5 TRANSMEMBRANE HELICES FORM THE SUGAR PATHWAY THROUGH THE NA+ GLUCOSE COTRANSPORTER/

To test the hypothesis that the C-terminal half of the Na+/glucose cot ransporter (SGLT1) contains the sugar permeation pathway, a cDNA const ruct (C-5) coding for rabbit SGLT1 amino acids 407-662, helices 10-14, was expressed in Xenopus oocytes, Expression and function of C-5 foll owed by Western blotting, electron microscopy, radioactive tracer, and electrophysiological methods. The C-5 protein was synthesized in 20-f old higher levels than SGLT1. The particle density in the protoplasmic face of the oocyte plasma membrane increased C-5-cRNA injection compa red with noninjected oocytes. The diameters of the C-5 particles were heterogeneous (4.8 +/- 0.3, 7.1 +/- 1.2, and 10.3 +/- 0.8 nm) in contr ast to the endogenous particles (7.6 +/- 1.2 nm), C-5 increased the al pha-methyl-D-glucopyranoside (alpha MDG) uptake up to 20-fold above th at of noninjected oocytes and showed an apparent K-0.5(alpha MDG) Of 5 0 mM and a turnover of similar to 660 s(-1). Influx was independent of Na+ with transport characteristics similar to those of SGLT1 in the a bsence of Na+:1) selective (alpha MDG > D-glucose > D-galactose much g reater than L-glucose approximate to D-mannose), 2) inhibited by phlor etin, K-i(PT) = similar to 500 mu M, and 3) insensitive to phlorizin, These results indicate that C-5 behaves as a specific low affinity glu cose uniporter. Preliminary studies with three additional constructs, hC(5) (the human equivalent of C-5), hC(4) (human SGLT1 amino acids 40 7-648, helices 10-13), and hN(13) (amino acids 1-648, helices 1-13), f urther suggest that helices 10-13 form the sugar permeation pathway fo r SGLT1.