Loo DD , Hirayama BA , Cha A , Bezanilla F , Wright EM .

DK19567 NIDDK NIH HHS , R01 DK019567 NIDDK NIH HHS

	Figure 1. . Time course of the raw or total currents (capacitive and hSGLT1 presteady-state currents) of an oocyte expressing hSGLT1. The experiment was performed using a two-electrode voltage clamp, and all data were obtained from a single oocyte. Membrane potential was held at −50 mV (Vh) and then stepped to a series of test values Vt (from +50 to −150 mV in 20-mV decrements; representative traces are shown for +50, −10, −50, −90, and −150 mV) before returning to Vh. The pulse duration was 100 ms in A and 500 ms in B, and the currents, which are the average of three sweeps, have been filtered at 500 Hz (A) and 50 Hz (B). An upwards deflection of the current trace represents an outward current.
	Figure 2. . Medium and slow components of the presteady-state currents of hSGLT1 (ON response). Shown are the current records for the ON pulse from Vh (−50 mV) to Vt +50, −10, −50, −90, and −150 mV for the 100-ms (A) and the 500-ms (B) duration pulses. The data were from the same oocyte as Fig. 1. To isolate the slow charge, the steady-state current was removed, and the transient current (for the 500 ms pulses) was fitted to a single exponential function. The fit was restricted to the region between 5τmed (τmed ≈ 3–20 ms) and 500 ms. The starting point was 25 ms at +50 mV, 60 ms at −10 mV, and 100 ms at Vt more negative than −90 mV. The fit was extrapolated to the peak of the total current trace (dashed lines in A and B), typically two data samples after onset of the voltage pulse (to 0.2 ms in A). The isolated slow charge is shown in D. To obtain the medium component, the slow component (dashed line) was subtracted from the 100-ms current records (A). The difference was fitted to I(t) = Icm exp(−t/τcm) + Imed exp(−t/τmed). Panel C shows the medium charge obtained after subtraction of the membrane capacitance (Icm exp(−t/τcm)). For clarity, the current trace at −50 mV (Vh) has been omitted in D.
	Figure 3. . Medium and slow components of the presteady-state currents of hSGLT1 (OFF response). Shown are the current records for the OFF pulse when membrane potential was stepped from the test potential (+50, −10, −50, −90, and −150 mV) back to the holding (−50 mV) for the 100 ms (A) and the 500 ms (B) duration pulses. The data were from the same oocyte as Fig. 1, and the protocol for isolation of the medium (C) and slow components of charge movement (D) is as described in Fig. 2. As τmed for OFF was independent of test voltage (τmed = 15 ms, see Fig. 4 A), the initial point for the fit (Fig. 3 B) was 96 ms for all the OFF pulses.
	Figure 4. . Kinetics of charge movement of hSGLT1: voltage dependence of the medium and slow components. Data was from the experiment of Fig. 1. (A) τ–V relations. The filled symbols represent the ON where membrane potential was stepped from Vh (−50 mV) to different test potentials (Vt). The open symbols represent the time constant of the relaxation of the OFF where membrane potential was returned from Vt to Vh (−50 mV). Error bars are standard errors (SE) of the fit when the SE exceeds the size of the symbol. The OFF responses were independent of the previous test potential (Vt), and open symbols represent the mean of 10 values with Vt varying between +50 and −150 mV. (B) Q–V relations for the medium charge. At each Vm, the medium charge (Q) was obtained as the time integral of the medium presteady-state current for the ON (circles) and OFF (squares) responses. The curve is the fit of the mean of the medium ON and OFF charge to the Boltzmann relation with Qmax = 8.7 ± 0.2 nC, zδ = 1.0 ± 0.1, and V0.5 = −33 ± 1 nC. (C) Q–V rela tions for slow charge. Slow charge (Q) was obtained from the time integral of the slow presteady-state current for the ON and OFF responses. The curve is the fit of the mean of the slow ON and OFF charge to the Boltzmann relation with Qmax = 7.2 ± 0.1 nC, zδ = 1.0 ± 0.1, and V0.5 = −67 ± 1 nC. (D) Q–V relations for total charge. Total charge is the sum of the medium and slow components (described in B and C). Filled and open symbols represent the total ON and OFF, respectively. The smooth curve is the fit of the total OFF charge to the Boltzmann relation with Qmax = 17.2 ± 0.3 nC, zδ = 0.9 ± 0.1, and V0.5 = −44 ± 1 mV.
