Functional expression of tagged human Na+-glucose cotransporter in Xenopuslaevis oocytes

1. High-affinity, secondary active transport of glucose in the intestine an d kidney is mediated by an integral membrane protein named SGLT1 (sodium gl ucose cotransporter). Though basic properties of the transporter are now de fined, many questions regarding the structure-function relationship of the protein, its biosynthesis and targeting remain unanswered. In order to bett er address these questions, we produced a functional hSGLT1 protein (from h uman) containing a reporter tag. 2. Six constructs, made from three tags (myc, haemaglutinin and poly-His) i nserted at both the C- and N-terminal positions, were thus tested using the Xenopus oocyte expression system via electrophysiology and immunohistochem istry. Of these, only the hSGLT1 construct with the myc tag inserted at the N-terminal position proved to be of interest, all other constructs showing no or little transport activity. A systematic comparison of transport prop erties was therefore performed beta een the myc-tagged and the untagged hSG LT1 proteins. 3. On the basis of both steady-state (affinities for substrate (glucose) an d inhibitor (phlorizin) as well as expression levels) and presteady-state p arameters (transient currents) we conclude that the two proteins are functi onally indistinguishable, at least under these criteria. Immunological dete ction confirmed the appropriate targeting of the tagged protein to the plas ma membrane of the oocyte with the epitope located at the extracellular sid e. 4. The myc-tagged hSGLT1 was also successfully expressed in polarized MDCK cells. alpha-Methyl glucose uptake studies on transfected cells showed an e xclusive apical uptake pathway, thus indicating that the expressed protein was correctly targeted to the apical domain of the cell. 5. These comparative studies demonstrate that the myc epitope inserted at t he N-terminus of hSGLT1 produces a fully functional protein while other epi topes of similar size inserted at either end of the protein inactivated the final protein.