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Department of Physiology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, CA 90095

Correspondence to Donald Loo: dloo{at}mednet.ucla.edu

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This study examines the conformations of the Na⁺/glucose cotransporter (SGLT1) during sugar transport using charge and fluorescence measurements on the human SGLT1 mutant G507C expressed in Xenopus oocytes. The mutant exhibited similar steady-state and presteady-state kinetics as wild-type SGLT1, and labeling of Cys507 by tetramethylrhodamine-6-maleimide had no effect on kinetics. Our strategy was to record changes in charge and fluorescence in response to rapid jumps in membrane potential in the presence and absence of sugar or the competitive inhibitor phlorizin. In Na⁺ buffer, step jumps in membrane voltage elicited presteady-state currents (charge movements) that decay to the steady state with time constants _med (3–20 ms, medium) and _slow (15–70 ms, slow). Concurrently, SGLT1 rhodamine fluorescence intensity increased with depolarizing and decreased with hyperpolarizing voltages (F). The charge vs. voltage (Q-V) and fluorescence vs. voltage (F-V) relations (for medium and slow components) obeyed Boltzmann relations with similar parameters: z (apparent valence of voltage sensor) 1; and V_0.5 (midpoint voltage) between –15 and –40 mV. Sugar induced an inward current (Na⁺/glucose cotransport), and reduced maximal charge (Q_max) and fluorescence (F_max) with half-maximal concentrations (K_0.5) of 1 mM. Increasing [MDG]_o also shifted the V_0.5 for Q and F to more positive values, with K_0.5's 1 mM. The major difference between Q and F was that at saturating [MDG]_o, the presteady-state current (and Q_max) was totally abolished, whereas F_max was only reduced 50%. Phlorizin reduced both Q_max and F_max (K_i 0.4 µM), with no changes in V_0.5's or relaxation time constants. Simulations using an eight-state kinetic model indicate that external sugar increases the occupancy probability of inward-facing conformations at the expense of outward-facing conformations. The simulations predict, and we have observed experimentally, that presteady-state currents are blocked by saturating sugar, but not the changes in fluorescence. Thus we have isolated an electroneutral conformational change that has not been previously described. This rate-limiting step at maximal inward Na⁺/sugar cotransport (saturating voltage and external Na⁺ and sugar concentrations) is the slow release of Na⁺ from the internal surface of SGLT1. The high affinity blocker phlorizin locks the cotransporter in an inactive conformation.

A.-K. Meinild's present address is August Krogh Institute, University of Copenhagen, Universitetsparken 13, Copenhagen 2100, Denmark.

Abbreviations used in this paper: au, arbitrary unit of fluorescence intensity; SGLT1, Na⁺/glucose cotransporter; hSGLT1, human Na⁺/glucose cotransporter; MDG, -methyl-D-glucopyranoside; TMR6M, tetramethylrhodamine-6-maleimide; MTSEA, 2-aminoethyl methanethiosulfonate hydrobromide.

	INTRODUCTION

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The Na⁺/glucose cotransporter, SGLT1, is a member of a large family of proteins (SLC5) that uses the Na⁺ electrochemical gradient to transport substrates (sugars, amino acids, osmolytes, neurotransmitters, ions, and water) against their concentration gradients. We have proposed that the protein functions by an alternating access mechanism, and a simplified six-state ordered kinetic model has been proposed (Parent et al., 1992b; Loo et al., 1998). In previous studies, we characterized the voltage-dependent partial reactions of SGLT1 in the absence of glucose using charge and fluorescence measurements on an hSGLT1 mutant Q457C where sugar transport is abolished after labeling of Cys457 by the fluorophore tetramethylrhodamine-6-maleimide (TMR6M) (Loo et al., 1998, 2005; Meinild et al., 2002). Our conclusion was that the presteady-state currents (in the absence of glucose) are associated with Na⁺ binding, and isomerization of the empty carrier involves a series of four conformational changes (Loo et al., 2005).

