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First published online 20 June 2006
doi: 10.1242/jcs.03035

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Ligands on the string: single-molecule AFM studies on the interaction of antibodies and substrates with the Na⁺-glucose co-transporter SGLT1 in living cells

¹ Institute for Biophysics, Johannes Kepler University of Linz, Altenbergerstr. 69, Linz, A-4040, Austria
² Department of Epithelial Cell Physiology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, Dortmund 44227, Germany

^* Author for correspondence (e-mail: peter.hinterdorfer{at}jku.at)

Accepted 3 May 2006

Atomic force microscopy (AFM) was used to probe topology, conformational changes and initial substratecarrier interactions of Na⁺-glucose co-transporter (SGLT1) in living cells on a single-molecule level. By scanning SGLT1-transfected Chinese hamster ovary (CHO) cells with AFM tips carrying an epitope-specific antibody directed against the extramembranous C-terminal loop 13, significant recognition events could be detected. Specificity was confirmed by the absence of events in nontransfected CHO cells and by the use of free antigen and free antibody superfusion. Thus, contrary to computer predictions on SGLT1 topology, loop 13 seems to be part of the extracellular surface of the transporter. Binding probability of the antibody decreased upon addition of phlorizin, a specific inhibitor of SGLT1, suggesting a considerable conformational change of loop 13 when the inhibitor occludes the sugar translocation pathway. Using an AFM tip carrying 1-thio-D-glucose, direct evidence could be obtained that in the presence of Na⁺ a sugarbinding site appears on the transporter surface. The binding site accepts the sugar residue of the glucoside phlorizin, free D-glucose, and D-galactose, but not free Lglucose and probably represents the first of several selectivity filters of the transporter. This work demonstrates the potential of AFM to study the presence and dynamics of plasma membrane transporters in intact cells on the single molecule level.

Key words: SGLT1, Na⁺-D-glucose co-transporter, Phlorizin, AFM, Force spectroscopy

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