	Figure 5. . Rising phase of charge movement. The experiment was performed on hSGLT1 using a two-electrode voltage clamp. Current records were obtained by phlorizin subtraction. Vh was −90 mV, data were digitized at 8 μs per sample, and pulse was 6 ms. (A) Presteady-state currents in 100 mM [Na+]o. (B) Dependence of the rising phase on [Na+]o. The current records were obtained at Vt = +50 mV in 100, 25, and 12 mM [Na+]o. (C) τ–V relations for the medium component in 100 and 0 mM [Na+]o. Data were obtained from one oocyte. The open symbols were obtained from the OFF response and represent the mean of 10 values (with test potential varying between +50 and −150 mV). (D) Q–V relations for the medium component in 100 and 0 mM [Na+]o. Q was obtained from the mean of the ON and OFF charges using 100-ms pulses in 100 mM Na+ and 30-ms pulses for 0 mM Na+. The curve (for 100 mM Na+) was drawn using the Boltzmann relation with Qmax = −20.3 ± 0.4 nC, zδ = 1.1 ± 0.1, and V0.5 = −47 ± 1 mV.
	Figure 6. . The rising phase of charge movement in hSGLT1-Q457C. The experiment was performed on TMR6M-labeled Q457C using the cut-open oocyte voltage clamp. The currents have been compensated for membrane capacitance and background current using the P/4 protocol with a Vshp of −150 mV. External and guard solutions contained 100 mM Na-methanesulfonate and internal solution contained 100 K-methanesulfonate. Data was digitized at 5 μs per sample. Vh was −80 mV. The Vt values were +50 and −150 mV. (A) Presteady-state currents in 100 mM [Na+]o. Current trace at +50 mV was averaged from 10 sweeps. The other records were single sweeps. (B) Presteady-state currents 0 [Na+]o. The records were single sweeps. Current and time scales are the same for A and B. (C) τ–V relations for fast charge. Data is from the experiment of A and B (Vh = −80 mV). The filled symbols are from the ON, and open symbols from the OFF response. The circles (•) are obtained from the decay of the presteady-state current with hyperpolarizing pulses in 100 mM [Na+]o. The triangles (▴) were from the rising phase of the presteady-state current with depolarizing pulses and were obtained using a two exponential fit with the constraint that the time constant of decay was the same as those obtained from the same oocyte with 100-ms pulses. The squares (▪) are the time constants of current decay in the absence of Na+.
	Figure 7. . Slow (ΔFslow) and medium (ΔFmed) components of ΔF. The experiment was performed using a two-electrode voltage clamp on a TMR6M-labeled Q457C. Bath solution contained 100 mM Na+. (A) Time course of ΔF for a 100-ms pulse. Vh was −50 mV and the Vt values are indicated next to the traces. (B) The corresponding ΔF records for a 500-ms pulse, and superimposition of the short and long (100- and 500-ms) pulses. The traces from the 100-ms pulses (from A) have been split to overlap with the 500-ms pulses at the onset of the ON and OFF. The dotted lines indicate the ΔF at 100 ms. (C) ΔFmed and ΔFslow. The time course of total ΔF (shown for Vt of +90 and −190 mV) was fitted (smooth curves) to two exponential components: ΔF = ΔFmed (1 − exp(−t/τmed)) + ΔFslow (1 − exp(−t/τslow)), where ΔFmed, ΔFslow, τmed, and τslow are the amplitudes and time constants of the medium and slow components. Parameters obtained from the fit were as follows: at +90 mV, ΔFmed = −1.17 au, τmed = 9.9 ms, ΔFslow = −0.38 au, τslow = 102 ms; at −190 mV, ΔFmed = −1.33 au, τmed = 7.9 ms, ΔFslow = −0.36 au, τslow = 91 ms. Dashed curves represent the ΔFmed and ΔFslow components (from the fit) with the time constants next to the traces. The τ's were independent of Vt (between +50 and −150 mV). τmed was 7.9 ms and 8.5 ms (n = 10) for the ON and OFF responses, respectively. τslow ranged between 60 and 150 ms with a mean of 92 ms for the ON, and 138 ms for the OFF. (D) ΔFmed–V (filled circles) and ΔFslow (open circles) relations. ΔF (at each Vt) was obtained from fitting the relaxation of ΔF to two exponential components (as in C). The ΔFmed–V and total ΔF–V relations (sum of ΔFmed and ΔFslow) were fitted with the Boltzmann relation with ΔFmax = 3.00 ± 0.15 au, zδ = 0.4 ± 0.1, V0.5 = −61 ± 4 mV; and ΔFmax = 3.86 ± 0.24 au, zδ = 0.4 ± 0.1, V0.5 = −61 ± 5 mV.
