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...

The porcine kidney epithelial cell line LLC-PK1 expresses a sodium-coupled glucose cotransporter (SGLT1) together with other differentiation markers of renal proximal tubule such as trehalase and gamma-glutamyltranspeptidase. Expression is regulated by cell density and exogenous differentiation inducers such as hexamethylene bisacetamide (HMBA). Northern blot and PCR analysis of clonal cell populations indicated SGLT1 mRNA was not detectable in subconfluent cultures, but 2.2 and 3.9 kb SGLT1 mRNA species appeared after cell confluence, accompanying expression of the transport activity. SGLT1 mRNA levels were significantly increased after treatment of confluent cultures with HMBA, paralleling increases in the transport activity and immunodetectable 75 kD cotransporter subunit. SGLT1 mRNA was also increased after treatment of cultures with the cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), an inducer of Na+/glucose cotransport activity. The 3.9 kb SGLT1 transcript showed the largest increase after either HMBA or IBMX treatment. HMBA treatment also resulted in increased mRNA levels of two other differentiation markers--trehalase and gamma-glutamyltranspeptidase. By contrast, trehalase and gamma-glutamyltranspeptidase mRNA levels were not increased by IBMX. Regulation of Na+/glucose symporter expression by either cell density, cyclic AMP elevation, or differentiation inducer treatment occurs, at least in part, at the level of SGLT1 mRNA and can be dissociated from regulation of other differentiation markers.

A full-length Na'/glucose cotransporter cDNA (SGLTl) from rabbit intestine was subcloned into the pMAMneo mammalian expression vector and transfected by Ca'+ precipitation into Madin-Darby canine kidney (MDCK) cells. Stable MDCK transfectants isolated after clonal isolation and selection in G418 exhibited dexamethasone-inducible Na'/glucose cotransport activity under regulation of the MMTV promoter of the vector. Transfectants expressed the recombinant 75 kDa Na'lglucose cotransporter subunit as shown by Western blot, and SGLTl mRNA as shown by Northern blot, but these were undetectable in untransfected MDCK cells. Over 93% of total recombinant transport activity was targeted to the apical membrane. This indicates that the primary amino acid sequence of SGLTl contains the information necessary to target this transporter to the apical membrane.

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