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Figure 1. Effects of D-glucose on the voltage-induced fluorescence changes of hSGLT1.A, schematic drawing of hSGLT1 construction for voltage-clamp fluorometry (VCF). Cysteine was introduced into the extracellular Gly-507 (orange) of the 12th transmembrane segment, and MTS-TAMRA was bound. Asparagine was introduced at Thr-287 (green) in the seventh transmembrane segment in the experiment in Figure 4. B, machine setup for VCF. Excitation light and fluorescence were bifurcated by a dichroic mirror. C, representative current trace of hSGLT1 G507 by administration of 10 mM sugars (black bars). An arrowhead indicates the zero level of the current. D, representative voltage-induced fluorescent traces of hSGLT1 measured in the presence of different concentrations (0, 5, and 25 mM) of D-glucose. The traces recorded at various membrane potentials shown in (E) are overlaid. Red, green, and blue traces indicate the fluorescence signal obtained by −200 mV, 0 mV, and +200 mV voltage step pulses, respectively. Changes in signal fluorescence value from the base fluorescence value were analyzed. E, pulse protocol for VCF. Membrane potential was held at −60 mV and determined at 200 ms step pulses of −200, −120 mV, −40 mV, 0 mV, +40 mV, +120 mV, and +200 mV. F, dose–response relationships of the normalized fluorescent intensity of hSGLT1 with D-glucose. Normalized fluorescence intensity (ΔF/F) was obtained by dividing the change in fluorescence under test pulses (ΔF) by the baseline fluorescence intensity at the holding potential (F), where the average values of the “baseline” and “signal” regions in (D) were used. Data were analyzed for −200 mV, 0 mV, and +200 mV. ΔF/F values at the end point region of test pulse in (D) were plotted (red, green, and blue circles) and fitted by the Hill equation. Data for 0, 1, 5, 11, 25, and 50 mM were plotted. Apparent Kd values were 1.8 mM for −200 mV, 1.2 mM for 0 mV, and 3.3 mM for +200 mV. ALL, D-allose; FRU, D-fructose; GAL, D-galactose; GLU, D-glucose; MTS-TAMRA, 2-((5(6)-tetramethyl-rhodamine)carboxylamino)ethyl methanethiosulfonate; SGLT, sodium–glucose cotransporter.
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Figure 2. Effects of sugars on the voltage-induced fluorescence changes of hSGLT1.A, representative voltage-induced fluorescent traces of hSGLT1 measured at different concentrations of D-glucose, D-galactose, D-fructose, D-allose, and D-ribose. Step pulses were applied as in Figure 1D. The red trace shows the fluorescence signal obtained by a −200 mV voltage step pulse. Fluorescence traces of D-glucose from Figure 1D were used for comparison. B, dose–response relationships of the normalized fluorescent intensity of hSGLT with D-glucose (red), D-galactose (blue), D-fructose (green), D-allose (pink), and D-ribose (black). Fluorescent values at the end point of the test pulse at the −200 mV step in (A) were plotted (colored circles). Hill-fitted curves are also shown. The apparent Kd value for D-allose was too high and thus was not reliable, and D-ribose could not even be fitted. Data were collected from the same oocytes described in (A), and data for 1, 5, 11, 25, and 50 mM were plotted. C, comparison of the apparent Kd values. Bars indicate means ± standard deviation (SD), and all data are plotted as black dots. Numbers in brackets indicate the number of experiments (n = 3–5). Data were statistically analyzed via one-way ANOVA with Tukey–Kramer’s post hoc test (N.S., p > 0.05). ALL, D-allose; FRU, D-fructose; GAL, D-galactose; GLU, D-glucose; RIB, D-ribose; SGLT, sodium–glucose cotransporter.
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Figure 3. Effects of sugars on the voltage-induced fluorescence changes of hSGLT1 T287N.A, representative voltage-induced fluorescent traces of the hSGLT1 T287N mutant with different concentrations (0, 20, 40, and 78 mM) of D-glucose, D-galactose, and D-fructose. Step pulses were applied as in Figure 1D. Red traces show each fluorescent signal obtained by the −200 mV step pulse. B, dose–response relationships of the fluorescent intensity of T287N with D-glucose (red), D-galactose (blue), and D-fructose (green). Fluorescent values at the end point of the test pulse at the −200 mV step in A were plotted for 20, 40, 78, 116, and 165 mM (colored circles), and Hill-fitted curves are also shown. C, comparison of the apparent Kd values. Bars indicate the means ± SD, and all data are plotted as black dots. Numbers in brackets indicate the number of experiments (n = 3–6). Data were statistically analyzed via one-way ANOVA and Tukey–Kramer’s post hoc test (∗p = 0.037 < 0.05). FRU, D-fructose; GAL, D-galactose; GLU, D-glucose; SGLT, sodium–glucose cotransporter.
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Figure 4. Effects of D-fructose on the sugar transport of hSGLT1.A, representative current trace of hSGLT1 WT by application of 10 mM D-galactose and D-fructose and both D-galactose and D-fructose. B, comparison of current amplitudes induced by 10 mM sugar. Bars indicate the means ± SD, and all data are plotted as black dots. Numbers in brackets indicate the number of experiments (n = 6–8). Data were statistically analyzed via one-way ANOVA with Tukey–Kramer’s post hoc test (∗∗∗p < 0.001). FRU, D-fructose; GAL, D-galactose; SGLT, sodium–glucose cotransporter.
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Figure 5. Effects of L-sorbose on the voltage-induced fluorescence changes of hSGLT1 WT and T287N.A, representative voltage-induced fluorescent traces of hSGLT1 measured with different concentrations of L-sorbose. Step pulses were applied as in Figure 1D. Red traces show each fluorescent signal obtained by the −200 mV step pulse. B, dose–response relationships of the fluorescent intensity of hSGLT1 WT and T287N. Fluorescent values at the end point of test pulse at the −200 mV step in A were plotted for 1, 5, 11, 25, and 50 mM in WT and for 20, 40, 78, 116, and 165 mM in T287N (orange circle), and Hill-fitted curves are also shown. C, comparison of the apparent Kd values. Bars indicate the means ± SD, and all data are plotted as black dots (n = 3–4). Data were statistically analyzed with student’s t test (∗∗p = 0.0025 < 0.01). SGLT, sodium–glucose cotransporter; SOR, L-sorbose.
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