The goal of the present study is to examine the conformations of the transporter under sugar transport conditions. Our kinetic model predicts that the conformational occupancy distribution of the transporter depends on the sugar concentration. We expressed hSGLT1 mutant G507C in Xenopus laevis oocytes and recorded changes in transporter currents (steady state and presteady state) and rhodamine fluorescence after step jumps in membrane voltage. The mutant transporter has similar steady-state and presteady-state kinetics as hSGLT1, before and after labeling of Cys507 by TMR6M. The charge and fluorescence measurements provide independent and complementary views of protein conformational changes. Our model predicts that the charge movements in the Na⁺/glucose cotransport cycle are associated with external Na⁺ binding and translocation of the empty transporter, whereas changes in fluorescence could originate from any (or all) of the conformational transitions of the transport cycle. By using a fully functional protein, we are now able to study the partial reactions involved in sugar binding and translocation. We expected that the fluorescence measurements would reveal conformations not detected by charge measurements. Our data do uncover a novel, slow conformational change with sugar transport that is not associated with charge movement, and simulations indicate that this is due to a slow electroneutral step in the transport cycle associated with the release of Na⁺ from SGLT1 in the internal membrane surface. We found that sugar shifted the conformations of SGLT1, resulting in a reduction in the occupancy probability in the outward-facing, and increasing the occupancy probability in the inward-facing, Na⁺-bound conformations. Phlorizin, the high affinity inhibitor, locked the transporter in the phlorizin-bound form and proportionally reduced the occupancy probabilities in all other conformations.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Preparation and Maintenance of Oocytes
Mature Xenopus laevis oocytes were isolated, defolliculated, and injected with hSGLT1 or hSGLT1 mutant G507C cRNA (see Loo et al., 1993, 1998). The G507C mutant was constructed using standard PCR techniques and confirmed by DNA sequencing. Oocytes expressing hSGLT1 G507C were labeled with TMR6M (Invitrogen; Loo et al., 1998; Meinild et al., 2002). We determined the maximal fluorescence change (F_max, see below) as a function of the incubating [TMR6M]_o (from 1 to 100 µM) and duration of labeling (from 1 to 30 min), and found that the extent of labeling of Cys 507C by the fluorophore was saturating in 100 µM [TMR6M]_o for 10 min in NaCl buffer at the normal resting membrane potential. We assume that all the functional transporters are labeled. After labeling, oocytes were washed free of dye and kept in NaCl buffer in the dark until use. MTSEA (2-aminoethyl methanethiosulfonate hydrobromide; Toronto Research Biochemicals) was prepared and used as previously described (Loo et al., 1998). Oocytes were bathed in a NaCl buffer containing (in mM) 100 NaCl, 2 KCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES, pH 7.4. MDG or phlorizin was added to the superfusing NaCl buffer at the reported concentrations. Na⁺ concentration was varied by equimolar replacement of Na⁺ with choline.

Uptake Experiments
Sugar uptake into oocytes was measured using 50 µM ¹⁴C--methyl-D-glucopyranoside (¹⁴C-MDG, Amersham Biosciences), a nonmetabolized sugar analogue that is transported by human SGLT1 (see Ikeda et al., 1989). The initial rates of sugar uptake into oocytes were measured at 20–23°C for 10–60 min. Experiments were repeated at least twice on oocytes isolated from different donor frogs. Uptake was expressed in pmol per oocyte per minute (mean ± SEM).

Combined Electrophysiological and Fluorescence Experiments
Electrophysiological and fluorescence experiments were performed simultaneously, using two-electrode voltage clamp fluorometry (Loo et al., 1998, 2005; Meinild et al., 2002). A standard pulse protocol was applied where membrane potential was held at –50 mV (V_h) and stepped to various test potentials (V_t from +90 to –150 mV in 20 mV decrements) for 100 (or 500) ms before returning to V_h. The current records were the averages of three sweeps, and the fluorescence records were averages of either 3 or 10 sweeps. To minimize the effects of photobleaching, the 500-ms fluorescence records were averages of three sweeps. Records were filtered at 500 or 50 Hz, depending on the sampling interval (0.1 or 0.5 ms per sample). Note that in the present studies, we do not record the fast components of F observed in our previous study using cut-open oocyte voltage clamp fluorometry (Loo et al., 2005). Fluorescence intensity is expressed as arbitary units (au). In TMR6M-labeled oocytes expressing mutant G507C bathed in NaCl buffer, F_max/F_total is 1%, where F_max is the maximal fluorescence intensity change and F_total is the total fluorescence intensity. Fluorescence data have been corrected for photobleaching and rundown (Meinild et al., 2002). All experiments were performed at room temperature (20–23°C).

Data Analysis
Steady-State Currents.
To obtain the current–voltage (I-V) relations of the sugar-induced current, the pulse protocol was applied with 100-ms pulses. The sugar-induced current was obtained by subtracting the current in NaCl buffer (measured at 100 ms) from the current with sugar (MDG) added to the external solution. The substrate-induced currents (at each voltage) were fitted to the equation

(1)

where I_max is the maximal current, [S]_o is the external substrate (MDG or Na⁺) concentration, K_0.5 is the half-maximal substrate concentration ([S]_o at 50% I_max), and n is the Hill coefficient. For the kinetics of sugar activation, n was fixed at 1 (Birnir et al., 1991; Parent et al., 1992a; Mackenzie et al., 1998).

