The Na ؉ -dependent, low affinity glucose transporter SGLT2 cloned from pig kidney is 76% identical (at the amino acid level) to its high affinity homologue SGLT1. Using two-microelectrode voltage clamp, we have characterized the presteady-state and steady-state kinetics of SGLT2 expressed in Xenopus oocytes. The kinetic properties of the steady-state sugar-evoked currents as a function of external Na ؉ and ␣-methyl-D-glucopyranoside (␣MG) concentrations were consistent with an ordered, simultaneous transport model in which Na ؉ binds first. Na ؉ binding was voltage-dependent and saturated with hyperpolarizing voltages. Phlorizin was a potent inhibitor of the sugar-evoked currents (K i Pz Ϸ 10 M) and blocked an inward Na ؉ current in the absence of sugar. SGLT2 exhibited Na ؉ -dependent presteadystate currents with time constants 3-7 ms. Charge movements were described by Boltzmann relations with apparent valence Ϸ 1 and maximal charge transfer Ϸ 11 nC, and were reduced by the addition of sugar or phlorizin. The differences between SGLT1 and SGLT2 were that (i) the apparent affinity constant (K 0.5 ) for ␣MG (Ϸ3 mM) was an order of magnitude higher for SGLT2; (ii) SGLT2 excluded galactose, suggesting discrete sugar binding; (iii) K 0.5 for Na ؉ was lower in SGLT2; and (iv) the Hill coefficient for Na ؉ was 1 for SGLT2 but 2 for SGLT1. Simulations of the six-state kinetic model previously proposed for SGLT1 indicated that many of the kinetic properties observed in SGLT2 are expected by simply reducing the Na ؉ /glucose coupling from 2 to 1.In the proximal tubule of the kidney, reabsorption of filtered glucose at the apical membrane is mediated by one or more Na ϩ /glucose cotransporters. The low affinity glucose transporter (SGLT2) 1 (1), cloned from a pig kidney cell line (2), bears 76% identity at the amino acid level to its high affinity homologue SGLT1 (3), yet exhibits functional characteristics (1) that clearly distinguish it from SGLT1.In the present study we examined the mechanisms of Na ϩ / glucose cotransport via the pig SGLT2 transporter expressed in Xenopus oocytes. We determined the dependence of the steadystate kinetics on Na ϩ and ␣MG concentrations, and examined presteady-state charge movements associated with SGLT2. We have also extended our analysis of the substrate selectivity of SGLT2, its cation activation, and the effects of phlorizin upon both the uncoupled and the sugar-coupled Na ϩ pathways through the transporter. EXPERIMENTAL PROCEDURESStage V-VI oocytes from Xenopus laevis were injected with pSGLT2 cRNA as described previously (1). Electrophysiological experiments were performed using a two-microelectrode voltage clamp (1, 4, 5). Oocytes were superfused at 20 -22°C with experimental medium in which the Na ϩ concentration varied between 0 and 100 mM by equimolar replacement with choline or Li ϩ . Test solutions (additionally containing sugars and/or phlorizin) were always washed out by superfusing the oocyte with substrate-free, choline chloride medium (pH 7.5). Sugar-evoked currents (I...

