A biophysical glance at the outer surface of the membrane transporter SGLT1

Abstract

Proteins mediating the transport of solutes across the cell membrane control the intracellular conditions in which life can occur. Because of the particular arrangement of spanning a lipid bilayer and the many conformations required for their function, transport proteins pose significant obstacles for the investigation of their structure-function relation. Crystallographic studies, if available, define the transmembrane segments in a "frozen" state and do not provide information on the dynamics of the extramembranous loops, which are similarly evolutionary conserved and thus as functionally important as the other parts of the protein. The current review presents biophysical methods that can shed light on the dynamics of transporters in the membrane. The techniques that are presented in some detail are single-molecule recognition atomic force microscopy and tryptophan scanning, which can report on the positioning of the loops and on conformational changes at the outer surface. Studies on a variety of symporters are discussed, which use gradients of sodium or protons as energy source to translocate (mainly organic) solutes against their concentration gradients into or out of the cells. Primarily, investigations of the sodium-glucose cotransporter SGLT1 are used as examples for this biophysical approach to understand transporter function.

Forces and Dynamics of Glucose and Inhibitor Binding to Sodium Glucose Co-transporter SGLT1 Studied by Single Molecule Force Spectroscopy

“…4,5 This implies that the opening of the vestibule and the initial hydrophilic sugar binding cavity is rather temperatureindependent as it occurs at the surface of the transporter. Similar conclusions can be drawn from tryptophan fluorescence studies with purified hSGLT1 (57). With an increase of the temperature to 25°C, all binding probabilities increase similar to the transport activity and turnover rate; at a higher turnover rate probably more SGLT1s in the outward open conformation face the membrane surface per time unit; thus, more binding events can occur.…”

“…It thus seems improbable that the observed effects are due to the difference in length of the linker PEG chains of the two probes, ϳ170 Å for phlorizin and ϳ700 Å for thioglucose. Moreover, the hydrophilic cavity of the transporter, which leads to the translocation, is only 7 Å deep (57).…”

Role of N-glycosylation in cell surface expression and protection against proteolysis of the intestinal anion exchanger SLC26A3

“…Physiological relevance of N-glycosylation of SLC26A3 in the intestine. It is believed that the extracellular loop domains and common outward-facing transmembrane domains of some transporters are important for substrate recognition and functional activity (6,59,64). Proteolytic activity of the gastrointestinal lumen is high, mainly due to the presence of various digestive enzymes.…”

“…This result suggests that deglycosylated HA-SLC26A3 is still expressed at the plasma membrane, and N-glcosylation of HA-SLC26A3 was not absolutely necessary for Cl Ϫ /HCO 3 Ϫ exchange activity. It is thought that the extracellular domains of some transporters and channels are important for substrate recognition and functional activity (6,59,64). In addition, N-glycosylation is known to play a regulatory role of some functional properties (15,40,57).…”

“…However, physiological functions and relevance of N-glycosylation of extracellular loop domains of these transporters and channels are largely unexplored. It is thought that the extracellular domains of some transporters and channels are important for substrate recognition and functional activity (6,59,64). The extracellular domains of transporters and channels in the apical membrane of intestinal epithelial cells are exposed to many luminal digestive enzymes under physiological conditions.…”