Isolation of Presteady-State Currents.
Total membrane current in response to a voltage pulse consisted of the bilayer capacitive transient, the presteady-state currents of SGLT1, and the steady-state currents. In the absence of external sugar, the steady-state currents consisted of the background (endogenous) currents of the oocyte and the Na⁺ leak (uniporter mode) mediated by SGLT1 (Parent et al., 1992b; Loo et al., 1998). We found previously that the presteady-state currents of hSGLT1 (in the absence of external sugar) contained three components, with time constants () ranging from fast (0.2–1.5 ms), medium (3–20 ms), and slow (30–160 ms), and the method of isolating the three components has been described in detail (Loo et al., 2005). The focus of this study is the medium component, as the fast component is beyond the resolution of the two-electrode voltage clamp (1 ms). Since the kinetics of the medium component (-V and Q-V relations) obtained from records corrected for the slow component (describe above) are similar to those obtained from uncorrected records (Loo et al., 2005), the medium component was estimated from uncorrected records using 100-ms voltage pulses (Loo et al., 1993; Hazama et al., 1997; Quick et al., 2001; Meinild et al., 2002). In these experiments, total current relaxation (I_tot) was fitted to the equation

(2)

where I_ss is the steady-state current, I_cmexp(–t/_cm) is the bilayer capacitance current with initial value I_cm and time constant _cm, and I_medexp(–t/_med) is the medium component of SGLT1 presteady-state current with initial value I_med and time constant _med. The medium component of presteady-state current (I_medexp(–t/_med) was isolated by subtraction of the steady-state (I_ss) and membrane bilayer capacitance (I_cmexp(–t/_cm)) from the total current. The medium charge movement associated with SGLT1 was obtained from the integral of the medium component of presteady-state current. This analysis was extended to the presteady-state current (or carrier transients) in the presence of sugar. At steady state, the current (I_ss) consisted of the oocyte background current, Na⁺-uniporter current (of SGLT1), and the Na⁺/sugar cotransport current.

In experiments where the slow and medium components were isolated (Fig. 2), 100- and 500-ms pulses were applied (Fig. 2, A and B). The slow time constant (_slow) was estimated from the 500-ms current records after the medium component has decayed (the period was five times the time constant of the medium component, see Fig. 2 of Loo et al., 2005 for detailed description). The early phase was obtained by extrapolation of the exponential fit to the peak of the capacitive transient, typically two sample points after onset of the voltage pulse. The slow charge was obtained from the integral of the slow component of presteady-state current. The medium charge was then estimated using 100-ms pulses after subtraction for the slow component and steady-state current.

Fitting of Q-V and F-V Relations.
The charge vs. voltage (Q-V_m) relations for medium and slow charge could, to a first approximation, be fitted to a single Boltzmann function (Loo et al., 1993; Hazama et al., 1997):

(3)

where Q_max = Q_dep – Q_hyp, Q_dep and Q_hyp are the Q (absolute value) at depolarizing and hyperpolarizing limits, V_m is membrane potential, F is the Faraday, R is the gas constant, T is the absolute temperature, V_0.5 is the membrane potential at 50% Q_max (or the midpoint voltage), and z is the maximum steepness factor for the dependence of Q on voltage, or the apparent valence of the voltage sensor. z is the product of the apparent valence of the movable charge (z), and the fraction of the membrane electric field traversed by the charge (). We denote z_Q for z obtained from charge movement.

The Boltzmann relation was also used to empirically fit the dependence of the change of fluorescence intensity (F) on membrane voltage (Loo et al., 1998; Meinild et al., 2002). The parameters obtained were the maximal fluorescence intensity change (F_max), the membrane voltage at 50% F_max (), and the apparent valence or voltage-steepness factor for fluorescence (z_F).

Fits of data to equations were performed using either Sigmaplot 2002 (SPSS), or Clampfit 8.1 (Axon Instruments, Inc.). On data obtained on a single oocyte, the statistics are given by the estimates and the standard error of the fit. When data is from a population, the statistics are given by the means and standard errors of the means with the number of samples. While data are shown for representative experiments, all experiments were performed on at least three oocytes from different batches.

Simulation of SGLT1
The differential equation for the eight-state model for Na⁺/glucose cotransport (Fig. 9) is shown as Eq. 4 (below). C_i is the occupancy probability in state i, and C₁ + C₂ + C₃ + C₄ + C₅ + C₆ + C_a + C_b = 1. Excluding the substrate-binding reactions, the rate constants k_ij for transitions from C_i C_j are given by k_ij = k_ij^o exp(–_ijF/RT), where k_ij^o is a voltage-independent rate, _ij is the equivalent charge movement (up to the transition state from C_i C_j), and F, R, and T have their usual physicochemical meanings (Parent et al., 1992b). Na⁺ and sugar binding to the protein on the external and internal membrane surfaces is represented by pseudo-rate constants k₁₂ = k₁₂^o [Na]_o² exp(–₁₂FV/RT), k₂₃ = k₂₃^o[MDG]_o, k₆₅ = k₆₅^o[Na]_i², and k₅₄ = k₅₄^o[MDG]_i. There are four components of presteady-state current associated with voltage-sensitive reactions (shaded region of Fig. 9): C₂C₁, C₁C_a, C_aC_b, and C_bC₆. The presteady-state current (I_ij) due to C_iC_j was calculated by I_ij = e(_ij + _ji)(k_ijC_ij – k_jiC_j), where e is the elementary charge (Parent et al., 1992b; Loo et al., 2005). The total SGLT1 current (I) is I = N_T (I₁₂ + I_1a + I_ab + I_b6), where N_T is the total number of transporters in the oocyte plasma membrane. The simulations shown in Figs. 10–13 and Table I were performed for wild-type hSGLT1 and TMR6M-labeled mutant G507C at 20°C using the parameters of Table II with [Na⁺]_o = 100 mM, [Na⁺]_i = 5 mM, [MDG]_i = 0, and N_T = 10¹⁰ transporters. All the simulations were performed at 0–100 mM [Na⁺]_o and 0–100 mM [sugar]_o.

