Gruber, H.

Hinterdorfer, P.

Kinne, R.

Puntheeranurak, T.

English

MPG.PuRe

Journal of the Korean Physical Society, Vol. 52, No. 5, May 2008, pp. 13361340Single-Molecule AFM Studies of Substrate Transport by Usingthe Sodium-Glucose Cotransporter SGLT1Theeraporn PuntheeranurakDepartment of Biology, Faculty of Science, Mahidol University and NANOTECCenter of Excellence at Mahidol University, Bangkok, 10400, ThailandRolf K. H. KinneDepartment of Epithelial Cell Physiology, Max Planck Institute of Molecular Physiology, Dortmund, 44227, GermanyHermann J. Gruber and Peter HinterdorferInstitute for Biophysics, Johannes Kepler University of Linz, Linz, A-4040, Austria(Received 15 July 2007)In an apical membrane of epithelial cells from the small intestine and the kidney, the high-anityNa+/D-glucose cotransporter type 1 (SGLT1) plays a crucial role in intestinal glucose absorp-tion and in renal glucose reabsorption. Here, the over-expression of rabbit SGLT1 in rbSGLT1-transfected Chinese hamster ovary (CHO) cells was rst characterized using the immuno-stainingmethod on non-permeabilized cells. The cells were then imaged with atomic force microscopy(AFM), revealing live and xed cells strongly attached to the glass surfaces. A bioconjugate chem-istry approach was employed to functionalize the surfaces of the AFM tips with D-glucose moleculesvia three dierent heterobifunctional crosslinkers. The D-glucose binding site and the translocationpathway of SGLT1 were investigated by studying the interaction forces between tip-bound D-glucoseand SGLT1 in live cells on the single-molecule level. Analysis of these forces suggested that a longcrosslinker with a small end group might be suitable for probing the D-glucose transport pathwayof SGLT1. We show that single-molecule AFM technology is a powerful method for investigatingtransmembrane proteins and transporter functions in live cells.PACS numbers: 07.79.Lh, 82.37.Rs, 87.15.Kg, 87.83.+aKeywords: SGLT1, Na+-D-glucose cotransporter, AFM, Force spectroscopyI. INTRODUCTIONMolecular recognition in biological systems betweenreceptors/ligands, proteins/antibodies and substrate/transporter is often the initiating step in a wide vari-ety of biological functions. A Na+-D-glucose cotrans-porter type 1 (SGLT1) is one of the most intensivelystudied membrane transporters [1,2]. SGLT1 transportsglucose into cells driven by the Na+ electrochemical po-tential dierence across the membrane [3]. Recently, ourgroup has pioneered an investigation of the topology andthe substrate binding of SGT1 on living cells by usingatomic force microscopy (AFM) [4]. The principle andthe operation of the AFM have been described exten-sively [5{7]. This approach is nowadays widely used be-cause the experiments can be performed on unalteredcells under near-physiological conditions without modi-cations of the membrane proteins [8,9]. Furthermore, theE-mail: sctpt@mahidol.ac.th; Fax: +66-2-354-7161high sensitivity of the AFM and the soft cantilevers allowfor the detection of pN forces of transporter-ligand com-plexes, providing a unique opportunity to detect molec-ular recognition of single molecular binding events underdierent experimental conditions [4,8{14].In the current study, we extended the molecular recog-nition force spectroscopy of the glucose-SGLT1 interac-tion by using novel crosslinkers for the coupling of D-glucose to AFM tips. Our studies demonstrate that anewly developed acrylamide crosslinker might be a pow-erful candidate for a detailed investigation of the sugartranslocation pathway of SGLT1.II. EXPERIMENTS1. Characterization of SGLT1 in the G6D3Cells: Fluorescent Immuno-stainingFreshly isolated G6D3 cells (CHO cells stably trans-fected with rbSGLT1) at passages below fteen were-1336-Single-Molecule AFM Studies of Substrate Transport   { Theeraporn Puntheeranurak et al. -1337-Fig. 1. Detection of SGLT1 on the surface of non-permeabilized G6D3 cells xed with paraformaldehyde. Spe-cic QIS30 antibodies were bound to SGLT1 on cell mem-branes and subsequently stained by incubation with anti-rabbit IgG linked to Cy3 uorophor (spreading white color).Nuclei are