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Volume 272, Number 33, Issue of August 15, 1997 pp. 20324-20327
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

COMMUNICATION:
Five Transmembrane Helices Form the Sugar Pathway through the Na⁺/Glucose Cotransporter

(Received for publication, April 29, 1997, and in revised form, June 10, 1997)

From the Departments of Physiology and ^§ Neurobiology, UCLA Medical Center, Los Angeles, California 90095-1751

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 -methyl-D-glucopyranoside (MDG) uptake up to 20-fold above that of noninjected oocytes and showed an apparent K_0.5^MDG of 50 mM and a turnover of ~660 s¹. Influx was independent of Na⁺ with transport characteristics similar to those of SGLT1 in the absence of Na⁺: 1) selective (MDG > 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.

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