External phlorizin was assumed to bind to SGLT1 in the Na⁺-bound conformation (Parent et al., 1992b): [CNa₂]'W[CNa₂Pz]', thus in the presence of phlorizin, there are nine states (Fig. 9). Simulations of the phlorizin-binding model (using the rate constants of Table II) were performed in a similar manner to the eight-state model, with modifications to include the phlorizin-bound state [CNa₂Pz]'(C₇): C₁ + C₂ + C₃ + C₄ + C₅ + C₆ + C_a + C_b + C₇ = 1; d/dt C₇ = –k₇₂C₇ + k₂₇C₂ ; and d/dt C₂ = –(k₂₁ + k₂₅ + k₂₃ + k₂₇)C₂ + k₁₂C₁ + k₅₂C₅ + k₃₂C₃ + k₇₂C₇.

Computer simulations were performed using Berkeley Madonna 8.0.1. The voltage pulse protocol was simulated by determining the initial occupancy probabilities at the holding potential (V_h = –50 mV), and as membrane potential was stepped to each test value (V_t ranging between +50 and –150 mV), the time course of the occupancy probabilities, cotransporter currents, and fluorescence (F) were obtained by numerically integrating the differential equation (Eq. 4) using the Runge-Kutta Method. Steady-state kinetic parameters were simulated by generating the I-V relations as functions of [Na⁺]_o and [sugar]_o. Kinetic parameters I_max, K_0.5, and n (at each V_m) were obtained by fitting the I vs. [sugar]_o (or [Na⁺]_o) relations to Eq. 1.

For presteady-state simulations, the transient cotransporter currents for the ON and OFF responses at each test voltage (V_t) were integrated to obtain the charge (Q). The Q vs. V_t relations were fitted with the Boltzmann relation (Eq. 3) to obtain the maximal charge (Q_max), apparent valence of the voltage sensor (z_Q), and midpoint voltage (V_0.5). The eigenvalues of the matrix (Eq. 4) were obtained using MATLAB 6.0 (The MathWorks Inc.). The time constants were the reciprocals of the eigenvalues (Loo et al., 2005).

(4)

Fluorescence experiments were simulated by assuming that changes of fluorescence intensity (F) with step jumps in membrane voltage are due to changes in occupancy probabilities: F qy₁C₁ + qy₂C₂ + qy_aC_a + qy_bC_b + qy₃C₃ + qy₄C₄ + qy₅C₅ + qy₆C₆, where qy_j is the apparent quantum yield of the fluorophore (TMR6M) when SGLT1 is in conformation C_j. The simulations were performed with qy₁ = 3, qy₂ = 1, qy_a = 3, qy_b = 3, qy₄ = 3, qy₅ = 3, and qy₆ 5–6. In practice, F was determined by the relative quantum yields qy₅/qy₂ 3, and qy₆/qy₂ 6 (see Discussion below).

The Dixon analysis (for the determination of the inhibitory constant K_i for phlorizin, Table I) was simulated by determining the intersection of the straight lines–1/I vs. [phlorizin]_o relations at [MDG]_o = 1 and 2 mM, where I is the sugar-coupled current (at –150 mV) at each [phlorizin]_o (see Panayotova-Heiermann et al., 1995). [Phlorizin]_o used was 0, 50, 100, 200, 500, 1,000, 5,000, and 10,000 nM.

	RESULTS

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Part I. Steady- and Presteady-State Kinetics
We first briefly describe the steady-state kinetic properties of hSGLT1 G507C, and the effects of MTSEA and TMR6M. The kinetics of the presteady-state currents in the absence of glucose and the fluorescence changes of the TMR6M-labeled mutant protein with step jumps in membrane voltage are then presented. These provide the basis for describing the conformations of SGLT1 under sugar-transporting conditions by charge and fluorescence measurements (Part II).