	Figure 8. . Dependence of the slow and medium components of ΔF on [Na+]. (A) Time course of ΔF when [Na+]o was 100, 25, and 0 mM. [Na+]o was varied by choline replacement. The records from 100- and 500-ms pulses are overlaid, with the 100-ms records split (at 100 ms) to align with the 500-ms records at the onset of the ON and OFF pulses (see Fig. 7 B). Data were collected from a single oocyte, and all three panels share the abscissa and ordinate scales. The 500-ms records were fitted to ΔF = ΔFmed (1 − exp(−t/τmed)) + ΔFslow (1 − exp(−t/τslow)). The τ's obtained are independent of Vt. For ON, respectively at 100, 50, 25, and 0 mM [Na+]o, τmed was 11.6 ± 0.2 ms (n = 9), 11.4 ± 0.3 ms (n = 10), 11.3 ± 0.4 ms (n = 9), and 10.9 ± 0.3 ms (n = 10); and τslow was 149 ± 21 ms (n = 7), 167 ± 28 ms (n = 6), 159 ± 44 ms (n = 6), and 146 ± 22 ms (n = 8). For OFF, τmed was 8.0 ± 0.2 ms (n = 10), 9.6 ± 0.4 ms (n = 10), 10.0 ± 0.4 ms (n = 9), and 10.2 ± 0.4 ms (n = 10); and τslow was 154 ± 41 ms (n = 7), 184 ± 38 ms (n = 8), 119 ± 45 ms (n = 7), and 146 ± 48 ms (n = 7). (B) ΔF–V relations for the medium component (ΔFmed). ΔFmed and ΔFslow were obtained by curve fitting (described in A). At each [Na+]o, the ΔFmed–V curves were fitted (smooth curves) to the Boltzmann relation. At 100 mM [Na+]o, zδ = 0.4 ± 0.03, and V0.5 = −30 ± 3 mV. At 50 mM [Na+]o, zδ = 0.4 ± 0.07, and V0.5 = −59 ± 6 mV. At 25 mM [Na+]o, zδ = 0.4 ± 0.15, and V0.5 = −99 ± 31 mV. For comparison, the curves have been normalized to the maximal extrapolated (slow-compensated) fluorescence change (ΔFmax) observed in 100 mM [Na+]o and have also been shifted to align at the extrapolated depolarizing limit (see Loo et al., 1993; Meinild et al., 2001). The dotted line at 0 Na+ was the Boltzmann relation with the same ΔFmax and zδ (0.4) as 100 mM [Na+], and V0.5 of −200 mV.
	Figure 9. . Fast fluorescence changes. Data was obtained using the cut-open oocyte on a TMR6M-labeled Q457C. External and guard solutions contained 100 mM Na-methanesulfonate and internal solution contained 100 mM K-methanesulfonate. Subtracting holding potential (Vshp) was −150 mV. The records were the averages of 20 sweeps. Data was digitized at 5 μs per sample (A) and 50 μs per sample (B). C and D show the time course of ΔF in Na+-free solution (choline replacement). Digitizing rate was 5 μs per sample (C) and 50 μs per sample (D).
	Figure 10. . Voltage dependence of the fast and medium components of ΔF. Time course of ΔF (for a 5-ms pulse) at Vt of +50 and −150 mV (Vh = −80 mV) in 100 mM [Na+]o (+Na) or 0 Na+ (−Na). Data is from the experiment of Fig. 9. The fluorescence records were fitted to two exponential components (denoted by fast and medium). The number next to each trace is the time constant of the fast component (τfast). A and B show the time courses of ΔF (for ON [A] and for OFF [B]). (C) τ–V relations for τfast (ON). Open symbols (0 Na+) were obtained from 5-ms pulses, and filled symbols (+Na) were from 40-ms pulses. (D) τ–V relations for τfast (OFF). Data were from 5-ms pulses. (E and F) τ–V relations for τmed (for ON [E] and for OFF [F]). Data were from 40-ms pulses.
	Figure 11. . Na+ dependence of the fast and medium components of ΔF. The fast and medium fluorescence amplitudes were obtained by fitting the time course of ΔF to two exponential components (compare, Fig. 7). (A) ΔF–V relation for the fast component. The curve is the fit of the data in 100 mM Na+ to the Boltzmann relation with ΔFmax = 7.0 au, z = 0.6, and V0.5 = −48 mV. The ΔF–V curve in 0 Na+ has been shifted to align with that of 100 mM Na+ at +50 mV. (B) ΔF–V relation for the medium component. The curve is the fit of the data in 100 mM Na+ with ΔFmax = 3.8 ± 1.9 au, z = 0.5 ± 0.3, and V0.5 = −100 ± 47 mV. The ΔF–V curve in 0 Na+ has been shifted to align with that of the 100 mM Na+ at +50 mV.
