
15). Although C5 functions as a facilitated sugar
transporter, it is not clear if the functional protein is monomeric or
oligomeric. Our freeze fracture electron microscopic analysis of
C5 in the oocyte plasma membrane suggests that the protein
exists as three populations of intramembrane particles with diameters
of 4.8, 7.1, and 10.3 nm. The smallest particle is probably a
C5 monomer, and the larger particles are probably
oligomers.
Analysis of glucose/galactose malabsorption SGLT1 mutants provides additional evidence that sugar affinity and transport are determined by the last five C-terminal helices of SGLT1. In two naturally occurring mutations in patients with glucose/galactose malabsorption, sugar transport is compromised in the absence of major effects on Na+ binding. In one, R499H in helix 12, the sugar affinity was markedly reduced (16); in the other, Q457R in helix 11, sugar binding was reduced by an order of magnitude, and there was no sugar translocation (17). Moreover, mutations in the N-terminal half of SGLT1, e.g. K321A in helix 7, dramatically decreased the apparent Na+ affinity without changes in sugar affinity (18). These results, together with our observations that C5, unlike SGLT1 (12), does not exhibit the Na+ leak pathway or voltage-induced fast current transients, may be taken as evidence that Na+ binding and translocation occur through the N-terminal half of the protein. Previous energy transfer measurements between extrinsic fluorescent probes covalently attached to the putative Na+ and sugar binding sites of the cotransporter indicated that the distance between the active sites was 35 Å (19).
Our conclusion is that five transmembrane helices (10-14) of SGLT1 retain sufficient tertiary structure to transport sugar downhill in a stereospecific, selective, phloretin-sensitive manner. Given that the 14th helix is absent from a number of functional SGLT family members (3, 20), e.g. the Na+/iodide and H+/proline cotransporters, and that partial function was retained by the human SGLT1 when the 14th helix was deleted (hN13), the present study suggests that the critical requirement for sugar transport pathway perhaps is formed by just four helices (10-13). The N-terminal region of SGLT1 (helices 1-9) may be required to couple Na+ and sugar transport, and studies are now in progress to test this hypothesis.
We thank Mike Kreman for carrying out the freeze fracture electron microscopy of oocyte plasma membranes, Manuela Contreras and Jason Lam for assistance with oocytes and mutagenesis, and Don Loo for critical suggestions.