Steady-State Kinetics
¹⁴C-MDG Uptake.
The rates of ¹⁴C-MDG (50 µM) uptake into oocytes were comparable for oocytes expressing the mutant transporter and wild-type hSGLT1. In a representative experiment on the same batch of oocytes (each determination being the mean of 8–10 oocytes and expressed as the mean ± SEM), in the presence of external Na⁺ (100 mM), uptake by oocytes injected with hSGLT1 G507C-cRNA was 143 ± 8 pmol/oocyte/h versus 176 ± 10 pmol/oocyte/h for oocytes injected with wild-type hSGLT1-cRNA. This uptake rate was 100-fold greater than that of noninjected oocytes (1.4 ± 0.1 pmol/oocyte/h). Uptake of MDG by hSGLT1 G507C was dependent on external Na⁺ and blocked by the SGLT1-specific inhibitor phlorizin. When external Na⁺ was replaced by choline, the rate of sugar uptake (1.6 ± 0.1 pmol/oocyte/h) was similar to that of noninjected control oocytes (0.6 ± 0.1 pmol/oocyte/h). Phlorizin (500 µM) in the external solution also reduced the rate of ¹⁴C-MDG uptake by the mutant to that of controls.

Na⁺ and Sugar Activation.
The total current from an oocyte expressing hSGLT1 G507C in NaCl buffer is shown in Fig. 1 A. Membrane potential (V_m) was held at –50 mV (V_h) and stepped to a series of test values for 100 ms (from +50 to –150 mV) before returning to V_h. The current relaxation consisted of an initial membrane capacitive transient (time constant 0.8 ms) followed by the decay of the presteady-state current to steady state (with 3–20 ms) (Loo et al., 1993, 2005). The current records when 1 mM MDG was added to the bathing medium are shown in Fig. 1 B. At each test voltage, MDG increased the steady-state current, and there was a shift in the profile of the presteady-state current records. This is particularly noticeable in the OFF response when the test voltage was returned to V_h (the effect of sugar on presteady-state current will be presented below).

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   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 MDG was added to the bath solution. (C) Steady-state I-V relations. The MDG-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 MDG-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 = 1.6 ± 0.1 mM. (E) Voltage dependence of . (F) Voltage dependence of . Experiment was performed by increasing [Na+]o (from 0 to 100 mM) with [MDG]o maintained at 25 mM. Errors bars are standard errors (SE) of the fit when SE exceeds the size of the symbol.

The dependence of the MDG-induced current on [Na⁺]_o was sigmoid. The Hill coefficient was 1.5 and independent of membrane voltage (unpublished data). Fig. 1 F shows the dependence of the half-maximal concentration for Na⁺ ( ) on voltage. At –50 mV, was 20 ± 2 mM and decreased to 2.8 ± 0.4 mM at –150 mV. In three experiments, at –50 mV was 18 ± 1 mM.

Effects of MTSEA and TMR6M.
The rate of ¹⁴C-MDG uptake for oocytes expressing hSGLT1 mutant G507C preincubated in MTSEA (1 mM for 15 min in NaCl buffer) was 81 ± 14% (n = 5) of the control nontreated oocytes, indicating that the mutant hSGLT1 G507C retained the ability to transport sugar after Cys507 was modified by MTSEA. We confirmed that the Cys507 was derivatized by MTSEA from the reduction in maximal fluorescence change (F_max, see Fig. 3 below) after the oocytes were preincubated in the reagent. In four experiments, compared with control nonpreincubated oocytes, F_max (measured with the oocytes bathed in NaCl buffer) was reduced 93% (from 9.1 ± 1.0 au to 0.06 ± 0.14 au). In control experiments on oocytes expressing wild-type hSGLT1, exposure to MTSEA had no effect on the kinetics of hSGLT1 (see also Loo et al., 1998), and after labeling by TMR6M, fluorescence changes induced by voltage jumps were not observed in noninjected oocytes and hSGLT1-expressing oocytes (unpublished data).

The affinity for sugar was unaffected by either MTSEA or TMR6M. In representative experiments on the same oocytes (at –50 mV), was 1.9 ± 0.1 mM before, 2.0 ± 0.2 mM after MTSEA, and 2.4 ± 0.3 mM after TMR6M. The I-V curves of the sugar-induced currents (at 20 mM MDG) between MTSEA-labeled and nonlabeled oocytes were similar, except for a slight reduction (6 ± 4%, n = 5) in the current at each voltage.

Presteady-State Kinetics
Charge Movement.
We have previously found that in two-electrode voltage clamp experiments on hSGLT1, the presteady-state currents contained medium and slow components with time constants of 3–20 and 30–100 ms (Loo et al., 2005). Similar time constants were also observed in mutant hSGLT1 G507C (Fig. 2). Like wild-type hSGLT1, the transient currents of the mutant did not quite reach steady state at 100 ms (Fig. 2 A), and there was a slow decay to steady state revealed in the 500-ms pulses (Fig. 2 B).

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   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) -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.

