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.
Atomic force microscopy (AFM) was used to probe topology, conformational changes and initial substrate-carrier 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 sugar-binding 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 L-glucose 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.
Puntheeranurak
Theeraporn
T
Institute for Biophysics, Johannes Kepler University of Linz, Altenbergerstrasse 69, A-4040 Linz, Austria.
Wildling
Linda
L
Gruber
Hermann J
HJ
Kinne
Rolf K H
RK
Hinterdorfer
Peter
P
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D001692:Biological Transport; D016466:CHO Cells; D002470:Cell Survival; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D005947:Glucose / Q000378:metabolism; D008024:Ligands; D018625:Microscopy, Atomic Force; D010695:Phlorhizin / Q000494:pharmacology; D011817:Rabbits; D012680:Sensitivity and Specificity; D012964:Sodium / Q000494:pharmacology; D051269:Sodium-Glucose Transporter 1 / Q000737:chemistry / Q000276:immunology* / Q000648:ultrastructure*; D013237:Stereoisomerism
D000906:Antibodies; D000939:Epitopes; D008024:Ligands; D051269:Sodium-Glucose Transporter 1; D012964:Sodium; D010695:Phlorhizin; D005947:Glucose