Mammalian Expi293 cells were grown and maintained in Expi293 Expression Medium at 37 °C and 8% CO2 under humidified conditions. Approximately 320 μg of the hSGLT2 plasmid and 160 μg of the MAP17 plasmid were premixed with 720 μl of FectoPRO in 60 ml of Opti-MEM for 10~20 min before transfection. Cells were transiently transfected with plasmids at a density of 2.0 × 10^6 cells ml^-1 using FectoPRO. For transfection, 60 ml of the mixture was added to 0.6 liters of the cell culture and incubated at 37 °C in the presence of 8% CO2 for 72 h before collection. The cells were collected via centrifugation (800g, 10 min, 4 °C) and stored at −80 °C before use. The detergent-solubilized proteins were analyzed via FSEC using an ACQUITY UPLC BEH450 SEC 2.5 µm column.

Sodium-glucose cotransporter 2 (SGLT2) is imporant in glucose reabsorption. SGLT2 inhibitors suppress renal glucose reabsorption, therefore reducing blood glucose levels in patients with type 2 diabetes. We and others have developed several SGLT2 inhibitors starting from phlorizin, a natural product. Using cryo-electron microscopy, we present the structures of human (h)SGLT2-MAP17 complexed with five natural or synthetic inhibitors. The four synthetic inhibitors (including canagliflozin) bind the transporter in the outward conformations, while phlorizin binds it in the inward conformation. The phlorizin-hSGLT2 interaction exhibits biphasic kinetics, suggesting that phlorizin alternately binds to the extracellular and intracellular sides. The Na+-bound outward-facing and unbound inward-open structures of hSGLT2-MAP17 suggest that the MAP17-associated bundle domain functions as a scaffold, with the hash domain rotating around the Na+-binding site. Thus, Na+ binding stabilizes the outward-facing conformation, and its release promotes state transition to inward-open conformation, exhibiting a role of Na+ in symport mechanism. These results provide structural evidence for the Na+-coupled alternating-access mechanism proposed for the transporter family.