Sugar binding to Na+/glucose cotransporters is determined by the carboxyl-terminal half of the protein.

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 K0.5 for sugar of approximately 2 mM, and a K0.5 for Na+ of approximately 1 mM. The high affinity SGLT1, found in the S3 segment, has a coupling ratio of 2, and K0.5 for sugar and Na+ of approximately 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 K0.5 for alpha-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 K0.5Na 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 alpha-phenyl derivatives of D-glucose as well as the inhibitors of sugar transport: phlorizin, deoxyphlorizin, and beta-D-glucopyranosylphenyl isothiocyanate are transported with high affinity (K0.5 for phlorizin was 5 microM). Thus, the pocket for organic substrate binding is increased from 10 x 5 x 5 (A) for SGLT1 to 11 x 18 x 5 (A) for the chimera.