	Figure 12. . Correlation between slow charge and fluorescence. Experiment was performed using a two-electrode voltage clamp on TMR6M Q457C. (A) Correlation between slow charge and fluorescence (red traces) in 100 mM [Na+]o. The charge record has been normalized to agree with ΔF at 40 and 750 ms. Shown are records at hyperpolarizing voltages of −110, −130, and −150 mV from Vh = −50 mV. ΔF contained two voltage-independent time constants of 9 ± 1 ms (n = 10) and 169 ± 19 ms (n = 7) for the ON, and 9 ± 1 ms (n = 10) and 154 ± 9 ms (n = 9) for the OFF response. τslow for charge (ON) was 104 ± 10 ms (n = 3). (B) Correlation between slow charge and slow fluorescence in Na+-free solution. Pulse duration was 300 ms, and Vh was −50 mV. The current records have been normalized to agree with the fluorescence at 50 and 300 ms (as in A). ΔF (for the ON pulse) contained two voltage-independent time constants of 11 ± 1 ms (n = 8) and 144 ± 15 ms (n = 8). τslow for charge (ON) was 63 ± 8 ms (n = 3).
	Figure 13. . Correlation between medium charge and fluorescence. Experiment was performed using a two-electrode voltage clamp on a TMR6M-labeled Q457C in 100 mM NaCl buffer. Vh was −50 mV. The time course of Q and ΔF are compared at +30 and −150 mV. Q was obtained from the total current by subtraction of the steady-state current and the oocyte membrane capacitive transient. The traces have been normalized to agree at the end of the voltage pulse (75 ms). The numbers next to the traces are the time constants of Q and ΔF.
	Figure 14. . Correlation between fast charge and fluorescence. The experiment was performed using the cut-open oocyte on TMR6M-labeled Q457C (from the oocyte of Fig. 6). Membrane potential was held at −80 mV and stepped to +50 mV. (A) Comparison of the rising phase of the presteady-state current (I) and fluorescence (ΔF) in 100 mM [Na+]o. I is from Fig. 6 A and ΔF is from Fig. 9 A. (B) Comparison of charge and ΔF in absence of Na+. I is from Fig. 6 B and ΔF is from Fig. 9 C.
	Figure 15. . Kinetic model for Na+/glucose cotransport (modified from Parent et al., 1992). (A) The transporter has six kinetic states consisting of the empty transporter C (states C1 and C6), the Na+-bound CNa2 (states C2 and C5) and the Na+- and sugar-bound SCNa2 (states C3 and C4) in the external and internal membrane surfaces. Two Na+ ions bind to the transporter 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↔C6); and Na+ binding/dissociation (C1↔C2). The simplifying assumption (for high external [Na+]) is that the two Na+ binding steps are lumped into one. The distribution of the conformations depends on membrane voltage. The transporter is in C2 at large hyperpolarizing voltages, and C6 at large depolarizing voltages. In the TMR6M-labeled Q457C, sugar transport is abolished, and since Na+ binding/dissociation at the cytoplasmic surface may be neglected because internal [Na+] is low and internal Na+ binding constant is high (see materials and methods), only the partial reactions in the shaded area are studied. (B) Seven-state model for presteady-state current. C1a and C1b represent intermediate states between C1 and C6, and C2a and C2b are the states with one Na+ bound. The rate constants (kij) for transition between two states (Ci→Cj) are defined by kij = kijo exp(−ɛijFV/RT), where kijo is a voltage-independent rate, ɛij is the voltage dependency, and F, R, and T have their usual physicochemical meanings. (C) A simplified five-state kinetic model for charge movement and the assumptions on the rate constants that were used for the simulation described in Fig. 16.
	Figure 16. . A model simulation for the presteady-state currents (A and B) and τ–V relations (C and D) in 100 and 0 mM external [Na+]. Simulations were performed on the kinetic scheme: C2↔C1↔C1a↔C1b↔C6 using the assumptions and rate constants summarized in Fig. 15 C. The simulation was performed with the membrane potential held at −80 mV at 20°C, and the voltage pulses (between +90 and −150 mV in 20 decrements) were applied for 10 to 500 ms. The differential equation relating the time evolution of states was solved numerically at each test voltage using Berkeley Madonna. The equation is The sum of the occupancy probabilities in all the states is 1 (C1+C2+C1a+C1b+C6=1). For simulation, the total number of transporters in the oocyte plasma membrane was 5 x 109 transporters (Zampighi et al., 1995). There are four components of charge movement (C2↔Cj) the charge (Iij) was calculated by Iij = \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}e\;({\varepsilon}_{ij}\;+\;{\varepsilon}_{ji})(k_{ij}C_{i}\;-\;k_{ji}C_{j})\end{equation*}\end{document}, where e is the elementary charge, ɛij is the voltage dependence, and kij is the rate constants for transition from Ci to Cj (see also Krofchick and Silverman, 2003). The eigenvalues (which are reciprocals of the time constants) of the matrix were obtained using MATLAB 6.0 (The MathWorks Inc.).

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