shown in the middle of the cells (DAPI staining).The scale bar represents 10 m.used for all experiments. In order to conrm theover-expression of SGLT1 on the surface of the G6D3cells used in this study, we employed the uorescentimmuno-staining method. The G6D3 cells forming a uni-form monolayer on poly-L-lysine-coated glass cover-slipswere xed with paraformaldehyde. For surface labeling,QIS30 antibody (specic antibody against loop 6-7 ofSGLT1 [15]) was used on non-permeabilized cells, fol-lowed by incubation with anti-rabbit IgG linked to Cy3uorophor. Nuclei were stained with 4'-6-diamidino-2-phenylindole (DAPI). As illustrated in Figure 1, SGLT1was clearly detected on the cell surface by using epiu-orescence microscopy. The incubation of the specimenslacking the primary antibody revealed no observable u-orescence (data not shown).2. AFM Imaging of G6D3 CellsThe AFM oers the potential for imaging the morphol-ogy of living cells in a non-destructive manner. Mono-layers of live G6D3 cells grown on poly-L-lysine-coatedglass cover-slips were imaged at room temperature in aKRH-Na+ medium. A contact mode AFM (PicoSPM II,Molecular Imaging, Tempe, AZ) at forces of less than 100pN with non-functionalized AFM cantilevers (cantileverfrom Veeco GmbH, Mannheim, Germany) of 0.03 to 0.5N/m nominal spring constants were utilized. Large-scaletopography and deection images of the living cells areshown in Figures 2(a) and 2(b), respectively. The re-gion surrounding the nucleus appeared at and assistedadhesion of the cells to the glass plate. Typical cellswere about 30 { 70 m in diameter, with a characteristicbright zone corresponding to the nuclei region appearingto be about 1 { 3 m in height. Due to the high ex-ibility and the softness of the cell membranes of livingcells, we were not able to achieve a higher resolution onFig. 2. AFM images of living and xed G6D3 cells: (a)Topographical image and (b) deection image of living cellsimaged in KRH-Na medium at a scan size of 80 m by usinga poly-L-lysine-coated glass cover-slip. The bright regions ofthe images correspond to the area of the nucleus. (c) Topo-graphical image of xed cells acquired at a scan size of 50m. (d) Low-scan size image (17 m) of the cell surface ofxed cells. The images show the substructures of the cellmembrane, which are most likely microvilli.the membrane surface. The surface morphology, such aslamellipodia and microvilli, could only be observed aftercell xation, as illustrated in Figures 2(c) and 2(d). Nev-ertheless, the AFM images revealed that the G6D3 cellswere strongly attached to the surface, which was a pre-requisite for atomic force microscopy. We achieved aboutan 80 % conuent-cell monolayer, which contained cellswith a maximum height of about 1 m. The G6D3 cellswere easily grown and could be maintained alive at roomtemperature for at least six hours, which was suitable forAFM force spectroscopy.3. Single-molecule Recognition of D-glucoseCoupled via Three Kinds of CrosslinkersRecently, we have demonstrated that AFM force spec-troscopy is a powerful method to probe the substrate-transporter interaction of SGLT1 in a live cell system[4]. We established a new method for the investigationof the membrane topology and the initial sugar-bindingsite of SGLT1. However, due to the bulky group of AFMcrosslinkers, we were only able to detect the interactionson the surfaces of the cells. Here, a new crosslinker witha smaller end group, i.e., acrylamide (AM) and a longerpolymer length was used for comparison with our stan-dard vinylsulfon (VS) crosslinker, which was shown to-1338- Journal of the Korean Physical Society, Vol. 52, No. 5, May 2008Fig. 3. Linkage of D-glucose to AFM tips. Sugars werecovalently coupled to AFM tips via heterobifunctional PEGderivatives.be apt for examining the initial sugar-binding site ofSGLT1 [4]. We coupled 1-thio--D-glucose to AFM tipsthrough three dierent exible PEG [poly(ethylene gly-col)] crosslinkers at a very dilute surface density. In brief,silicon tips were rst functionalized with ethanolamineand the NHS (N-hydroxysuccinimide) end of the PEGlinkers (NHS-PEG800-VS, NHS-PEG800-AM and