The -V relation for slow charge was similar to the medium (Fig. 2 C). _slow for ON was maximal at hyperpolarizing voltages ( = 70 ms at –150 mV) and decreased as the test voltage was made positive ( = 15 ms at +50 mV). _slow for OFF was also independent of voltage and was 39 ± 5 ms (n = 10). The Q_slow-V curve obeyed the Boltzmann relation with a Q_max of 7 nC, z_Q of 1.0, and a V_0.5 of –62 mV (Fig. 2 E). When the medium and slow charges were added to obtain the total charge (Q_total), the Q_total-V curve followed the Boltzmann relation with a Q_max of 19 nC, z_Q of 1.0, and V_0.5 of –50 mV (Fig. 2 F).

We focused our attention on the medium component and estimated medium charge from 100-ms pulses uncompensated for slow charge. Previously we found the _med-V and Q_med-V relations obtained from current records corrected for slow charge were similar to those obtained from uncorrected records (Loo et al., 2005). For the medium component, the maximal charge (Q_max) ranged from 5 to 30 nC, depending on the level of expression of mutant G507C in the oocyte plasma membrane. z_Q was 1.0 ± 0.1 (n = 6). V_0.5 ranged between –30 and –47 mV, with a mean of –38 ± 3 (n = 6).

TMR6M did not affect the kinetics of the presteady-state currents. In a representative experiment on the same oocyte where we monitored the medium charge movement before and after TMR6M, z_Q, V_0.5, and Q_max were 0.9 ± 0.1, –30 ± 1 mV, and 18 ± 1 nC before, and 0.9 ± 0.1, –28 ± 1 mV, and 18 ± 1 nC after TMR6M. The population means for z_Q and V_0.5 after labeling by TMR6M were 1.0 ± 0.1 (n = 6), and –35 ± 3 mV (n = 6). V_0.5 ranged between –28 and –45 mV. The -V relations (for medium charge) were the same before and after labeling by TMR6M (unpublished data).

Fluorescence.
The time course of the fluorescence signal (F) from a TMR6M-labeled hSGLT1 G507C-expressing oocyte in response to a series of voltage pulses (from V_h –50 mV) is shown in Fig. 3. Fluorescence intensity increased with depolarizing voltages and decreased with hyperpolarizing voltages. Fluorescence intensity returned to baseline when the test pulse was stepped back to V_h. Like charge movement, the fluorescence records consisted of medium and slow components. The slow component was much more apparent in the depolarizing direction, especially the OFF response (Fig. 3, A vs. B).

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Changes of fluorescence intensity (F) 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 F observed with selected 100-ms pulses. (B) Time course of F 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) Fmed-V relation. The line was the fit of the data with the Boltzmann relation. (D) Fslow-V relation. As in C, the line was the fit with the Boltzmann relation. (E) -V relation for the medium component of F. (F) -V relation for the slow component of F. The open symbols (in E and F) are the time constants for the OFF pulses.

The main difference between the medium and slow components was their amplitude. Maximal fluorescence change for the medium component (F_max^med) was 64 ± 1% (n = 7) of the total maximal fluorescence change (F_total^max).

The dependence of the relaxation time constants for F on voltage differed between medium and slow components. For medium, was 15 ± 1 ms at –150 mV and decreased to 8.7 ± 0.1 ms at +90 mV for ON (filled symbols, Fig. 3 E). For OFF, was independent of test voltage (open symbols, Fig. 3 E) and was 14.4 ± 0.3 ms (n = 12). For slow F (Fig. 3 F), (139 ± 8 ms, n = 10) was relatively independent of voltage, for ON and OFF = 150 ± 14 ms.

Part II. Presteady-State Kinetics in Sugar
Sugar increased the steady-state current (due to Na⁺/sugar cotransport, see Fig. 1) and shifted the profile of the presteady-state currents. Fig. 4 A shows the total current records for the OFF-pulse when membrane potential was stepped from various test values (V_t, from +50 to –150 mV) back to the holding potential (–50 mV) from an oocyte in 0, 1, and 10 mM MDG. The most pronounced effect of sugar was the reduction in presteady-state current with hyperpolarizing voltages. For example, at 10 mM [MDG]_o, the OFF currents for hyperpolarizing voltages was completely inhibited (Fig. 4 A). As in the absence of sugar, the ON and OFF charges were equal and opposite, and the Q-V relations at each [MDG]_o fitted the Boltzmann relation (unpublished data). With increasing [MDG]_o (from 0 to 100 mM), there was a reduction and eventual elimination in the maximal charge (Q_max) with a K_0.5 of 1.1 mM (Fig. 6 and Fig. 7 A). The midpoint voltage (V_0.5) shifted to more positive values (maximal shift was +60 mV, from –45 mV in NaCl alone to +15 mV in 10 mM MDG) with a K_0.5 of 1.3 mM (Fig. 7 C). z_Q (=1.0) was unaffected by MDG.

