Five transmembrane helices form the sugar pathway through the Na+/glucose cotransporter.

To test the hypothesis that the C-terminal half of the Na+/glucose cotransporter (SGLT1) contains the sugar permeation pathway, a cDNA construct (C5) coding for rabbit SGLT1 amino acids 407-662, helices 10-14, was expressed in Xenopus oocytes. Expression and function of C5 was followed by Western blotting, electron microscopy, radioactive tracer, and electrophysiological methods. The C5 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 C5-cRNA injection compared with noninjected oocytes. The diameters of the C5 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). C5 increased the alpha-methyl-D-glucopyranoside (alphaMDG) uptake up to 20-fold above that of noninjected oocytes and showed an apparent K0.5alphaMDG of 50 mM and a turnover of approximately 660 s-1. Influx was independent of Na+ with transport characteristics similar to those of SGLT1 in the absence of Na+: 1) selective (alphaMDG > D-glucose > D-galactose >> L-glucose approximately D-mannose), 2) inhibited by phloretin, KiPT = approximately 500 microM, and 3) insensitive to phlorizin. These results indicate that C5 behaves as a specific low affinity glucose uniporter. Preliminary studies with three additional constructs, hC5 (the human equivalent of C5), hC4 (human SGLT1 amino acids 407-648, helices 10-13), and hN13 (amino acids 1-648, helices 1-13), further suggest that helices 10-13 form the sugar permeation pathway for SGLT1.