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Figure 1. Steady-state kinetics of G507C. (A) Total current records from an hSGLT1 G507C cRNA-injected oocyte that had been labeled with TMR6M. Membrane potential (Vm) was held at −50 mV (Vh), and then stepped to a test value (Vt, starting at +50 mV and ending at −150 mV in 20-mV decrements) for 100 ms before returning to Vh. The bath solution was NaCl buffer. (B) Total current records when 1 mM αMDG was added to the bath solution. (C) Steady-state I-V relations. The αMDG-induced current (0.1–10 mM) was obtained by subtracting the current (measured at 100 ms) with sugar added from baseline current in Na+ alone. (D) Dose–response relation for αMDG-induced current. [Na+]o = 100 mM, and Vm = −50 mV. The curve was the fit (Eq. 1) to the data: Imax = 778 ± 13 nA, and \documentclass[10pt]{article}
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\begin{equation*}{\mathrm{K}}_{0.5}^{{\alpha}{\mathrm{MDG}}}\end{equation*}\end{document} = 1.6 ± 0.1 mM. (E) Voltage dependence of \documentclass[10pt]{article}
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\begin{equation*}{\mathrm{K}}_{0.5}^{{\alpha}{\mathrm{MDG}}}\end{equation*}\end{document} . (F) Voltage dependence of \documentclass[10pt]{article}
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\begin{equation*}{\mathrm{K}}_{0.5}^{{\mathrm{Na}}}\end{equation*}\end{document} . Experiment was performed by increasing [Na+]o (from 0 to 100 mM) with [αMDG]o maintained at 25 mM. Errors bars are standard errors (SE) of the fit when SE exceeds the size of the symbol.
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Figure 2. Presteady-state kinetics of hSGLT1 G507C (nonlabeled). (A) Current record for 100-ms test voltage pulses. Vh was −50 mV, and the current records at selected Vt = +30, −10, −50, −90, and −150 mV are shown for the ON and OFF pulses. (B) The corresponding current records for 500-ms pulses. (C) τ-V relation for the medium and slow components. The OFF responses were independent of the previous test potential (Vt), and the open symbol represents the mean of 10 values with Vt varying between +50 and −150 mV. (D) Q-V relation for medium charge. (E) Q-V relation for slow charge. (F) Q-V relation for total charge. The curve in D was obtained from fitting the data with the Boltzmann relation (Eq. 3). In E and F, the Boltzmann fits were obtained under the constraint z = 1.0.
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Figure 3. Changes of fluorescence intensity (ΔF) of TMR6M-labeled G507C hSGLT1 with step jumps in membrane voltage. Membrane was held at −50 mV and then stepped to various test voltages. (A) Time course of ΔF observed with selected 100-ms pulses. (B) Time course of ΔF observed with selected 500-ms pulses. To minimize photobleaching, the 500-ms records were averages of three records compared with 10 for 100-ms records, hence the greater apparent noise level of the former. (C) ΔFmed-V relation. The line was the fit of the data with the Boltzmann relation. (D) ΔFslow-V relation. As in C, the line was the fit with the Boltzmann relation. (E) τ-V relation for the medium component of ΔF. (F) τ-V relation for the slow component of ΔF. The open symbols (in E and F) are the time constants τ for the OFF pulses.
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Figure 4. hSGLT1 G507C OFF currents in the presence of sugar (A) and phlorizin (B). Membrane potential was held at −50 mV (Vh), and shown are the selected total OFF current records (in response to a series of test voltage pulses from +50 to −150 mV) of an oocyte expressing G507C (unlabeled). The numbers beside the traces indicate the test voltage (Vt). (A) Effect of αMDG. OFF current records in Na+ buffer (top), with 0.1 (middle) and 1 mM αMDG (bottom) added to the external solution. At Vh, the αMDG-induced currents were 14 and 99 nA at 0.1 and 1 mM αMDG. (B) Effect of phlorizin. OFF current records in Na+ buffer, and with 0.5 and 1 μM phlorizin added to the external solution. Dashed lines represent zero current.
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Figure 5. Effect of sugar on fluorescence. Time course of ΔF with step jumps in membrane voltage in NaCl buffer with various [αMDG]o added (0, 0.5, 1, and 10 mM). Membrane potential was held at Vh (−50 mV) and stepped to test values (from +90 to −150 mV) for 100 ms before returning to Vh. The dashed lines represent baseline fluorescence (ΔF = 0) at Vh. Abscissa and ordinate scales are the same for all panels. In this experiment, five [αMDG]o were examined (0, 0.5, 1, 5, and 10 mM). Between each [sugar]o, fluorescence records were obtained in sugar-free NaCl buffer to allow for compensation for photobleaching (Meinild et al., 2002). The apparent greater noise at 10 mM [αMDG]o is due to the compensation for photobleaching.
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Figure 6. Comparison of charge and fluorescence (ΔF) records in the presence and absence of saturating [αMDG]o (100 mM). Presteady-state current records for ON and OFF (compensated for oocyte membrane capacitance) and ΔF. Vh was −50 mV and the test voltage varied from +50 to −150 mV. Numbers beside the traces are the test voltages. Dashed lines are the zero current or zero ΔF levels. (A) In NaCl buffer. (B) In NaCl buffer with 100 mM [αMDG]o.