View larger version (12K): [in this window] [in a new window] [Download PPT slide]   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 MDG. OFF current records in Na+ buffer (top), with 0.1 (middle) and 1 mM MDG (bottom) added to the external solution. At Vh, the MDG-induced currents were 14 and 99 nA at 0.1 and 1 mM MDG. (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.

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Effect of sugar on fluorescence. Time course of F with step jumps in membrane voltage in NaCl buffer with various [MDG]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 (F = 0) at Vh. Abscissa and ordinate scales are the same for all panels. In this experiment, five [MDG]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 [MDG]o is due to the compensation for photobleaching.

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Figure 6. Comparison of charge and fluorescence (F) records in the presence and absence of saturating [MDG]o (100 mM). Presteady-state current records for ON and OFF (compensated for oocyte membrane capacitance) and F. 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 F levels. (A) In NaCl buffer. (B) In NaCl buffer with 100 mM [MDG]o.

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Figure 7. Effect of sugar and phlorizin on charge and fluorescence. (A) Dependence of Qmax and Fmax on [MDG]o. The line was the fit of the charge data to Eq. 1. The line through the F 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 [MDG]o. (B) Dependence of Qmax and Fmax on [phlorizin]o. The lines were fits of the data. (C) Dependence of the shift of V0.5 for charge (V0.5Q) and fluorescence (V0.5F) on [MDG]o. The line was obtained by fitting all the data. (D) Dependence of V0.5 for charge and F on [phlorizin]o. The dashed lines represent the mean of the data points.

The time constant () of the medium component of F was also unaffected by MDG. On average, for ON, at +30 mV, was 10.7 ± 0.2 ms (n = 5). At –150 mV, F was too low for reliable estimates of (compare Fig. 5). On average, the time constant for OFF was 17.1 ± 0.3 ms (n = 5). When large depolarizing voltages were applied at saturating sugar concentrations (>10 mM, compare Fig. 5 and Fig. 6 B), the time course of F was slow, with ranging between 600 and 900 ms. In three experiments at 100 mM MDG where 500-ms pulses were applied, the mean was 800 ± 100 ms.

Blockade by Phlorizin
Phlorizin reduced, and at high concentrations eliminated, the presteady-state current, but did not alter the profiles of the OFF current records (Fig. 4 B). The maximal charge (Q_max) was reduced with [phlorizin]_o, with an inhibitory constant (K_i) of 0.3 µM (Fig. 7 B). There was no change in the V_0.5 (Fig. 7 D) and z_Q (not depicted)

Fig. 8 shows the effect of phlorizin on the fluorescence records. The inhibitor reduced F at every test voltage. At the highest concentration applied (100 µM), F was completely inhibited. The K_i for the reduction of F_max with [phlorizin]_o was 0.4 µM, similar to the K_i for the inhibition of charge (Fig. 7 B). There was also no effect of phlorizin on the V_0.5 (Fig. 7 D) and z for fluorescence (not depicted).

View larger version (9K): [in this window] [in a new window] [Download PPT slide]   Figure 8. Effect of phlorizin on fluorescence. Shown is the time course of F 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.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The goal of this study is to examine sugar interactions with SGLT1 and to place this in context of a kinetic model for Na⁺/sugar cotransport. We used a rhodamine-labeled mutant G507C of SGLT1 that transports sugar with similar kinetics as wild type. The kinetics of wild-type hSGLT1 and mutant G507C determined from the electrical and optical measurements (steady-state sugar-induced current, presteady-state current, and F measurements) are summarized in Table I. Apart from the slight increase in for mutant G507C compared with wild-type hSGLT1 (1.7 vs. 0.3–0.5 mM), the kinetics of hSGLT1 are similar to those of the TMR6M-labeled G507C. The values were identical from electrical and optical measurements.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   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 (C1CaCbC6), and Na+ binding/dissociation (C1C2). Rate constants (kij) for transitions between states (CiCj) are kij = kijo exp(–ijFV/RT), where kijo is a voltage-independent rate, ij 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.

Kinetic Model of SGLT1
Previously, we have developed and refined a model to account for the observed steady-state and presteady-state kinetics of SGLT1 (Parent et al., 1992b; Loo et al., 2002) Basically, this is a six-state, ordered, nonrapid equilibrium, alternating-access model with mirror symmetry and a Na⁺/glucose transport stoichiometry of 2. We postulated that the ligand-free carrier is negatively charged (valence –2). There are six kinetic states, empty (C₁ and C₆), Na⁺ bound (C₂ and C₅), and Na⁺- and sugar-bound (C₃ and C₄) states in the external and internal membrane surfaces (Fig. 9). The Na⁺ ions bind to the protein before glucose, and the substrates are transported simultaneously. To simplify the model we assumed the Na⁺ ions bind to two identical Na⁺ binding sites with the same rate constant. The only voltage-dependent steps are translocation of the ligand-free protein between the two sides of the membrane and the binding of external Na⁺. The effects of voltage on these reaction steps were described by the Eyring rate theory. The presteady-state currents are associated with the voltage-dependent steps: binding of external Na⁺ (C₁C₂, 30% of total charge; Loo et al., 1993; Hazama et al., 1997) and translocation of the empty carrier (C₁C₆, 70% of total charge).

