The Human Sodium-Glucose Cotransporter (hSGLT1) Is a Disulfide-Bridged Homodimer with a Re-Entrant C-Terminal Loop

Na-coupled cotransporters are proteins that use the trans-membrane electrochemical gradient of Na to activate the transport of a second solute. The sodium-glucose cotransporter 1 (SGLT1) constitutes a well-studied prototype of this transport mechanism but essential molecular characteristics, namely its quaternary structure and the exact arrangement of the C-terminal transmembrane segments, are still debated. After expression in Xenopus oocytes, human SGLT1 molecules (hSGLT1) were labelled on an externally accessible cysteine residue with a thiol-reactive fluorophore (tetramethylrhodamine-C5-maleimide, TMR). Addition of dipicrylamine (DPA, a negatively-charged amphiphatic fluorescence "quencher") to the fluorescently-labelled oocytes is used to quench the fluorescence originating from hSGLT1 in a voltage-dependent manner. Using this arrangement with a cysteine residue introduced at position 624 in the loop between transmembrane segments 12 and 13, the voltage-dependent fluorescence signal clearly indicated that this portion of the 12-13 loop is located on the external side of the membrane. As the 12-13 loop begins on the intracellular side of the membrane, this suggests that the 12-13 loop is re-entrant. Using fluorescence resonance energy transfer (FRET), we observed that different hSGLT1 molecules are within molecular distances from each other suggesting a multimeric complex arrangement. In agreement with this conclusion, a western blot analysis showed that hSGLT1 migrates as either a monomer or a dimer in reducing and non-reducing conditions, respectively. A systematic mutational study of endogenous cysteine residues in hSGLT1 showed that a disulfide bridge is formed between the C355 residues of two neighbouring hSGLT1 molecules. It is concluded that, 1) hSGLT1 is expressed as a disulfide bridged homodimer via C355 and that 2) a portion of the intracellular 12-13 loop is re-entrant and readily accessible from the extracellular milieu.