"Structure-function relationship of loop 13 of Na+/glucose cotransporter SGLT1 investigated by atomic force microscopy and surface plasmon resonance spectroscopy"
, in Johannes Kepler Universität Linz, 5-2005
Structure-function relationship of loop 13 of Na+/glucose cotransporter SGLT1 investigated by atomic force microscopy and surface plasmon resonance spectroscopy
Sprache des Titels:
In order to obtain information on the role of a C-terminal large extramembranous peptide loop of the sodium/D-glucose cotransporter SGLT1 (loop 13) in the interaction of ligands and inhibitors with the transporter, loop 13 containing N- and C-terminal His6 [His tief 6]-tags was immobilised in a defined orientation on lipid layers. The interactions of the fixed peptide with several ligands were investigated using Atomic Force Microscopy (AFM) and Plasmon Surface Resonance Spectroscopy (BIACORE).
During BIACORE experiments using peptides in immobilised state, loop 13 interacted with PAN-3 antibody in a highly specific way. This interaction was facilitated by sodium.
Highly reproducible blocking effects of several sugars on PAN-3 binding to membrane-fixed loop 13 suggested interactions between the peptide and D-glucose resp. D-glucose analoga. Characteristic features of whole SGLT1 regarding glucose binding were detected such as the preference of [beta]-D-glucose compared to [alpha]-D-glucose and especially compared to L-glucose.
These results demonstrate the presence of a highly selective sugar binding site on loop 13 close to a part of the PAN-3 binding site.
In addition binding of the classical inhibitor of SGLT1, phlorizin, to immobilised loop 13 could be observed directly by BIACORE. Sodium and several sugars were shown not only to have an influence on PAN-3 binding to the fixed loop 13 but also on phlorizin interaction with this part of SGLT1.
For the intact SGLT1 these findings suggest that, in addition to the translocation sites for sugars and sodium located inside the C- and N-terminal transmembrane helices, binding and recognition sites are present on the surface of the transporter in the extramembranous loops.
Thus, cotransport of sugars and sodium by SGLT1 is a multi-step process involving a chain of binding and translocation events that achieve the high selectivity and energetic coupling of the substances.