All steady-state and presteady-state parameters (I/V curves, K_0.5 and I_max values for Na⁺ and sugar as a function of membrane voltage and given cis and trans concentrations) were computer generated simultaneously with numerical values assigned for the 12 of the 14 rate constants, and the results were compared and contrasted with the experimental datasets. Optimization of the simulations was accomplished by progressively adjusting each rate constant to obtain a global fit to the experimental results. A single set of rate constants, reasonable but not necessarily unique, was obtained that accounted for the overall behavior of the cotransporter, including the I/V curves in the presence and absence of sugar, the steady-state kinetics of inward Na⁺/sugar cotransport as a function of the external ligand concentrations and voltage (Parent et al., 1992b).

Subsequently, we tested and refined the model by detailed investigations of: (a) the presteady-state behavior of SGLT1 as a function of external Na⁺ concentration, membrane voltage, and temperature (Loo et al., 1993, 2005, Hazama et al., 1997); (b) the stoichiometry of Na⁺ and glucose cotransport (Mackenzie et al., 1998); (c) Na⁺ transport in the absence of substrate (Loo et al., 1999); (d) the kinetics of reverse Na⁺/glucose cotransport (Quick et al., 2003; Eskandari et al., 2005); (e) the conformational states of SGLT1 as a function of ligand concentrations and membrane voltage using optical techniques on a TMR6M-labeled hSGLT1 mutant (Q457C) where Na⁺/sugar cotransport is abolished after labeling (Loo et al., 1998, 2005; Meinild et al., 2002) and (f) electron microscopic techniques to determine the density of SGLT1 in the membrane (Zampighi et al., 1995). These comprehensive studies support the validly of the model but require the presence of two additional states (C_a and C_b) between C₁ and C₆ to account for the presteady-state behavior (shaded region of Fig. 9; Loo et al., 2005). The major discrepancies between the model and the observed behavior are the fast presteady-state kinetics in the submillisecond range, and these are thought to be due to assumptions about Na⁺ binding to two identical sites with similar rate constants (see Loo et al., 2005 and below). In this study we have employed the eight-state model (Fig. 9) along with electrical and optical methods to examine the conformational states during the Na⁺/glucose cotransport cycle. Since the binding of the high-affinity inhibitor phlorizin is Na⁺ dependent, this is incorporated into the model as C₂C₇ ([CNa₂]' + Pz[CNa₂Pz]') with ON and OFF rates k₂₇ and k₇₂ (Fig. 9). Phlorizin binding to the cytosolic surface of SGLT1 ([CNa₂]''+ Pz[CNa₂Pz]'') is neglected owing to the low affinity (K_i > 1 mM; Quick et al., 2003; Eskandari et al., 2005).

Model Simulations
As a starting point, we used the rate constants for the voltage-dependent steps from Loo et al. (2005). The rate constants for the sugar binding and translocation steps (k₂₃, k₃₂, k₃₄, and k₄₃) are from Hirayama et al. (1996). The rate constants for Na⁺ and sugar binding on the internal membrane are from Eskandari et al. (2005). We extend the model to mutant G507C by reducing the rates for sugar binding (k₂₃, k₅₄) for mutant G507C to account for the lower (Table I). A set of rate constants was obtained (Table II) that accounts qualitatively and quantitatively for the steady-state and presteady-state experimental data (see below). As a working hypothesis we assume that changes in fluorescence intensity (F) associated with SGLT1 were due to differences in apparent quantum yield of the bound fluorophore in different conformations: F qy₁C₁ + qy₂C₂ + qy₃C₃ + qy₄C₄ + qy₅C₅ + qy₆C₆ + qy_aC_a + qy_bC_b. This hypothesis is supported by the findings that in the SGLT homologue (vSGLT) from Vibrio parahaemolyticus (Veenstra et al., 2004), and hSGLT1 mutant Q457C (unpublished data), the sugar-induced quenches of fluorescence of covalently linked extrinsic fluorophores are not accompanied by shifts in absorption or emission spectra. As discussed below, since the occupancy probabilities of conformations C₁, C₃, C₄, C_a, and C_b are negligible, the fluorescence signal is reduced to F qy₂C₂ + qy₅C₅ + qy₆C₆.

Occupancy Probabilities.
Ligands and voltage alter the occupancy probability (P_o) of each state (Fig. 10 A). In external NaCl buffer, the transporter is predominantly in C₂ at negative membrane voltages (98% occupancy in C₂ at –150 mV), and in C₆ at positive voltages (e.g., 73% in C₆ at +50 mV). External sugar alters the di