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.
Panayotova-Heiermann
M
M
Department of Physiology, UCLA School of Medicine, Los Angeles, California 90095-1751, USA.
Loo
D D
DD
Kong
C T
CT
Lever
J E
JE
Wright
E M
EM
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D015640:Ion Channel Gating; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000378:metabolism*; D008757:Methylglucosides / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009865:Oocytes; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D051269:Sodium-Glucose Transporter 1; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013552:Swine; D014982:Xenopus laevis
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D051269:Sodium-Glucose Transporter 1; C027020:methylglucoside; D012964:Sodium; D005947:Glucose