NHS-PEG5000-AM) was covalently bound to amines on thetip surface, followed by the attachment of sugar via afree SH group (Figure 3).For the glucose/SGLT1 recognition, force-distance cy-cles in KRH-Na+ buer were performed by using ligand-coated cantilevers (rectangular, Veeco GmbH) with0.02-N/m nominal spring constants in the conventionalcontact-force-calibration-plot mode. Force-distance cy-cles were recorded on cell surfaces with the assistanceof a CCD camera for the localization of the AFM can-tilever on isolated cells or cell monolayers. The princi-ple of single-molecule-recognition force detection of thesugar-transporter complex on living G6D3 cells by usingAFM is illustrated in Figure 4(a). In the force-distancemode, the deection angle of the cantilever is measuredas a function of the vertical position of the cantilever.At a xed lateral position, the tip rst approached thesurface of the cells (dotted trace). Far away from thesurface (1), no bending of the cantilever is visible andthe cantilever deection (z) and thus, the force (f) arealmost zero (the force is calculated according to Hook'slaw; i.e., f = kz, where k is the spring constant ofthe cantilever). As the cantilever reaches the cell sur-face (2), the cantilever is deected upwards due to therepulsive tip-sample interaction. Subsequently, the tip isretracted (retrace) and relaxation of the repulsive forceFig. 4. Single-molecule recognition event detected withAFM. (a) Schematic representation of a force-distance cy-cle on live cells. The tip was moved towards the cell sur-face (dotted line, 1-2) and then retracted (solid line) at aconstant lateral position. During tip approach, the ligandspecically binds with a receptor that leads to a force signalwith a distinct shape (3) during tip retraction. The force in-creases until bond-rupture occurs (4) at the unbinding force(fu). (b) Typical force curves showing specic interactionsbetween glucose and SGLT1 upon tip-surface retraction byusing PEG5000-AM and PEG800-VS (inset) crosslinkers.in the contact region is observed (2 to 3). In the casewhen the sugar on the tip has bound to a transporteron the cell surface, an attractive force develops (down-ward deection) and the crosslinker via which the sugaris coupled to the tip will be stretched more and morewith increasing tip-surface separation, leading to a forcesignal of distinct shape (3). Finally, the sugar will detachfrom the transporter (3 to 4) at a particular unbindingforce, fu (see arrow) and the cantilever jumps back tozero deection (4). The sweep-amplitude of the force-distance cycles was 1000 nm at a 1-Hz sweep rate. Up to500 { 1000 force-distance cycles were performed for eachlocation on the surface of cells and up to four locationsSingle-Molecule AFM Studies of Substrate Transport   { Theeraporn Puntheeranurak et al. -1339-Fig. 5. Inhibition of D-glucose binding with a competitivephlorizin inhibitor. Binding probabilities of D-glucose cou-pled to AFM tips via (a) PEG800-VS, (b) PEG800-AM and(c) PEG5000-AM crosslinkers and of the block by phlorizinare shown. Values are means SEM (standard error of themean); n = 2000 { 4000. Phlorizin is abbreviated by Phlz. *= p-value < 0.05 and *** = p-value < 0.005.(dierent cells) for each condition, i.e., initial condition,ligand addition and washout condition.Glucose/SGLT1 interaction was observed with allthree crosslinkers. Figure 4(b) shows typical force curves(retraction) of a single molecule sugar-SGLT1 recogni-tion event obtained by using glucose tips coupled viaPEG5000-AM and PEG800-VS (inset). The specicityof the recognition of SGLT1 by glucose was conrmedby blocking experiment through the addition of phlo-rizin, a specic high-anity competitive inhibitor thatbinds to the external membrane surface of SGLT1. Thebinding probability (probability of nding an unbindingevent in force-distance cycles) was reduced signicantlyfrom 9.0  0.3 % to 3.2  1.3 % (Figure 5(a)), from7.5  0.8 % to 1.7  0.4 % (Figure 5(b)) and from9.7  3.1 % to 1.5  0.4 % (Figure 5(c)) for D-glucosecoupled to AFM tips via PEG800-VS, PEG800-AM andPEG5000-AM, respectively. After phlorizin was subse-quently