The Na+ activation and substrate specificity of human, rabbit, and rat Na+-glucose cotransporter (SGLT-1) isoforms were characterized using the Xenopus oocyte expression system and the two-electrode voltageclamp method. We find that there are differences, major and minor, in both the kinetics and substrate specificities between these isoforms; the substrate concentration at half-maximal current (K0.5) for hexoses varies from 0.2 to > 40 mM, depending on the species and sugar; the affinity constant (Ki) for phlorizin, the classic competitive inhibitor of SGLT-1, varies lover two orders of magnitude (rat Ki = 0.03 microM vs. rabbit Ki = 1.4 microM); and some glucoside inhibitors of the rabbit isoform, p-nitrophenyl glucose and beta-naphthyl glucose, are transported by the human and rat transporters. Na+ activation is more sensitive to membrane potential in the human and rat isoforms compared with rabbit. The rabbit isoform has a higher apparent affinity for alpha-methylglucose and 3-O-methylglucose by a factor of two than either human or rat. These results can be quantitatively fitted by our six-state kinetic model of SGLT-1, providing insight into the processes involved in these changes. For example, the model predicts that Na+ binding (rate constant, k12) in human and rat SGLT-1 is similar but is fourfold larger than in rabbit, whereas sugar binding (k23) in rabbit and rat is similar but double the value in human SGLT-1. The differences in the primary amino acid sequences between these three homologous proteins must account for the kinetic and substrate specificity differences, and comparisons of the functional properties and amino acid sequences of SGLT-1 isoforms provide useful information about structure/function relationships.

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 ϳ300 -500 nA. The apparent affinity constants for sugar (␣-methyl-D-glucopyranoside; ␣MDG) (K 0.5 ␣MDG ) and sodium (K 0.5 Na ) at a membrane potential of ؊150 mV were 0.2 mM and 4 mM. The K 0.5 Na increased continuously with depolarizing potentials reaching 40 mM at ؊30 mV. K 0.5 ␣MDG 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 Ϸ D-galactose > 3-OMe-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 i Pz ) 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 ϳ1, a potential for 50% maximal charge transfer (V 0.5 ) of ؊43 mV, and a maximal charge (Q max ) of 9 nanocoulombs. The apparent turnover number for the rat SGLT1 was 30 s ؊1. 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 ϩ , the Na ϩ /glucose cotransporter (SGLT) 1 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 0.5 ␣MDG ϳ 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 0.5 ␣MDG ϳ2 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. MATERIALS AND METHODSThe pBluescript II SK plasmid containing the coding sequence for rat SGLT1 (4) was linearized with SalI and transcribed in vitro with T3 ...

To test the hypothesis that the C-terminal half of the Na ؉ /glucose cotransporter (SGLT1) contains the sugar permeation pathway, a cDNA construct (C 5 ) coding for rabbit SGLT1 amino acids 407-662, helices 10 -14, was expressed in Xenopus oocytes. Expression and function of C 5 was followed by Western blotting, electron microscopy, radioactive tracer, and electrophysiological methods. The C 5 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 C 5 -cRNA injection compared with noninjected oocytes. The diameters of the C 5 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). C 5 increased the ␣-methyl-D-glucopyranoside (␣MDG) uptake up to 20-fold above that of noninjected oocytes and showed an apparent K 0.5 ␣MDG of 50 mM and a turnover of ϳ660 s ؊1. Influx was independent of Na ؉ with transport characteristics similar to those of SGLT1 in the absence of Na2) inhibited by phloretin, K i PT ‫؍‬ ϳ500 M, and 3) insensitive to phlorizin. These results indicate that C 5 behaves as a specific low affinity glucose uniporter. Preliminary studies with three additional constructs, hC 5 (the human equivalent of C 5 ), hC 4 (human SGLT1 amino acids 407-648, helices 10 -13), and hN 13 (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 ϩ , H ϩ , and Li ϩ ) 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 ϩ -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 Cterminal 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 uniport...

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 0.5 for sugar of ϳ2 mM, and a K 0.5 for Na ؉ of ϳ1 mM. The high affinity SGLT1, found in the S3 segment, has a coupling ratio of 2, and K 0.5 for sugar and Na ؉ of ϳ0.2 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 steadystate kinetics were characterized by a two-electrode voltage-clamp. The K 0.5 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 0.5 Na 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 0.5 for phlorizin was 5 M). 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 (1-3). In the S1 and S2 segments of the renal convoluted tubule, reabsorption is mediated to 90% by a low affinity Na ϩ /glucose cotransporter (SGLT2) 1 with an apparent K 0.5 for sugar of 6 mM and a Na ϩ :glucose coupling ratio of 1. The reabsorption process is completed in the S3 segment by another Na ϩ /glucose cotransporter (SGLT1) with significantly higher apparent affinity for sugar (K 0.5 ϳ0.35 mM) than SGLT2 and a Na ϩ /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 ϩ /glucose transport activity (apparent K 0.5 D-GLC ϳ0.28 mM; Refs. 5 and 6) located at the apical surface (7). The presence of SGLT1 (2:1 Na ϩ /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 ϩ -dependent glucose transporter, the rat, human, and rabbit SGLT1s (11-16). All three isoforms show an apparent K 0.5 ␣MDG between 0.17 and 0.49 mM, an apparent K 0.5 Na of 2-7 mM, and a Hil...

