Sugar Binding to Na+/Glucose Cotransporters Is Determined by the Carboxyl-terminal Half of the Protein

Abstract

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...

Three Surface Subdomains Form the Vestibule of the Na+/Glucose Cotransporter SGLT1

“…Therefore, we hypothesized that this region in subdomain II either lies close to the sugar-binding site, which appears only in the presence of Na ϩ , or represents part of the initial binding pocket (vestibule), which subsequently transfers glucose to the translocation pathway. Several groups have proposed that the glucose translocation region lies between helices 10 -13 of SGLT1, however, with low affinity to D-glucose and lost sensitivity to phlorizin (10,13). In this study, we suggest that the residues between aa 339 and 356 located between helix 8 and 9 also contribute to efficient high affinity D-glucose transport.…”

“…Many researchers have attempted to localize a substrate translocation pathway in SGLT1. Studies on chimera proteins of SGLT1/SGLT2 and a truncated protein (residues 407-648) showed that residues from 381-662 of SGLT1 are important for sugar transport, however, with less sugar specificity and lower sensitivity to the competitive inhibitor phlorizin (10,13). Also residues 457, 468, and 499 have been shown to play an important role in controlling the sugar binding of SGLT1 (14,15).…”

Molecular Analysis of the SGLT2 Gene in Patients with Renal Glucosuria

“…If transferred to the brush border membrane at all, then the protein predicted by this mutation would have only the seven N-terminally located transmembranous domains, whereas domains 8 to 14 would be missing. Such a mutation has been shown to result in a nonfunctional glucose transporter because it lacks the transmembranous domains 10 to 13 that have been demonstrated to be essential for sugar binding and sugar translocation (15,19,20). It is interesting, however, that one patient (patient 06-1) homozygous for a truncating mutation showed a considerably lower glucose excretion, suggesting some glucose reabsorption by an unknown alternative mechanism.…”

Replacement of Ala-166 with Cysteine in the High Affinity Rabbit Sodium/Glucose Transporter Alters Transport Kinetics and Allows Methanethiosulfonate Ethylamine to Inhibit Transporter Function

“…A set of glycosylation mutants has yielded information on topology, and a chimera of the high affinity and low affinity isoforms has suggested that sugar binding is determined by the C-terminal half of the protein (8). From comparisons of SGLT1 homologues cloned from different species, certain residues have been hypothesized to be important in determining the kinetic differences between these isoforms (9).…”