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Figure 7. Effect of sugar and phlorizin on charge and fluorescence. (A) Dependence of Qmax and ΔFmax on [αMDG]o. The line was the fit of the charge data to Eq. 1. The line through the ΔF data was drawn using Eq. 1 with a K0.5 of 1.1 mM and a maximal change of 0.5 observed at 100 mM [αMDG]o. (B) Dependence of Qmax and ΔFmax on [phlorizin]o. The lines were fits of the data. (C) Dependence of the shift of V0.5 for charge (ΔV0.5Q) and fluorescence (ΔV0.5F) on [αMDG]o. The line was obtained by fitting all the data. (D) Dependence of V0.5 for charge and ΔF on [phlorizin]o. The dashed lines represent the mean of the data points.
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Figure 8. Effect of phlorizin on fluorescence. Shown is the time course of ΔF in NaCl buffer with [phlorizin]o (0, 0.1, 1, and 100 μM) in one oocyte. Membrane potential was held at Vh −50 mV and stepped for 100 ms to Vt (from +50 to −150 mV), before returning to Vh. Abscissa and ordinate scales are the same for all panels.
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Figure 9. An eight-state kinetic model for SGLT1. This model is an extension of the six-state model proposed by Parent et al. (1992b) with inclusion of intermediate states (Ca and Cb) between C1 and C6 (Loo et al., 2005). Kinetic states of the transporter consist of the empty transporter C (states C1 and C6), Na+-bound CNa2 (states C2 and C5), Na+- and sugar-bound SCNa2 (states C3 and C4) in the external (′) and internal membrane surfaces (′′). Two Na+ ions bind to the protein before the sugar molecule. The shaded region represents the voltage-dependent steps: conformational change of the empty transporter between the external and internal membrane surfaces (C1⇄Ca⇄Cb⇄C6), and Na+ binding/dissociation (C1⇄C2). Rate constants (kij) for transitions between states (Ci→Cj) are kij = kijo exp(−ɛijFV/RT), where kijo is a voltage-independent rate, ɛij is the equivalent charge movement, and F, R, and T have their usual physicochemical meanings (see Simulation of SGLT1 in Materials and methods). In Fig. 9, the reduced membrane voltage u = FV/RT. The rate constants obey the microscopic reversibility conditions (compare Parent et al., 1992b): k52o k21o k1ao kabo kb6o k65o = k12o k25o k56o k6bo kbao ka1o, and k54o k43o k32ok25o = k45o k52o k23o k34o.
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Figure 10. Simulation predictions on occupancy probabilities (Po). The simulations were performed in 100 mM external Na+. A, B, and D show Po as a function of voltage in the presence of 0, 1, and 10 mM [αMDG]o. C and E show the distribution of conformations in the presence of external phlorizin (0.4 and 1 μM). Cpz is the phlorizin-bound state [CNa2Pz]′. For clarity, states with occupancy probabilities <0.1 were not plotted. The states were as follows: C1 in A, C, and E; and C1, C2, C3, and C4 in B and D.
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Figure 11. Simulated OFF currents. The simulations were performed in 100 mM external Na+. (A, B, and D) OFF currents with 0, 1, and 10 mM αMDG in the external medium. (C and E) OFF currents in the presence of 0.4 and 1 μM external phlorizin. Vh was −50 mV, and currents shown (for test voltages at +50, +10, −30, −50, −70, −90, and −150 mV) have been filtered at 500 Hz. Using the cut-open oocyte voltage clamp, we have previously found that there are two fast components of presteady-state currents with time constants of 0.18 and 1.3 ms (Loo et al., 2005). In the two-electrode voltage clamp, they are hidden by the oocyte membrane capacitance.
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Figure 12. Comparison of fluorescence data and simulation of the eight-state model (Fig. 9). Simulation was performed using the kinetic parameters of Table II. Representative fluorescence traces at test voltages +50 and −150 mV from Vh (−50 mV) are from Fig. 5. The dashed lines are the simulated ΔF. The apparent quantum yields used for the simulation were qy2 = 1, qy6 = 5, and qy1 = qya = qyb = qy3 = qy4 = qy5 = 3. Abscissa and ordinate scales are the same for all panels.
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Figure 13. Simulated fluorescence records. The simulations were performed in 100 mM external Na+. (A, B, and D) Time course of fluorescence intensity change (ΔF) with 0, 1, and 10 mM αMDG in the external solution. (C and E) Effect of phlorizin (0.4 and 4 μM). Total fluorescence (F) associated with SGLT1 is defined by F ≈ qy1C1 + qy2C2 + qyaCa + qybCb + qy3C3 + qy4C4 + qy5C5 + qy6C6, where qyj is the apparent quantum yield of the fluorophore (TMR6M) when SGLT1 is in conformation Cj. Fluorescence records were simulated with qy1 = 3, qy2 = 1, qya = 3, qyb = 3, qy4 = 3, qy5 = 3, qy6 = 6. Shown are the predicted ΔF records when Vm is stepped from −50 mV to Vt (+50, +10, −30, −50, −70, −90, and −150 mV). Steady-state fluorescence levels have been removed. The simulation for ΔF in the presence of phlorizin is independent of the quantum yield of the phlorizin-bound state ([CNa2Pz]′). See Table I for comparison of simulated and observed presteady-state kinetic parameters.
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Figure 14. Simulated conformational change C5≡C6. The simulations were performed with 300-ms voltage pulses in 100 mM external Na+. (A and B) Components of fluorescence change (ΔF) when Vm was stepped from −50 to +50 mV with 0 and 10 mM αMDG in the external solution. The bold lines are the total ΔF, and the broken lines are changes of fluorescence due to C2, C5, and C6. (C and D) Time course of occupancy probability (Po) in 0 and 10 mM αMDG.
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