To investigate the role of charged intramembrane residues in the function of the rabbit Na+lglucose cotransporter (rbSGLT1) we substituted arginine-427 (R427) by alanine in the putative domain M9 SGLT1. This residue is conserved in all the members of the SGLT1 family. The mutant protein (R427A) was expressed in Xenopus oocytes and, although Western blot analysis revealed that it was produced in amounts comparable to wildtype, no function was measured. Freeze-fracture analysis showed that R427A SGLT1 was not in the plasma membrane while immunocytochemical experiments localized the transporter to just beneath it. These results indicate that arginine-427 plays a critical role in SGLT1 trafficking to the plasma membrane.

Our goal was to identify pairs of charged residues in the membrane domains of the Na+/glucose cotransporter (SGLT1) that form salt bridges, to obtain information about packing of the transmembrane helices. The strategy was to neutralize Glu225, Asp273, Asp294, and Lys321 in helices 6-8, express the mutants in oocytes, measure [14C]-alphaMDG uptake, and then attempt to find second-site mutations of opposite charge that restored function. alphaMDG uptake by E225A was identical to that by SGLT1, whereas transport was reduced by over 90% for D273A, D294A, and K321A and was not restored in the double mutants D273A/K321A or D294A/K321A. This suggested that K321 did not form salt bridges with D273 or D294 and that E225 was not involved in salt-bridging. Neutralization of K321 dramatically changed the Na+ uniport and Na+/glucose cotransport kinetics. The maximum rate of uniport in K321A increased 3-5-fold with a decrease in the apparent affinity for Na+ (70 vs 3 mM) and no change in apparent H+ affinity (0.5 microM). The change in Na+ affinity caused a +50 mV shift in the charge/voltage (Q/V) and relaxation time constant (tau)/voltage curves in the presteady-state kinetics. The presteady-state kinetics in H+ remained unchanged. The lower Na+ affinity resulted also in a 200-fold increase in the apparent K0.5 for alphaMDG and phlorizin. Replacements of K321 with alanine, valine, glutamine, arginine, or glutamic acid residues changed the steady-state kinetics in a similar way. Therefore, we suggest that K321 determines, directly or indirectly, (i) the rate and selectivity of SGLT1 uniport activity and (ii) the apparent affinities of SGLT1 for Na+, and indirectly sugar in the cotransport mode.

A truncated human Na + /glucose cotransporter (C 5 , residues 407^664) was expressed and purified from Escherichia coli using a GST fusion vector and glutathione affinity chromatography. The truncated transporter (C 5 ) was cleaved from GST-C 5 by Factor Xa proteolysis and purified by gel filtration chromatography. Up to 1 mg of purified GST-C 5 was obtained from 1 l bacterial culture. Reconstitution of both GST-C 5 and C 5 proteins into lipid vesicles resulted in 2.5-fold higher initial uptake rates of [ 3 H]D-glucose into C 5 -proteoliposomes than into liposomes. Transport was stereospecific, saturable, and inhibited by phloretin. These properties are similar to those obtained for C 5 in Xenopus laevis oocytes, and provide additional evidence that the five C-terminal transmembrane helices in SGLT1 form the sugar translocation pathway.z 1999 Federation of European Biochemical Societies.