Real Time Imaging of SGLT1 Location and Activity in Mammalian Cell Lines

Abstract

Real time imaging of SGLT1 location and activity in mammalian cell lines.In polarized epithelial cells with apical and basal membranes SGLT translocation to the apical surface is a complex and poorly understood process. We have engineered SGLT1 constructs linked to YFP or CFP to investigate transporter insertion into plasma membrane of COS, CHO and HEK cells. Activity was assessed in parallel, using the patch clamp technique, a FRET based glucose sensor and radioactive uptake. Insertion is both variable and highly regulated. In COS cells SGLT1 is constitutively inserted into the plasma membrane and active. In HEK cells SGLT1 is mainly in the endoplasmic reticulum, and its activity varies from cell to cell. In CHO cells SGLT1 is degraded, but localizes to mitochondria when co-expressed with EGFR (not endogenously present in WT CHO cells), or after incubation with the proteasome inhibitor III (MG-262, 1 μM). Incubation of HEK and CHO cells with the cholesterol inhibitor MβCD (15 mM) increases both insertion and activity, suggesting that SGLT1 internalization is lipid raft mediated. To test whether lipid raft mediated internalization involves caveolin, experiments were carried out in HEK cells co-expressing SGLT1 with caveolin WT, or the caveolin dominant negative P132L. Our data suggest that P132L may increase insertion of SGLT1 into the plasma membrane. Activity will have to be assessed to confirm this preliminary data. Finally, the lack of effect of the dominant negative dynamin (K44A) rules out a clathrin-dependent process and further supports lipid raft involvement. We propose that SGLT1 translocation to the apical surface involves a lipid raft-dependent route with stopover at the basal membrane. Accordingly, a decrease in membrane cholesterol content would cause accumulation of SGLT1 in the basal membrane and decrease insertion into the apical membrane.