Forces and dynamics of glucose and inhibitor binding to sodium glucose co-transporter SGLT1 studied by single molecule force spectroscopy

Abstract

Single molecule force spectroscopy was employed to investigate the dynamics of the sodium glucose co-transporter (SGLT1) upon substrate and inhibitor binding on the single molecule level. CHO cells stably expressing rbSGLT1 were probed by using atomic force microscopy tips carrying either thioglucose, 2'-aminoethyl β-d-glucopyranoside, or aminophlorizin. Poly(ethylene glycol) (PEG) chains of different length and varying end groups were used as tether. Experiments were performed at 10, 25 and 37 °C to address different conformational states of SGLT1. Unbinding forces between ligands and SGLT1 were recorded at different loading rates by changing the retraction velocity, yielding binding probability, width of energy barrier of the binding pocket, and the kinetic off rate constant of the binding reaction. With increasing temperature, width of energy barrier and average life time increased for the interaction of SGLT1 with thioglucose (coupled via acrylamide to a long PEG) but decreased for aminophlorizin binding. The former indicates that in the membrane-bound SGLT1 the pathway to sugar translocation involves several steps with different temperature sensitivity. The latter suggests that also the aglucon binding sites for transport inhibitors have specific, temperature-sensitive conformations.

Keywords: Atomic Force Microscopy (AFM); Glucose Translocation Pathway; Glucose Transport; Kinetics; Ligand-binding Protein; Off Rate Constant; Phlorizin Binding; SGLT1; Spectroscopy; Width of Energy Barrier.

FIGURE 1.

Linkage of substrates and inhibitors of SGLT1to the AFM tip via heterobifunctional PEG linker by a three-step protocol. Thio-glc, hex-glc, and AcS-aminophlorizin (amino-phl) (A) were coupled to cross-linkers varying in lengths having a maleimide end group (∼8 nm) or an acrylamide-end group (∼30–40 nm) (B). C, amino groups were generated on the silicon nitride AFM cantilever tip using aminopropyltriethoxysilane (step 1) to attach the flexible distensible PEG-linker covalently via its NHS end to the tip (step 2). The ligand was linked to the free thio-reactive end of the PEG chain (step 3). Thioglucose was coupled to the acrylamide-linker, hexyl-glucose, and aminophlorizin were bound to the maleimide-linker. Circles in B and C indicate the thio-reactive group (TRG). In C the dotted line circles the ligand, which is coupled to the PEG linker.

FIGURE 2.

Single molecule recognition of SGLT1 on the surface of living cells. A, schematic representation of a force-distance cycle, where the cantilever approached the cell surface and subsequently retracted (solid line). The deflection angle of the cantilever remained zero (1) until the tip contacted the sample surface from where the cantilever was bent upward (2). Upon retraction a force signal with a distinct shape occurred when an interaction between the ligand and a receptor occurred (3). The cantilever was increasingly bent downward until the bond broke at a defined unbinding force f_u required to break a single ligand-receptor interaction (4). B, force curves show specific interactions between thioglucose coupled via an acrylamide-linker and SGLT1 upon tip-surface retraction. The specific recognition disappeared in the presence of phlorizin (inset).

FIGURE 3.

Specific interaction of glucose and inhibitors at 37 °C. Data of one typical experiment are presented for each cantilever, probed in different conditions: initial condition (first column), block condition (second column), and washout condition (third column). Values are the mean ± S.E. for n = 2000–3000, where 2–3 different cells were probed at each condition. The specificity of the recognition events could be demonstrated by a significantly reduced binding probability in the presence of phlorizin when compared with control levels (initial condition) with p < 0.005. After a careful washout procedure the interaction increased again. amino-phl^, 3-aminophlorizin.

FIGURE 4.

Binding probabilities of glucose, hexyl-glucose, and aminophlorizin at different temperatures. The interaction between thioglucose on AA-PEG₅₀₀₀ (A), hexyl-glucose on mal-PEG₁₃₀₀ (B), and aminophlorizin (C) on mal-PEG₁₃₀₀ was studied at 10 °C, 25 °C, and 37 °C using the same cantilever and the same cells. Values are the mean ± S.E. of three to five different cantilevers and two different cells for one cantilever. On each cell up to 1000 force-distance cycles were recorded.

FIGURE 5.

Dynamic force spectroscopy spectra of thioglucose on AA-PEG₅₀₀₀ probed at different temperatures. Glucose/SGLT1 recognition was investigated at different tip-retraction velocities to gain dynamic aspects of bond rupture in various temperatures, i.e. 10 °C (A), 25 °C (B), and 37 °C (C). By applying a maximum likelihood approach the most likely and accurate values for the length scale x_β and the kinetic off rate k_off were estimated. A, at 10 °C x_β of thioglucose on AA-PEG₅₀₀₀ was lowest, and the sugar dissociated fastest. B, at 25 °C x_β as well as k_off were increased. C, notably, at 37 °C x_β was even further increased, whereas the dissociation was slower than at 10 °C. Data were obtained from merging results from six different cantilevers.

FIGURE 6.

Dynamic force spectra of the different SGLT1 ligand complexes at different loading rates. The most probable unbinding forces were plotted against the logarithm of the loading rate. Comparisons of force spectra with thioglucose on AA-PEG₅₀₀₀, hexyl-glucose on mal-PEG₁₃₀₀, and aminophlorizin on mal-PEG₁₃₀₀ at 10 °C (A), 25 °C (B), and 37 °C (C) are shown.