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

Abstract

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 α-methyl-D- glucopyranoside (αMDG) uptake up to 20-fold above that of noninjected oocytes and showed an apparent K0.5/(αMDG) of 50 mM and a turnover of ~660 s-1. Influx was independent of Na+ with transport characteristics similar to those of SGLT1 in the absence of Na+:1) selective (αMDG > D- glucose > D-galactose >> L-glucose ≃ D-mannose), 2) inhibited by phloretin, K(i)/(PT) = ~500 μM, 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.