'Active' Sugar Transport In Eukaryotes

Sugar transporters in prokaryotes and eukaryotes belong to a large family of membrane proteins containing 12 transmembrane alpha-helices. They are divided into two classes: one facilitative (uniporters) and the other concentrative (cotransporters or symporters). The concentrative transporters are energised by either H+ or Na+ gradients, which are generated and maintained by ion pumps. The facilitative and H+-driven sugar transporters belong to a gene family with a distinctive secondary structure profile. The Na+-driven transporters belong to a separate, small gene family with no homology at either the primary or secondary structural levels. It is likely that the Na+-and H+-driven sugar cotransporters share common transport mechanisms. To explore these mechanisms, we have expressed cloned eukaryote Na+/sugar cotransporters (SGLT) in Xenopus laevis oocytes and measured the kinetics of sugar transport using two-electrode voltage-clamp techniques. For SGLT1, we have developed a six-state ordered model that accounts for the experimental data. To test the model we have carried out the following experiments. (i) We measured pre-steady-state kinetics of SGLT1 using voltage-jump techniques. In the absence of sugar, SGLT1 exhibits transient carrier currents that reflect voltage-dependent conformational changes of the protein. Time constants for the carrier currents give estimates of rate constants for the conformational changes, and the charge movements, integrals of the transient currents, give estimates of the number and valence of SGLT1 proteins in the plasma membrane. Ultrastructural studies have confirmed these estimates of SGLT1 density. (ii) We have perturbed the kinetics of the cotransporter by site-directed mutagenesis of selected residues. For example, we have identified a charged residue which dramatically changes the kinetics of charge transfer. (iii) We have examined the kinetics of sugar and Na+ analogs. The Vmax of sugar transport decreases dramatically with bulky phenyl glucosides and increases when H+ replaces Na+. These results permit us to extend and refine our model for transport. The model has been useful in the analysis of mutant SGLT1 proteins: in the case of a D176A mutant, the primary effect is to alter rates of conformational changes of the unloaded protein, and with the glucose/galactose malabsorption syndrome mutant D28N SGLT1, the mutation disrupts the delivery of SGLT1 glycosylated protein from the endoplasmic reticulum to the plasma membrane.