washed out, the binding probability increasedagain (Figures 5(a)-(c)). By constructing an empiricalprobability density function of the unbinding forces, themaximum forces of the distribution from several experi-ments were found to be 40.1, 49.6 pN and 65.7 pN for theglucose coupled to AFM tips via PEG800-VS, PEG800-AM and PEG5000-AM, respectively (data not shown). Astudent t-test analysis unequivocally revealed that thedierences between D-glucose coupled via PEG800-VSand PEG5000-AM were statistically signicant (p-value< 0.005). The higher forces measured when D-glucosewas coupled to the AFM tip via acrylamide suggest thatD-glucose might be able to penetrate further into SGLT1in this conguration and, thus, partly image the sugartransport pathway of SGLT1. In contrast and as al-ready mentioned earlier, the vinylsulfon crosslinker con-tains a bulky group, most likely allowing only for super-cial binding and obstructing glucose transport. There-fore, the acrylamide crosslinkers appear to be suitablecrosslinkers for further investigations of the sugar trans-port pathway of SGLT1.III. CONCLUSIONThese studies demonstrate that AFM force spec-troscopy is a useful method for exploring the functionaldynamics of SGLT1 in live cells on a single-moleculelevel. Specic glucose/SGLT1 interactions were ob-served using 1-thio--D-glucose coupled to AFM tipsvia crosslinkers carrying vinylsulfon and acrylamide endgroups. The strikingly dierent unbinding force val-ues measured using these crosslinkers might arise fromthe dierences in the accessibility of glucose to trans-membrane parts of SGLT1. We, thus, hypothesize thata long, thin acrylamide-PEG crosslinker possibly al-lows glucose to penetrate deeper into the transporterthan the other two crosslinkers. Therefore, we suggestthat this crosslinker might be a promising candidate forfurther single-molecule studies elucidating the glucose-translocation pathway.ACKNOWLEDGMENTSWe are indebted to Linda Wildling for her helpfulsynthesis of the AFM crosslinkers. This work was sup-ported in part by the Thailand Research Fund (T.P.) andby the National Nanotechnology Center (NANOTEC),NSTDA, Ministry of Science and Technology, Thailand,through its program of Center of Excellence Network.The Max Planck Institute of Molecular Physiology, Dort-mund, Germany and the Austrian Science FoundationProject P-14549 (P.H.) are gratefully acknowledged.REFERENCES[1] M. A. Hediger and D. B. Rhoads, Physiol. Rev. 74, 993(1994).-1340- Journal of the Korean Physical Society, Vol. 52, No. 5, May 2008[2] E. M. Wright, Am. J. Physiol. Renal Physiol. 280, F10-8(2001).[3] R. K. Crane, Rev. Physiol. Biochem. Pharmacol. 78, 99(1977).[4] T. Puntheeranurak, L. Wildling, H. J. Gruber, R. K.Kinne and P. Hinterdorfer, J. Cell Sci. 119, 2960 (2006).[5] R. Lal and S. A. John, Am. J. Physiol. 266, C1 (1994).[6] G. Binnig, C. F. Quate and C. Gerber, Phys. Rev. Lett.56, 930 (1986).[7] J. H. Hoh and P. K. Hansma, Trends Cell Biol. 2, 208(1992).[8] G. Pster, C. M. Stroh, H. Perschinka, M. Kind, M.Knoach, P. Hinterdorfer and G. Wick, J. Cell Sci. 118,1587 (2005).[9] V. Dupres, F. D. Menozzi, C. Locht, B. H. Clare, N.L. Abbott, S. Cuenot, C. Bompard, D. Raze and Y. F.Dufrene, Nat. Methods 2, 515 (2005).[10] E. L. Florin, V. T. Moy and H. E. Gaub, Science 264,415 (1994).[11] P. Hinterdorfer, W. Baumgartner, H. J. Gruber, K.Schilcher and H. Schindler, Proc. Natl. Acad. Sci. U.S.A.93, 3477 (1996).[12] G. U. Lee, L. A. Chrisey and R. J. Colton, Science 266,771 (1994).[13] S. Wielert-Badt, P. Hinterdorfer, H. J. Gruber, J. T. Lin,D. Badt, B. Wimmer, H. Schindler and R. K. Kinne,Biophys. J. 82, 2767 (2002).[14] F. Liu, F. T. Arce, S. Ramachandran and R. Lal, J. Biol.Chem. 281, 23207 (2006).[15] H. Kipp, S. Khoursandi, D. Scharlau and R. K. Kinne,Am. J. Physiol. Cell Physiol. 285, C737 (2003).

Single-Molecule AFM Studies of Substrate Transport by Using the Sodium-Glucose Cotransporter SGLT1

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Single-Molecule AFM Studies of Substrate Transport by Using the Sodium-Glucose Cotransporter SGLT1

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